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1.  Restoration of MiR-17/20a in Solid Tumor Cells Enhances the Natural Killer Cell Antitumor Activity by Targeting Mekk2 
Cancer immunology research  2014;2(8):789-799.
Aberrant microRNA (miRNA) expression has been identified in various human solid cancers. However, whether the levels of miRNA expression in tumor cells have any effect on tumor progression has not been determined. In this proof-of-concept study, the restoration of high-level expression of the miR-17~92 cluster of miRNAs reveals its function as a tumor suppressor in murine solid cancer cells. Specifically, genetically engineered expression of higher levels of miR-17/20a in the miR-17~92 cluster in both murine breast cancer and colon cancer cells triggered natural killer (NK) cell recognition by inhibiting the expression of MHC class I (H-2D) through the Mekk2/Mek5/Erk5 pathway. Results from the mouse tumor studies were recapitulated using samples of human solid tumors. Together, these data indicate that miR-17/20amiRnas functions as a tumor suppressor by reprogramming tumor cells for NK cell-mediated cytotoxicity.
doi:10.1158/2326-6066.CIR-13-0162
PMCID: PMC4396632  PMID: 24801835
miR-17/20a; miR-17~92 cluster; MHC class I; Mekk2/Erk5 pathway; colon cancer; breast cancer; NK cell tumor cytotoxicity
2.  Polo-like kinase 2 activates an antioxidant pathway to promote the survival of cells with mitochondrial dysfunction 
We previously reported that Polo-like kinase 2 (PLK2) is highly expressed in cells with defective mitochondrial respiration and is essential for their survival. Although PLK2 has been widely studied as a cell cycle regulator, we have uncovered an antioxidant function for this kinase that activates the GSK3-NRF2 signaling pathway. Here, we report that the expression of PLK2 is responsive to oxidative stress and that PLK2 mediates antioxidant signaling by phosphorylating GSK3, thereby promoting the nuclear translocation of NRF2. We further show that the antioxidant activity of PLK2 is essential for preventing p53-dependent necrotic cell death. Thus, the regulation of redox homeostasis by PLK2 promotes the survival of cells with dysfunctional mitochondria, which may have therapeutic implications for cancer and neurodegenerative diseases.
doi:10.1016/j.freeradbiomed.2014.05.022
PMCID: PMC4115326  PMID: 24887096
Polo-like kinase 2; Antioxidant; Mitochondrial dysfunction; Oxidative stress; GSK3; NRF2; p53; Necrosis
3.  MiR-34a regulates blood–tumor barrier function by targeting protein kinase Cε 
Molecular Biology of the Cell  2015;26(10):1786-1796.
It is shown for the first time that overexpression of miR-34a increases blood–tumor barrier permeability by targeting PKCε, which is activated by p-PKCε and directly regulates the expression of tight junction–related proteins.
MicroRNA-34a (miR-34a) functions to regulate protein expression at the posttranscriptional level by binding the 3′ UTR of target genes and regulates functions of vascular endothelial cells. However, the role of miR-34a in regulating blood–tumor barrier (BTB) permeability remains unknown. In this study, we show that miR-34a overexpression leads to significantly increased permeability of BTB, whereas miR-34a silencing reduces the permeability of the BTB. In addition, miR-34a overexpression significantly down-regulates the expression and distribution of tight junction–related proteins in glioma endothelial cells (GECs), paralleled by protein kinase Cε (PKCε) reduction. Moreover, luciferase reporter gene analysis shows that PKCε is the target gene of miR-34a. We also show that cotransfection of miR-34a and PKCε inversely coregulates BTB permeability and protein expression levels of tight junction–related proteins. Pretreatment of ψεRACK, a PKCε-specific activator, decreases BTB permeability in miR-34a–overexpressed GECs and up-regulates expression levels of tight junction proteins. In contrast, pretreatment of εV1-2, a specific PKCε inhibitor, gives opposite results. Collectively, our findings indicate that miR-34a regulates BTB function by targeting PKCε; after phosphorylation, PKCε is activated and contributes to regulation of the expression of tight junction–related proteins, ultimately altering BTB permeability.
doi:10.1091/mbc.E14-10-1474
PMCID: PMC4436826  PMID: 25788289
4.  Characterization of Chromosome Inheritance of the Intergeneric BC2 and BC3 Progeny between Saccharum spp. and Erianthus arundinaceus 
PLoS ONE  2015;10(7):e0133722.
Erianthus arundinaceus (E. arundinaceus) has many desirable agronomic traits for sugarcane improvement, such as high biomass, vigor, rationing ability, tolerance to drought, and water logging, as well as resistance to pests and disease. To investigate the introgression of the E. arundinaceus genome into sugarcane in the higher generations, intergeneric BC2 and BC3 progeny generated between Saccharum spp. and E. arundinaceus were studied using the genomic in situ hybridization (GISH) technique. The results showed that the BC2 and BC3 generations resulted from n + n chromosome transmission. Furthermore, chromosome translocation occurred at terminal fragments from the E. arundinaceus chromosome in some progeny of Saccharum spp. and E. arundinaceus. Notably, the translocated chromosomes could be stably transmitted to their progeny. This study illustrates the characterization of chromosome inheritance of the intergeneric BC2 and BC3 progeny between Saccharum spp. and E. arundinaceus. This work could provide more useful molecular cytogenetic information for the germplasm resources of E. arundinaceus, and may promote further understanding of the germplasm resources of E. arundinaceus for sugarcane breeders to accelerate its progress in sugarcane commercial breeding.
doi:10.1371/journal.pone.0133722
PMCID: PMC4510360  PMID: 26196281
5.  Host-Guest Carbon Dots for Enhanced Optical Properties and Beyond 
Scientific Reports  2015;5:12354.
Carbon dots, generally small carbon nanoparticles with various forms of surface passivation, have achieved the performance level of semiconductor quantum dots in the green spectral region, but their absorption and fluorescence in red/near-IR are relatively weaker. Conceptually similar to endofullerenes, host-guest carbon dots were designed and prepared with red/near-IR dyes encapsulated as guest in the carbon nanoparticle core. Beyond the desired enhancement in optical properties, the host-guest configuration may significantly broaden the field of carbon dots.
doi:10.1038/srep12354
PMCID: PMC4508828  PMID: 26196598
6.  Microsecond Scale Vibrational Spectroscopic Imaging by Multiplex Stimulated Raman Scattering Microscopy 
Real-time vibrational spectroscopic imaging is desired for monitoring cellular states and cellular processes in a label-free manner. Raman spectroscopic imaging of highly dynamic systems is inhibited by relatively slow spectral acquisition on millisecond to second scale. Here, we report microsecond scale vibrational spectroscopic imaging by lock-in free parallel detection of spectrally dispersed stimulated Raman scattering signal. Using a homebuilt tuned amplifier array, our method enables Raman spectral acquisition, within the window defined by the broadband pulse, at the speed of 32 microseconds and with close to shot-noise limited detection sensitivity. Incorporated with multivariate curve resolution analysis, our platform allows compositional mapping of lipid droplets in single live cells, observation of intracellular retinoid metabolism, discrimination of fat droplets from protein-rich organelles in Caenorhabditis elegans, spectral detection of fast flowing tumor cells, and monitoring drug diffusion through skin tissue in vivo. The reported technique opens new opportunities for compositional analysis of cellular compartment in a microscope setting and high-throughput spectral profiling of single cells in a flow cytometer setting.
doi:10.1038/lsa.2015.38
PMCID: PMC4498251  PMID: 26167336
Optical microscopy; Raman scattering; Vibrational spectroscopy
7.  A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging 
Science (New York, N.Y.)  2015;348(6239):1160-1163.
Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.
doi:10.1126/science.aaa1356
PMCID: PMC4494668  PMID: 25931448
8.  Cold-inducible RNA-binding Protein Mediates Neuroinflammation in Cerebral Ischemia 
Biochimica et biophysica acta  2014;1840(7):2253-2261.
Background
Neuroinflammation is a key cascade after cerebral ischemia. Excessive production of proinflammatory mediators in ischemia exacerbates brain injury. Cold-inducible RNA- binding protein (CIRP) is a newly discovered proinflammatory mediator that can be released into the circulation during hemorrhage or septic shock. Here, we examine the involvement of CIRP in brain injury during ischemic stroke.
Methods
Stroke was induced by middle cerebral artery occlusion (MCAO). In vitro hypoxia was conducted in a hypoxia chamber containing 1% oxygen. CIRP and tumor necrosis factor-α (TNF-α) levels were assessed by RT-PCR and Western blot analysis.
Results
CIRP is elevated along with an upregulation of TNF-α expression in mouse brain after MCAO. In CIRP-deficient mice, the brain infarct volume, induction of TNF-α, and activation of microglia are markedly reduced after MCAO. Using microglial BV2 cells, we demonstrate that hypoxia induces the expression, translocation, and release of CIRP, which is associated with an increase of TNF-α levels. Addition of recombinant murine (rm) CIRP directly induces TNF-α release from BV2 cells and such induction is inhibited by neutralizing antisera to CIRP. Moreover, rmCIRP activates the NF-κB signaling pathway in BV2 cells. The conditioned medium from BV2 cells exposed to hypoxia triggers the apoptotic cascade by increasing caspase activity and decreasing Bcl-2 expression in neural SH-SY5Y cells, which is inhibited by antisera to CIRP.
Conclusion
Extracellular CIRP is a detrimental factor in stimulating inflammation to cause neuronal damage in cerebral ischemia.
General significance
Development of an anti-CIRP therapy may benefit patients with brain ischemia.
doi:10.1016/j.bbagen.2014.02.027
PMCID: PMC4061249  PMID: 24613680
Cerebral ischemia; Hypoxia; Cold shock protein; Inflammation; Microglia; Neural cells
9.  Bone regeneration via novel macroporous CPC scaffolds in critical-sized cranial defects in rats 
Objectives
Calcium phosphate cement (CPC) is promising for dental and craniofacial applications due to its ability to be injected or filled into complex-shaped bone defects and molded for esthetics, and its resorbability and replacement by new bone. The objective of this study was to investigate bone regeneration via novel macroporous CPC containing absorbable fibers, hydrogel microbeads and growth factors in critical-sized cranial defects in rats.
Methods
Mannitol porogen and alginate hydrogel microbeads were incorporated into CPC. Absorbable fibers were used to provide mechanical reinforcement to CPC scaffolds. Six CPC groups were tested in rats: (1) Control CPC without macropores and microbeads; (2) Macroporous CPC + large fiber; (3) Macroporous CPC + large fiber + nanofiber; (4) Same as (3), but with rhBMP2 in CPC matrix; (5) Same as (3), but with rhBMP2 in CPC matrix + rhTGF-β1 in microbeads; (6) Same as (3), but with rhBMP2 in CPC matrix + VEGF in microbeads. Rats were sacrificed at 4 and 24 weeks for histological and micro-CT analyses.
Results
The macroporous CPC scaffolds containing porogen, absorbable fibers and hydrogel microbeads had mechanical properties similar to cancellous bone. At 4 weeks, the new bone area fraction (mean ± sd; n = 5) in CPC control group was the lowest at (14.8 ± 3.3)%, and that of group 6 (rhBMP2 + VEGF) was (31.0 ± 13.8)% (p < 0.05). At 24 weeks, group 4 (rhBMP2) had the most new bone of (38.8 ± 15.6)%, higher than (12.7 ± 5.3)% of CPC control (p < 0.05). Micro-CT revealed nearly complete bridging of the critical-sized defects with new bone for several macroporous CPC groups, compared to much less new bone formation for CPC control.
Significance
Macroporous CPC scaffolds containing porogen, fibers and microbeads with growth factors were investigated in rat cranial defects for the first time. Macroporous CPCs had new bone up to 2-fold that of traditional CPC control at 4 weeks, and 3-fold that of traditional CPC at 24 weeks, and hence may be useful for dental, craniofacial and orthopedic applications.
doi:10.1016/j.dental.2014.03.008
PMCID: PMC4312701  PMID: 24768062
Calcium phosphate cement; macroporous scaffold; strength and toughness; bone regeneration; growth factors; critical-sized cranial defects
10.  Changes in short-chain acyl-coA dehydrogenase during rat cardiac development and stress 
This study was designed to investigate the expression of short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme of fatty acid β-oxidation, during rat heart development and the difference of SCAD between pathological and physiological cardiac hypertrophy. The expression of SCAD was lowest in the foetal and neonatal heart, which had time-dependent increase during normal heart development. In contrast, a significant decrease in SCAD expression was observed in different ages of spontaneously hypertensive rats (SHR). On the other hand, swim-trained rats developed physiological cardiac hypertrophy, whereas SHR developed pathological cardiac hypertrophy. The two kinds of cardiac hypertrophy exhibited divergent SCAD changes in myocardial fatty acids utilization. In addition, the expression of SCAD was significantly decreased in pathological cardiomyocyte hypertrophy, however, increased in physiological cardiomyocyte hypertrophy. SCAD siRNA treatment triggered the pathological cardiomyocyte hypertrophy, which showed that the down-regulation of SCAD expression may play an important role in pathological cardiac hypertrophy. The changes in peroxisome proliferator-activated receptor α (PPARα) was accordant with that of SCAD. Moreover, the specific PPARα ligand fenofibrate treatment increased the expression of SCAD and inhibited pathological cardiac hypertrophy. Therefore, we speculate that the down-regulated expression of SCAD in pathological cardiac hypertrophy may be responsible for ‘the recapitulation of foetal energy metabolism’. The deactivation of PPARα may result in the decrease in SCAD expression in pathological cardiac hypertrophy. Changes in SCAD are different in pathological and physiological cardiac hypertrophy, which may be used as the molecular markers of pathological and physiological cardiac hypertrophy.
doi:10.1111/jcmm.12541
PMCID: PMC4511364  PMID: 25753319
short-chain acyl-CoA dehydrogenase; peroxisome proliferator-activated receptor α; heart development; pathological cardiac hypertrophy; physiological cardiac hypertrophy
11.  Analysis of clinical efficacy of CyberKnife® treatment for locally advanced pancreatic cancer 
OncoTargets and therapy  2015;8:1427-1431.
Objective
To evaluate the efficacy and safety of CyberKnife® treatment for locally-advanced pancreatic cancer (LAPC).
Methods
The efficacy of CyberKnife® treatment was analyzed in 59 LAPC patients treated between October 2006 and September 2014. The median tumor volume was 27.1 mL (13.0–125.145 mL). The median prescribed dose was 45 Gy (35–50 Gy), delivered in 5 fractions (3–8 fractions). The overall survival (OS) rates and freedom from local progression (FFLP) rates were estimated using the Kaplan–Meier survival curve.
Results
The median follow-up for all patients was 10.9 months (3.2–48.7 months) and 15.6 months (3.9–37.6 months) among surviving patients. The median OS was 12.5 months, and the 1-year and 2-year survival rates were 53.9% and 35.1%, respectively. The 1-year FFLP rate was 90.8% based on the computed tomography (CT) evaluation. Grade 1–2 acute and late-stage gastrointestinal (GI) reactions were observed in 61% of the patients. One patient experienced grade 3 toxicity.
Conclusion
Excellent clinical efficacy was obtained after treatment of LAPC using CyberKnife®, with minimal toxicity.
doi:10.2147/OTT.S81939
PMCID: PMC4472027  PMID: 26109866
pancreatic cancer; stereotactic body radiotherapy; CyberKnife®; local control; toxicity
12.  Phylogenetic relationships of subfamilies in the family Hesperiidae (Lepidoptera: Hesperioidea) from China 
Scientific Reports  2015;5:11140.
Hesperiidae is one of the largest families of butterflies. Our knowledge of the higher systematics on hesperiids from China is still very limited. We infer the phylogenetic relationships of the subfamilies of Chinese skippers based on three mitochondrial genes (cytochrome b (Cytb), the NADH dehydrogenase subunit 1 (ND1) and cytochrome oxidase I (COI)). In this study, 30 species in 23 genera were included in the Bayesian and maximum likelihood analyses. The subfamily Coeliadinae, Eudaminae, Pyrginae and Heteropterinae were recovered as a monophyletic clade with strong support. The subfamily Hesperiinae formed a clade, but support for monophyly was weak. Our results imply that the five subfamilies of Chinese Hesperiidae should be divided into: Coeliadinae, Eudaminae, Pyrginae, Heteropterinae and Hesperiinae. The relationships of the five subfamilies should be as follows: Coeliadinae + (Eudaminae + (Pyrginae + (Heteropterinae + Hesperiinae))).
doi:10.1038/srep11140
PMCID: PMC4461911  PMID: 26059470
13.  Temporal MRI characterization, neurobiochemical and neurobehavioral changes in a mouse repetitive concussive head injury model 
Scientific Reports  2015;5:11178.
Single and repeated sports-related mild traumatic brain injury (mTBI), also referred to as concussion, can result in chronic post-concussive syndrome (PCS), neuropsychological and cognitive deficits, or chronic traumatic encephalopathy (CTE). However PCS is often difficult to diagnose using routine clinical, neuroimaging or laboratory evaluations, while CTE currently only can be definitively diagnosed postmortem. We sought to develop an animal model to simulate human repetitive concussive head injury for systematic study. In this study, mice received single or multiple head impacts by a stereotaxic impact device with a custom-made rubber tip-fitted impactor. Dynamic changes in MRI, neurobiochemical markers (Tau hyperphosphorylation and glia activation in brain tissues) and neurobehavioral functions such as anxiety, depression, motor function and cognitive function at various acute/subacute (1-7 day post-injury) and chronic (14-60 days post-injury) time points were examined. To explore the potential biomarkers of rCHI, serum levels of total Tau (T-Tau) and phosphorylated Tau (P-Tau) were also monitored at various time points. Our results show temporal dynamics of MRI consistent with structural perturbation in the acute phase and neurobiochemical changes (P-Tau and GFAP induction) in the subacute and chronic phase as well as development of chronic neurobehavioral changes, which resemble those observed in mTBI patients.
doi:10.1038/srep11178
PMCID: PMC4461921  PMID: 26058556
14.  Epigenetic state network approach for describing cell phenotypic transitions 
Interface Focus  2014;4(3):20130068.
Recent breakthroughs of cell phenotype reprogramming impose theoretical challenges on unravelling the complexity of large circuits maintaining cell phenotypes coupled at many different epigenetic and gene regulation levels, and quantitatively describing the phenotypic transition dynamics. A popular picture proposed by Waddington views cell differentiation as a ball sliding down a landscape with valleys corresponding to different cell types separated by ridges. Based on theories of dynamical systems, we establish a novel ‘epigenetic state network’ framework that captures the global architecture of cell phenotypes, which allows us to translate the metaphorical low-dimensional Waddington epigenetic landscape concept into a simple-yet-predictive rigorous mathematical framework of cell phenotypic transitions. Specifically, we simplify a high-dimensional epigenetic landscape into a collection of discrete states corresponding to stable cell phenotypes connected by optimal transition pathways among them. We then apply the approach to the phenotypic transition processes among fibroblasts (FBs), pluripotent stem cells (PSCs) and cardiomyocytes (CMs). The epigenetic state network for this case predicts three major transition pathways connecting FBs and CMs. One goes by way of PSCs. The other two pathways involve transdifferentiation either indirectly through cardiac progenitor cells or directly from FB to CM. The predicted pathways and multiple intermediate states are supported by existing microarray data and other experiments. Our approach provides a theoretical framework for studying cell phenotypic transitions. Future studies at single-cell levels can directly test the model predictions.
doi:10.1098/rsfs.2013.0068
PMCID: PMC3996583  PMID: 24904734
gene regulatory network; nonlinear dynamics; non-equilibrium steady state
15.  A Network Meta-Analysis of Efficacy and Evaluation of Safety of Subcutaneous Pegylated Interferon Beta-1a versus Other Injectable Therapies for the Treatment of Relapsing-Remitting Multiple Sclerosis 
PLoS ONE  2015;10(6):e0127960.
Subcutaneous pegylated interferon beta-1a (peginterferon beta-1a [PEG-IFN]) 125 μg every two or four weeks has been studied in relapsing-remitting multiple sclerosis (RRMS) patients in the pivotal Phase 3 ADVANCE trial. In the absence of direct comparative evidence, a network meta-analysis (NMA) was conducted to provide an indirect assessment of the relative efficacy, safety, and tolerability of PEG-IFN versus other injectable RRMS therapies. Systematic searches were conducted in MEDLINE, Embase, and the Cochrane Library, and conference proceedings from relevant annual symposia were hand-searched. Included studies were randomized controlled trials evaluating ≥1 first-line treatments including interferon beta-1a 30, 44, and 22 μg, interferon beta-1b, and glatiramer acetate in patients with RRMS. Studies were included based on a pre-specified protocol and extracted by a team of independent reviewers and information scientists, utilizing criteria from NICE and IQWiG. In line with ADVANCE findings, NMA results support that PEG-IFN every 2 weeks significantly reduced annualized relapse rate, and 3- and 6-month confirmed disability progression (CDP) versus placebo. There was numerical trend favoring PEG-IFN every 2 weeks versus other IFNs assessed for annualized relapse rate, and versus all other injectables for 3- and 6-month CDP (6-month CDP was significantly reduced versus IFN beta-1a 30 μg). The safety and tolerability profile of PEG-IFN beta-1a 125 μg every 2 weeks was consistent with that of other evaluated treatments. Study limitations for the NMA include variant definitions of relapse and other systematic differences across trials, assumptions that populations were sufficiently similar, and inability to perform NMA of adverse events. With similar efficacy compared to other RRMS treatments in terms of annualized relapse rate and 3- and 6-month CDP, a promising safety profile, and up to 93% reduction in number of injections (which may improve adherence), PEG-IFN every 2 weeks offers a valuable alternative treatment option for patients with RRMS.
doi:10.1371/journal.pone.0127960
PMCID: PMC4454514  PMID: 26039748
16.  Transcriptome Responses of the Host Trichoplusia ni to Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus 
Journal of Virology  2014;88(23):13781-13797.
ABSTRACT
Productive infection of Trichoplusia ni cells by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) leads to expression of ∼156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni. We used an ∼60-Gb RNA sequencing (RNA-seq) data set from infected and uninfected T. ni cells to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes (assembled transcripts), representing the 48-h infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were downregulated after 6 h postinfection (p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4,028) of the T. ni unigenes were upregulated during the early period (0 to 6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i. but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathway members that were upregulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure.
IMPORTANCE Baculoviruses are insect-specific DNA viruses that are highly pathogenic to their insect hosts. In addition to their use for biological control of certain insects, baculoviruses also serve as viral vectors for numerous biotechnological applications, such as mammalian cell transduction and protein expression for vaccine production. While there is considerable information regarding viral gene expression in infected cells, little is known regarding responses of the host cell to baculovirus infection. In these studies, we assembled a cell transcriptome from the host Trichoplusia ni and used that transcriptome to analyze changes in host cell gene expression throughout the infection cycle. The study was performed in parallel with a prior study of changes in viral gene expression. Combined, these studies provide an unprecedented new level of detail and an overview of events in the infection cycle, and they will stimulate new experimental approaches to understand, modify, and utilize baculoviruses for a variety of applications.
doi:10.1128/JVI.02243-14
PMCID: PMC4248991  PMID: 25231311
17.  Evaluation of antibacterial and remineralizing nanocomposite and adhesive in rat tooth cavity model 
Acta biomaterialia  2014;10(6):2804-2813.
Antibacterial and remineralizing dental composites and adhesives were recently developed to inhibit biofilm acids and combat secondary caries. It is not clear what effect these materials will have on dental pulps in vivo. The objectives of this study were to investigate the antibacterial and remineralizing restorations in a rat tooth cavity model, and determine pulpal inflammatory response and tertiary dentin formation. Nanoparticles of amorphous calcium phosphate (NACP) and antibacterial dimethylaminododecyl methacrylate (DMADDM) were synthesized and incorporated into a composite and an adhesive. Occlusal cavities were prepared in the first molars of rats and restored with four types of restoration: Control composite and adhesive; control plus DMADDM; control plus NACP; and control plus both DMADDM and NACP. At 8 or 30 days (d), rat molars were harvested for histological analysis. For inflammatory cell response, regardless of time periods, NACP group and DMADDM+NACP group showed lower scores (better biocompatibility) than control group (p = 0.014 for 8 d, p = 0.018 for 30 d). For tissue disorganization, NACP and DMADDM+NACP had better scores than control (p = 0.027) at 30 d. At 8 d, restorations containing NACP had tertiary dentin thickness (TDT) that was 5-6 fold that of control. At 30 d, restorations containing NACP had TDT that was 4-6 fold that of control. In conclusion, novel antibacterial and remineralizing restorations were tested in rat teeth in vivo for the first time. Composite and adhesive containing NACP and DMADDM exhibited milder pulpal inflammation and much greater tertiary dentin formation, than control adhesive and composite. Therefore, the novel composite and adhesive containing NACP and DMADDM are promising as a new therapeutic restorative system to not only combat oral pathogens and biofilm acids as shown previously, but also facilitate the healing of the dentin-pulp complex.
doi:10.1016/j.actbio.2014.02.033
PMCID: PMC4312698  PMID: 24583320
Dental nanocomposite; bonding agent; calcium phosphate nanoparticles; quaternary ammonium methacrylate; rat tooth cavity; in vivo properties
18.  Oxytocin inhibits the activity of acid-sensing ion channels through the vasopressin, V1A receptor in primary sensory neurons 
British Journal of Pharmacology  2014;171(12):3065-3076.
BACKGROUND AND PURPOSE
A growing number of studies have demonstrated that oxytocin (OT) plays an analgesic role in modulation of nociception and pain. Most work to date has focused on the central mechanisms of OT analgesia, but little is known about whether peripheral mechanisms are also involved. Acid-sensing ion channels (ASICs) are distributed in peripheral sensory neurons and participate in nociception. Here, we investigated the effects of OT on the activity of ASICs in dorsal root ganglion (DRG) neurons.
EXPERIMENTAL APPROACH
Electrophysiological experiments were performed on neurons from rat DRG. Nociceptive behaviour was induced by acetic acid in rats and mice lacking vasopressin, V1A receptors.
KEY RESULTS
OT inhibited the functional activity of native ASICs. Firstly, OT dose-dependently decreased the amplitude of ASIC currents in DRG neurons. Secondly, OT inhibition of ASIC currents was mimicked by arginine vasopressin (AVP) and completely blocked by the V1A receptor antagonist SR49059, but not by the OT receptor antagonist L-368899. Thirdly, OT altered acidosis-evoked membrane excitability of DRG neurons and significantly decreased the amplitude of the depolarization and number of action potentials induced by acid stimuli. Finally, peripherally administered OT or AVP inhibited nociceptive responses to intraplantar injection of acetic acid in rats. Both OT and AVP also induced an analgesic effect on acidosis-evoked pain in wild-type mice, but not in V1A receptor knockout mice.
CONCLUSIONS AND IMPLICATIONS
These results reveal a novel peripheral mechanism for the analgesic effect of OT involving the modulation of native ASICs in primary sensory neurons mediated by V1A receptors.
doi:10.1111/bph.12635
PMCID: PMC4055206  PMID: 24641084
oxytocin; acid-sensing ion channel; proton-gated current; vasopressin; V1A receptor; dorsal root ganglion neuron; electrophysiology; pain
19.  Fast Vibrational Imaging of Single Cells and Tissues by Stimulated Raman Scattering Microscopy 
Accounts of Chemical Research  2014;47(8):2282-2290.
Conspectus
Traditionally, molecules are analyzed in a test tube. Taking biochemistry as an example, the majority of our knowledge about cellular content comes from analysis of fixed cells or tissue homogenates using tools such as immunoblotting and liquid chromatography–mass spectrometry. These tools can indicate the presence of molecules but do not provide information on their location or interaction with each other in real time, restricting our understanding of the functions of the molecule under study. For real-time imaging of labeled molecules in live cells, fluorescence microscopy is the tool of choice. Fluorescent labels, however, are too bulky for small molecules such as fatty acids, amino acids, and cholesterol. These challenges highlight a critical need for development of chemical imaging platforms that allow in situ or in vivo analysis of molecules. Vibrational spectroscopy based on spontaneous Raman scattering is widely used for label-free analysis of chemical content in cells and tissues. However, the Raman process is a weak effect, limiting its application for fast chemical imaging of a living system.
With high imaging speed and 3D spatial resolution, coherent Raman scattering microscopy is enabling a new approach for real-time vibrational imaging of single cells in a living system. In most experiments, coherent Raman processes involve two excitation fields denoted as pump at ωp and Stokes at ωs. When the beating frequency between the pump and Stokes fields (ωp – ωs) is resonant with a Raman-active molecular vibration, four major coherent Raman scattering processes occur simultaneously, namely, coherent anti-Stokes Raman scattering (CARS) at (ωp – ωs) + ωp, coherent Stokes Raman scattering (CSRS) at ωs – (ωp – ωs), stimulated Raman gain (SRG) at ωs, and stimulated Raman loss (SRL) at ωp. In SRG, the Stokes beam experiences a gain in intensity, whereas in SRL, the pump beam experiences a loss. Both SRG and SRL belong to stimulated Raman scattering (SRS), in which the energy difference between the pump and Stokes fields is transferred to the molecule for vibrational excitation. The SRS signal appears at the same wavelengths as the excitation fields and is commonly extracted through a phase-sensitive detection scheme. The detected intensity change because of a Raman transition is proportional to Im[χ(3)]IpIs, where χ(3) represents the third-order nonlinear susceptibility, Ip and Is stand for the intensity of the pump and Stokes fields.
In this Account, we discuss the most recent advances in the technical development and enabling applications of SRS microscopy. Compared to CARS, the SRS contrast is free of nonresonant background. Moreover, the SRS intensity is linearly proportional to the density of target molecules in focus. For single-frequency imaging, an SRS microscope offers a speed that is ∼1000 times faster than a line-scan Raman microscope and 10 000 times faster than a point-scan Raman microscope. It is important to emphasize that SRS and spontaneous Raman scattering are complementary to each other. Spontaneous Raman spectroscopy covers the entire window of molecular vibrations, which allows extraction of subtleties via multivariate analysis. SRS offers the speed advantage by focusing on either a single Raman band or a defined spectral window of target molecules. Integrating single-frequency SRS imaging and spontaneous Raman spectroscopy on a single platform allows quantitative compositional analysis of objects inside single live cells.
doi:10.1021/ar400331q
PMCID: PMC4139189  PMID: 24871269
20.  Association of the CYP24A1-rs2296241 polymorphism of the vitamin D catabolism enzyme with hormone-related cancer risk: a meta-analysis 
OncoTargets and therapy  2015;8:1175-1183.
Background
The evidence for vitamin D reducing cancer risk is inconsistent, and it is not clear whether this reduction is related to variation in cytochrome P450 (CYP)24A1, the only enzyme known to degrade active vitamin D. We focused on evaluating the association of CYP24A1-rs2296241 polymorphism with hormone-related cancer risk by conducting a meta-analysis.
Methods
A systematic literature search was conducted in April 2014 (updated in December 2014) to identify eligible studies. A random-effects model was used to pool the odds ratio (OR).
Results
Eleven studies including 5,145 cases and 5,136 controls were considered for the allelic model, and eight studies of 3,959 cases and 3,560 controls were utilized for the additive, recessive, and dominant models. There was no significant association between CYP24A1-rs2296241 and hormone-related cancer risk in any of the models, yet substantial heterogeneity was observed. Subgroup analyses indicated that CYP24A1-rs2296241 variation reduced the prostate cancer risk in the additive (OR 0.91, 95% confidence interval 0.85–0.97) and recessive (OR 0.80, 95% confidence interval 0.67–0.95) models, with no evidence of heterogeneity.
Conclusion
This meta-analysis indicated that CYP24A1-rs2296241 polymorphism reduced the androgen-related prostate cancer risk in additive and recessive models. More genetic loci are needed to confirm the effect of CYP24A1 variation on the risk of prostate cancer.
doi:10.2147/OTT.S80311
PMCID: PMC4447172  PMID: 26045671
CYP24A1; hormone-related cancer; meta-analysis; polymorphism; vitamin D
21.  Metabolomics for Biomarker Discovery: Moving to the Clinic 
BioMed Research International  2015;2015:354671.
To improve the clinical course of diseases, more accurate diagnostic and assessment methods are required as early as possible. In order to achieve this, metabolomics offers new opportunities for biomarker discovery in complex diseases and may provide pathological understanding of diseases beyond traditional technologies. It is the systematic analysis of low-molecular-weight metabolites in biological samples and has become an important tool in clinical research and the diagnosis of human disease and has been applied to discovery and identification of the perturbed pathways. It provides a powerful approach to discover biomarkers in biological systems and offers a holistic approach with the promise to clinically enhance diagnostics. When carried out properly, it could provide insight into the understanding of the underlying mechanisms of diseases, help to identify patients at risk of disease, and predict the response to specific treatments. Currently, metabolomics has become an important tool in clinical research and the diagnosis of human disease and becomes a hot topic. This review will highlight the importance and benefit of metabolomics for identifying biomarkers that accurately screen potential biomarkers of diseases.
doi:10.1155/2015/354671
PMCID: PMC4452245  PMID: 26090402
22.  Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation 
Nature Communications  2015;6:7023.
DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1–USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1–USP7 interaction surface for therapeutic applications.
DNMT1 is a methyl-transferase involved in maintaining tissue-specific patterns of DNA methylation. Here the authors solve the structure of a DNMT1-USP7 complex and demonstrate the mechanism by which DNMT1 stability is regulated through acetylation by preventing association with the deubiquitinase USP7.
doi:10.1038/ncomms8023
PMCID: PMC4432644  PMID: 25960197
23.  Progesterone reduces inflammation and apoptosis in neonatal rats with hypoxic ischemic brain damage through the PI3K/Akt pathway 
A neonatal rat model with hypoxic ischemic brain damage (HIBD) was established. Forty 7-day-old neonatal Wistar rats were randomly divided into four groups: sham operation, model, progesterone and Akt inhibitor. Electron microscopy revealed that the neonatal rats with HIBD showed neuronal changes. The protein expression levels of pAkt, Nuclear factor κB (NF-κB) and Bcl-2 in the hippocampus were detected by immunohistochemistry and Western blot. The neuronal structure was normal in the sham operation group after HIBD for 24 h. Cavitation change due to hypoxic ischemic brain damage was observed in the neurons of the model group. Progesterone treatment improved neuronal damage and cavitation. Neuronal cavitation was clearly changed in the Akt inhibitor group. The protein expression levels of hippocampal pAkt and Bcl-2 did not significantly change after HIBD, whereas that of NF-κB increased. Progesterone pre-treatment increased the expression levels of pAkt and Bcl-2 but decreased that of NF-κB. The protein expression levels of pAkt and Bcl-2 decreased in the Akt inhibitor group, whereas that of NF-κB increased. This result indicates that progesterone can decrease inflammation in HIBD, inhibit apoptosis and protect the brain by activating the Phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway.
PMCID: PMC4509338  PMID: 26221393
Progesterone; hypoxic-ischemic brain damage; PI3K/Akt; NF-κB; Bcl-2
24.  Origin of tensile strength of a woven sample cut in bias directions 
Royal Society Open Science  2015;2(5):140499.
Textile fabrics are highly anisotropic, so that their mechanical properties including strengths are a function of direction. An extreme case is when a woven fabric sample is cut in such a way where the bias angle and hence the tension loading direction is around 45° relative to the principal directions. Then, once loaded, no yarn in the sample is held at both ends, so the yarns have to build up their internal tension entirely via yarn–yarn friction at the interlacing points. The overall fabric strength in such a sample is a result of contributions from the yarns being pulled out and those broken during the process, and thus becomes a function of the bias direction angle θ, sample width W and length L, along with other factors known to affect fabric strength tested in principal directions. Furthermore, in such a bias sample when the major parameters, e.g. the sample width W, change, not only the resultant strengths differ, but also the strength generating mechanisms (or failure types) vary. This is an interesting problem and is analysed in this study. More specifically, the issues examined in this paper include the exact mechanisms and details of how each interlacing point imparts the frictional constraint for a yarn to acquire tension to the level of its strength when both yarn ends were not actively held by the testing grips; the theoretical expression of the critical yarn length for a yarn to be able to break rather than be pulled out, as a function of the related factors; and the general relations between the tensile strength of such a bias sample and its structural properties. At the end, theoretical predictions are compared with our experimental data.
doi:10.1098/rsos.140499
PMCID: PMC4453249  PMID: 26064655
fabric strength; bias directions; yarn pullout and breakage; failure types; sample width effect
25.  eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells 
PLoS ONE  2015;10(5):e0125389.
Objective
We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs).
Methods
Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.
Results
Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.
Conclusions
eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.
doi:10.1371/journal.pone.0125389
PMCID: PMC4427111  PMID: 25962137

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