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1.  Effect of perinatal asphyxia and carbamazepine treatment on cortical dopamine and DOPAC levels 
One of the most important manifestations of perinatal asphyxia is the occurrence of seizures, which are treated with antiepileptic drugs, such as carbamazepine. These early seizures, combined with pharmacological treatments, may influence the development of dopaminergic neurotransmission in the frontal cortex. This study aimed to determine the extracellular levels of dopamine and its main metabolite DOPAC in 30-day-old rats that had been asphyxiated for 45 min in a low (8%) oxygen chamber at a perinatal age and treated with daily doses of carbamazepine. Quantifications were performed using microdialysis coupled to a high-performance liquid chromatography (HPLC) system in basal conditions and following the use of the chemical stimulus.
Significant decreases in basal and stimulated extracellular dopamine and DOPAC content were observed in the frontal cortex of the asphyxiated group, and these decreases were partially recovered in the animals administered daily doses of carbamazepine. Greater basal dopamine concentrations were also observed as an independent effect of carbamazepine.
Perinatal asphyxia plus carbamazepine affects extracellular levels of dopamine and DOPAC in the frontal cortex and stimulated the release of dopamine, which provides evidence for the altered availability of dopamine in cortical brain areas during brain development.
PMCID: PMC4335632
Perinatal asphyxia; Carbamazepine; Dopamine; Frontal cortex; Microdialysis
2.  Effects of human blood red cells on the haemolytic capability of clinical isolates of Candida tropicalis 
Candida tropicalis is an increasingly important human pathogen associated with high mortality rates; however, little is known regarding the virulence properties of C. tropicalis, particularly the production of haemolytic factor. Although Candida spp may acquire iron from human blood red cells (RBCs) by producing a haemolytic factor that promotes cell lyses, at present there are no data regarding the effect of RBCs on the production of haemolytic molecules. The present study was undertaken to evaluate the role of human red blood cells on the production haemolytic factor by C. tropicalis; in addition, the transcription levels of a putative haemolysin-like protein gene (HLPt) were also analysed.
C. tropicalis isolates produced a haemolytic factor following growth in either the absence or presence of RBCs; however, distinct levels of haemolysis were observed, with 60% of the isolates exhibiting a significant increase in the production of haemolytic factor when grown in the presence of human RBCs. All isolates in which the putative HLPt gene was up-regulated in presence of human RBCs, ranging from 1.044 to 6.965-fold, also exhibited higher haemolytic activity following growth in the presence of RBCs compared to that observed in the absence of RBCs.
We propose that human RBCs may induce changes in the phenotypic expression of haemolytic factor and in transcriptional levels of the putative C. tropicalis HLPt gene in an isolate-dependent fashion.
PMCID: PMC4329199
Haemolytic activity; Putative HLPt gene; Erythrocytes
3.  Differential regulation of protein expression in response to polyunsaturated fatty acids in the liver of apoE-knockout mice and in HepG2 cells 
Polyunsaturated fatty acids (PUFAs) are nutrients necessary for life. The liver is the essential metabolic center, which aids in maintaining health via diverse biological actions. In the present work, a proteomics study was conducted with an aim to provide new insights into PUFA-regulated hepatic protein expression in apoE-knockout mice. Additionally, we investigated how n-3 PUFAs influence cytokine-challenge by using HepG2 cells as a model.
Through the proteomic analysis using 2-dimensional electrophoresis and mass spectrometry, we found that 28, 23, 14, and 28 hepatic proteins were up-regulated at least a two-fold difference in intensity compared with the control group in mice treated with the docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid, and linoleic acid, respectively. In contrast, 12 hepatic proteins were down-regulated with a ratio value of less than 0.5 compared to their control counterparts by these four fatty acids. All of the altered proteins were then sorted according to their biochemical properties related to metabolism, redox stress/inflammation, enzymatic reactions, and miscellaneous functions. The results provide evidence that PUFAs may act as either pro-inflammatory or anti-inflammatory agents. Cytokine-challenged HepG2 cells were used to reveal the anti-inflammatory function of n-3 PUFAs. The results showed that interleukin (IL)-1β combined with IL-6 induced C-reactive protein (CRP) mRNA expression and its protein secretion by HepG2 cells. The CRP promoter activity was significantly increased in the IL-6-treated cells, whereas IL-1β alone had no effect. However, IL-1β and IL-6 acted synergistically to further enhance CRP promoter activities. Furthermore, n-3 PUFAs inhibited nuclear factor-κB (NF-κB) activation and the phosphorylation of the nuclear signal transducer and activator of transcription 3 (STAT3) during cytokine-induced CRP production.
This study indicates that PUFAs induced changes in the hepatic protein profile in vivo. Furthermore, n-3 PUFAs exert their anti-inflammatory properties through differential molecular mechanisms in hepatic cells. These results provide novel information regarding the roles of PUFAs in the liver at the tissue and cellular levels.
PMCID: PMC4331445
Polyunsaturated fatty acids (PUFAs); Proteomics; Inflammation; C-reactive protein (CRP); Nuclear factor-κB (NF-κB) pathway
4.  Aberrant gene expression profiles, during in vitro osteoblast differentiation, of telomerase deficient mouse bone marrow stromal stem cells (mBMSCs) 
Telomerase deficiency has been associated with inadequate differentiation of mesenchymal stem cells. However, the effect of telomerase deficiency on differential regulation of osteoblast specific genes, based on functional gene grouping, during in vitro osteoblast differentiation has not been reported before.
To examine these effects, Terc-/- BMSCs (bone marrow stromal stem cells) were employed which exhibited reduced proliferation during in vitro osteogenesis along with increased population doubling time and level compared to wild type (WT) BMSCs during the normal culture. Osteogenic super array at day 10 of osteoblast differentiation revealed that telomerase deficiency strongly affected the osteoblast commitment by down-regulating Runx2, Twist and Vdr – known transcription regulators of osteogenesis. Similarly, in Terc-/- BMSCs a marked reduction in other genes engaged in various phases of osteoblast differentiation were observed, such as Fgfr2 involved in bone mineralization, Phex and Dmp1 engaged in ossification, and Col11a1 and Col2a1 involved in cartilage condensation. A similar trend was observed for genes involved in osteoblast proliferation (Tgfb1, Fgfr2 and Pdgfa) and bone mineral metabolism (Col1a1, Col2a1, Col1a2 and Col11a1). More profound changes were observed in genes engaged in extracellular matrix production: Col1a1, Col1a2, Mmp10, Serpinh1 and Col4a1.
Taken together, these data suggest that telomerase deficiency causes impairment of BMSCs differentiation into osteoblasts affecting commitment, proliferation, matrix mineralization and maturation. Thus, modulating telomerase in BMSCs with advanced aging could improve BMSCs responsiveness towards osteoblast differentiation signals, optimal for osteoblast commitment, proliferation and maturation processes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-015-0116-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4318164  PMID: 25633569
Telomerase; Telomeres; BMSCs; Mesenchymal stem cells; Aging; Osteoblast
5.  Protective effects of polyunsatutared fatty acids supplementation against testicular damage induced by intermittent hypobaric hypoxia in rats 
Intermittent hypobaric hypoxia (IHH) induces changes in the redox status and structure in rat testis. These effects may be present in people at high altitudes, such as athletes and miners. Polyunsaturated fatty acids (PUFA) can be effective in counteracting these oxidative modifications due to their antioxidants properties. The aim of the work was to test whether PUFA supplementation attenuates oxidative damage in testis by reinforcing the antioxidant defense system. The animals were divided into four groups (7 rats per group): normobaric normoxia (~750 tor; pO2 156 mmHg; Nx); Nx + PUFA, supplemented with PUFA (DHA: EPA = 3:1; 0.3 g kg−1 of body weight per day); hypoxic hypoxia (~428 tor; pO2 90 mmHg; Hx) and, Hx + PUFA. The hypoxic groups were exposed in 4 cycles to 96 h of HH followed by 96 h of normobaric normoxia for 32 days. Total antioxidant capacity (FRAP) and lipid peroxidation (malondialdehyde, MDA) in plasma and reduced (GSH)/oxidized glutathione (GSSG) ratio, tissue lipid peroxidation (TBARS) and antioxidant enzymes activity were assessed at the end of the study in testis. Also, SIRTUIN 1 and HIF-1 protein expression in testis were determined.
IHH increased lipid peroxidation in plasma and HIF-1 protein levels in testis. In addition, IHH reduced FRAP levels in plasma, antioxidant enzymes activities and SIRTUIN 1 protein levels in testis. PUFA supplementation attenuated these effects, inducing the increases in FRAP, in the antioxidant enzymes activity and HIF-1 levels.
These results suggest that the IHH model induces a prooxidant status in plasma and testis. The molecular protective effect of PUFA may involve the induction of an antioxidant mechanism.
PMCID: PMC4307138  PMID: 25613908
Intermittent hypoxia; Testis; Oxidative stress; PUFA; SIRTUIN 1
6.  Expression profile and down-regulation of argininosuccinate synthetase in hepatocellular carcinoma in a transgenic mouse model 
Argininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter.
We established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a post-transcriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx)-transgenic mice, the Tg (ASS-EGFP, HBx) double transgenic mice developed HCC in which ASS expression was down-regulated, as in clinical samples.
The BAC transgenic mouse model described is a valuable tool for studying ASS gene expression. Moreover, this mouse model is a close reproduction of clinical behavior of ASS in HCC and is useful in testing arginine-depleting agents and for studies of the role of ASS in tumorigenesis.
PMCID: PMC4308890  PMID: 25616743
Argininosuccinate synthetase; Transgenic mouse model; Hepatocellular carcinoma; Embryo expression map; Brain expression map; Ventricular zone; Subventricular zone; Post-transcriptional regulation; Bacterial artificial chromosome; GFP reporter gene
7.  Regulation of cancer metastasis by microRNAs 
MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that have been found highly conserved among species. MiRNAs are able to negatively regulate gene expression through base pairing of 3’ UTRs of their target genes. Therefore, miRNAs have been shown to play an important role in regulating various cellular activities. Over the past decade, substantial evidences have been obtained to show that miRNAs are aberrantly expressed in human malignancies and could act as “OncomiRs” or “Tumor suppressor miRs”. In recent years, increasing number of studies have demonstrated the involvement of miRNAs in cancer metastasis. Many studies have shown that microRNAs could directly target genes playing a central role in epithelia-mesenchymal-transition (EMT), a cellular transformation process that allows cancer cells to acquire motility and invasiveness. EMT is considered an essential step driving the early phase of cancer metastasis. This review will summarize the recent findings and characterization of miRNAs that are involved in the regulation of EMT, migration, invasion and metastasis of cancer cells. Lastly, we will discuss potential use of miRNAs as diagnostic and prognostic biomarkers as well as therapeutic targets for cancer.
PMCID: PMC4318216  PMID: 25614041
miRNAs; Breast cancer; Metastasis; Migration; Invasion; EMT
8.  Combination of proteasome and HDAC inhibitor enhances HPV16 E7-specific CD8+ T cell immune response and antitumor effects in a preclinical cervical cancer model 
Bortezomib, a proteasome inhibitor and suberoylanilide hydroxamic acid (SAHA, also known as Vorinostat), a histone deacetylase inhibitor, have been recognized as potent chemotherapeutic drugs. Bortezomib and SAHA are FDA-approved for the treatment of cutaneous T cell lymphoma and multiple myeloma/mantle cell lymphoma, respectively. Furthermore, the combination of the bortezomib and SAHA has been tested in a variety of preclinical models and in clinical trials and may be ideal for the treatment of cancer. However, it remains unclear how this treatment strategy affects the host immune response against tumors.
Here, we used a well-defined E6/E7-expressing tumor model to examine how the immune system can be motivated to act against tumor cells expressing tumor antigens. We demonstrate that the combination of bortezomib and SAHA elicits potent antitumor effects in TC-1 tumor-bearing mice. Additionally, we are the first to show that treatment with bortezomib and SAHA leads to tumor-specific immunity by rendering tumor cells more susceptible to killing by antigen-specific CD8+ T cells than treatment with either drug alone.
The current study serves an important foundation for the future clinical application of both drugs for the treatment of cervical cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-014-0111-1) contains supplementary material, which is available to authorized users.
PMCID: PMC4298946  PMID: 25591912
Bortezomib; SAHA; Vorinostat; Antitumor; Host immunity
9.  Biological roles of CCAAT/Enhancer-binding protein delta during inflammation 
CCAAT/enhancer-binding protein delta (CEBPD) belongs to the CCAAT/enhancer-binding protein family, and these proteins function as transcription factors in many biological processes, including cell differentiation, motility, growth arrest, proliferation, cell death, metabolism and immune responses. The functional diversity of CEBPD depends, in part, on the cell type and cellular context, which indicates that CEBPD could interpret a variety of cues to adjust cellular responses in specific situations. Here, we review the regulation of the CEBPD gene and its function in response to inflammatory stimuli. We also address its effects in inflammation-related diseases through a discussion of its recently discovered downstream targets. Regarding to the previous discoveries and new insights in inflammation-associated diseases, suggesting CEBPD could also be a central gene in inflammation. Importantly, the results of this study indicate that the investigation of CEBPD could open a new avenue to help better understand the inflammatory response.
PMCID: PMC4318212  PMID: 25591788
10.  Molecular regulation of the expression of leptin by hypoxia in human coronary artery smooth muscle cells 
Leptin, produced mainly by white adipose tissue, is a hormone that promotes vascular smooth muscle cell (VSMC) migration and proliferation, a process involved in the pathophysiology of atherosclerosis. Leptin expression in human coronary artery smooth cell (HCASMC) is induced by hypoxia. However, our understanding of the process of atherosclerosis in HCASMC is only emerging. Since the mechanisms by which hypoxia regulates leptin in HCASMC are as yet unknown, this study aims to investigate the mechanics of molecular regulation of leptin expression in HCASMC under hypoxia. We subjected cultured HCASMCs to hypoxia for varying periods of time. Through use of different signal pathway inhibitors, we were able to sort out and identify the pathway through which hypoxia-induced leptin expression occurs.
Leptin mRNA and protein levels increased after 2.5% hypoxia for 2-to-4 hours, with earlier expression of angiotensin II (AngII) and reactive oxygen species (ROS). The addition before hypoxia of the c-Jun N-terminal kinase (JNK) pathway inhibitor (SP600125), JNK small interfering RNA (siRNA), AngII receptor blockers (ARBs; losartan), or N-acetyl-L-cysteine (NAC, an ROS scavenger), had the effect of inhibiting JNK phosphorylation and leptin expression. Gel shift assay and luciferase promoter study showed that leptin/activator protein 1 (AP-1) binding and transcriptional activity to the leptin promoter increased after hypoxia, and SP600125, JNK siRNA, losartan, and NAC abolished the binding and transcriptional activity induced by hypoxia. The use of SP600125, JNK siRNA, losartan, and NAC effectively inhibited the binding and transcriptional activity induced by hypoxia. Migration and proliferation, ROS generation, and the presence of leptin in the nuclei of HCASMCs also increased under hypoxia.
Hypoxia in HCASMCs increases leptin expression through the induction of AngII, ROS, and the JNK pathway to enhance atherosclerosis in HCASMCs.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-014-0109-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4298872  PMID: 25573199
Leptin; Human coronary artery smooth muscle cell; Hypoxia; Angiotensin II; c-Jun N-terminal kinase pathway
11.  EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex 
Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation.
Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was −30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells.
The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-014-0108-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4307906  PMID: 25576037
EpCAM; Aptamer; PEI-EpApt-SiEp; siRNA delivery; Cancer targeting
12.  Profiling the circulating miRNAs in mice exposed to gram-positive and gram-negative bacteria by Illumina small RNA deep sequencing 
We profiled the expression of circulating microRNAs (miRNAs) in mice using Illumina small RNA deep sequencing in order to identify the miRNAs that may potentially be used as biomarkers to distinguish between gram-negative and gram-positive bacterial infections.
Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14 + Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection.
This study identified mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p as potential circulating miRNAs for gram-positive bacterial infections.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-014-0106-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4300083  PMID: 25563241
microRNAs (miRNAs); Circulating microRNAs; Gram-positive bacteria; Gram-negative bacteria; Small RNA deep sequencing
13.  KUD773, a phenylthiazole derivative, displays anticancer activity in human hormone-refractory prostate cancers through inhibition of tubulin polymerization and anti-Aurora A activity 
Hormone-refractory prostate cancer (HRPC), which is resistant to hormone therapy, is a major obstacle in clinical treatment. An approach to inhibit HRPC growth and ultimately to kill cancers is highly demanded.
KUD773 induced the anti-proliferative effect and subsequent apoptosis in PC-3 and DU-145 (two HRPC cell lines); whereas, it showed less active in normal prostate cells. Further examination showed that KUD773 inhibited tubulin polymerization and induced an increase of mitotic phosphoproteins and polo-like kinase 1 (PLK1) phosphorylation, indicating a mitotic arrest of the cell cycle through an anti-tubulin action. The kinase assay demonstrated that KUD773 inhibited Aurora A activity. KUD773 induced an increase of Cdk1 phosphorylation at Thr161 (a stimulatory phosphorylation site) and a decrease of phosphorylation at Tyr15 (an inhibitory phosphorylation site), suggesting the activation of Cdk1. The data were substantiated by an up-regulation of cyclin B1 (a Cdk1 partner). Furthermore, KUD773 induced the phosphorylation and subsequent down-regulation of Bcl-2 and activation of caspase cascades.
The data suggest that KUD773 induces apoptotic signaling in a sequential manner. It inhibits tubulin polymerization associated with an anti-Aurora A activity, leading to Cdk1 activation and mitotic arrest of the cell cycle that in turn induces Bcl-2 degradation and a subsequent caspase activation in HRPCs.
PMCID: PMC4304192  PMID: 25563361
Hormone-refractory prostate cancer; Phenylthiazole derivative; Tubulin depolymerization; Aurora A kinase; Mitochondria-involved apoptosis
14.  MicroRNA miR-466 inhibits Lymphangiogenesis by targeting prospero-related homeobox 1 in the alkali burn corneal injury model 
Lymphangiogenesis is one of the major causes of corneal graft rejection. Among the lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and -D are considered to be the most potent. Both bind to VEGF receptor 3 (VEGFR3) to activate Prospero homeobox 1 (Prox1), a transcription factor essential for the development and maintenance of lymphatic vasculature. MicroRNAs (miRNAs) bind to the 3' untranslated regions (3' UTRs) of target genes in a sequence-specific manner and suppress gene expression. In the current study, we searched for miRNAs that target the pro-lymphangiogenic factor Prox1.
Among the miRNAs predicted by the bioinformatic analysis to seed match with the 3' UTR of Prox-1, we chose 3 (miR-466, miR-4305, and miR-4795-5p) for further investigation. Both the miR-466 and miR-4305 mimics, but not the miR-4795-5p mimic, significantly reduced the luciferase activity of the Prox-1 3' UTR reporter vector. In primary lymphatic endothelial cells (HDLEC), miR-466 mimic transfection suppressed Prox1 mRNA and protein expression, while miR-4305 mimic transfection did not. Experiments using mutated reporter constructs of the two possible seed match sites on the 3' UTR of Prox1 suggested that the target site 2 directly bound miR-466. HDLEC transfected with the miR-466 mimic suppressed tube formation as compared to the scrambled control. Furthermore, HDLEC transfected with a miR-466 inhibitor showed enhanced tube formation as compared to control inhibitor transfected cells, and this inhibitory effect was counteracted by Prox1 siRNA. The miR-466 mimic reduced angiogenesis and lymphangiogenesis resulting in clearer corneas in an cornea injury rat model compared to the scrambled control.
Our data suggest that miR-446 may have a protective effect on transplanted corneas by suppressing Prox1 expression at the post-transcriptional level. The results of the current study may provide insights into the mechanisms of lymphangiogenesis resulting from corneal graft rejection and alkali-burn injuries, as well as into the development of new treatments for lymphangiogenic eye diseases.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-014-0104-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4304626  PMID: 25573115
MicroRNA; Prox1; miR-466; miR-181; Tube Formation; Lymphangiogenesis; Cornea transplantation; Alkali burn
15.  Effects of tobacco smoking during pregnancy on oxidative stress in the umbilical cord and mononuclear blood cells of neonates 
Although cigarette smoke is known to be a complex mixture of over 4000 substances that can lead to damage through active or passive smoking, its mechanisms and biochemical consequences in pregnancy and neonates are not yet fully understood. Therefore, in the present study, we propose to study the impact of smoking during gestation on the viability of blood mononuclear cells (MNC) from umbilical cords of newborns to assess the degree of oxidative stress and cell viability. After childbirth, the cord blood and the umbilical cord were immediately collected in public hospitals in Greater Vitoria, ES, Brazil. Flow cytometry was used to analyze the cord blood followed by biochemical and histological tests to analyze possible changes in the umbilical cord.
Pregnant smokers had a reduction of MNC viability from the umbilical cord (10%), an increase in the production of reactive oxygen species (ROS) and an increase in cell apoptosis (~2-fold) compared to pregnant non-smokers. In the umbilical cord, it was observed an increase of advanced oxidation protein products - AOPP (~2.5-fold) and a loss of the typical architecture and disposition of endothelial cells from the umbilical artery.
These data suggest that maternal cigarette smoking during pregnancy (even in small amounts) may compromise the viability of MNC cells and damage the umbilical cord structure, possibly by excessive ROS bioavailability.
PMCID: PMC4302517  PMID: 25547987
Cigarette smoke; Cord blood; Oxidative stress; Pregnant women; Apoptosis
16.  The tryptophan kynurenine pathway, neopterin and IL-6 during vulvectomy and abdominal hysterectomy 
Surgery has wide ranging immunomodulatory properties of which the mechanism is poorly understood. In order to investigate how different types of surgery influence inflammation, we designed a longitudinal observational study investigating two inflammatory profiles of two separate patient groups undergoing gynaecological operations of differing severity. In addition to measuring the well known inflammatory markers neopterin and IL-6, we also determined the kynurenine/tryptophan ratio.
This study was a prospective, single center, two-armed observational study involving 28 female patients. Plasma levels of tryptophan, kynurenine, neopterin and IL-6 were determined from samples taken at: 24hrs pre-operative, prior to induction, ten minutes before the operation was expected to end, and at 24 and 96 hours post operative in patients undergoing abdominal hysterectomy and vulvectomy.
There were 15 and 13 patients included in the vulvectomy and abdominal hysterectomy groups, respectively. In this study we show that anesthesia and surgery significantly increases the enzyme activity of indoleamine 2, 3 dioxygenase (IDO) as measured by the kynurenine/tryptophan ratio (P=0.003), while maintaining stable neopterin levels. However, abdominal hysterectomy causes a considerable IL-6 increase (P<0.001).
Surgery and associated anesthesia cause a significant tryptophan level decrease while significantly increasing IDO activity. Both types of surgery produce nearly identical neopterin time curve relationships, with no significant change occurring in either group. However, even though neopterin is unaffected by the severity of surgery, IL-6 responded to surgical invasiveness by revealing a significant increase during abdominal hysterectomy.
PMCID: PMC4300209  PMID: 25526661
Kynurenine; Tryptophan; Indoleamine 2,3-dioxygenase; Neopterin
17.  Improving clinical efficacy of adeno associated vectors by rational capsid bioengineering 
Adeno associated vectors (AAV) have shown considerable promise to treat various genetic disorders in both preclinical and clinical settings mainly because of its safety profile. However, efficient use of AAV to deliver genes in immune-competent sites like muscles and liver requires very high doses which are associated with concomitant cellular immune response against the viral capsids leading to destruction of the transduced cells. Coupled with that, there are enough evidences that at high doses, AAV particles are subjected to increased cellular phosphorylation/uniquitination leading to proteasome mediated degradation and loss of the viral particles. The presence of preexisting immunity against AAV further adds on to the problem which is acting as a major roadblock to efficiently use it as a gene therapy vector in the clinics. To overcome this, rational bioengineering of AAV capsid becomes a prime tool by which specific amino acid residue(s) can be suitably modified/replaced by compatible residue(s) to create vectors having lower host immune response and higher intracellular trafficking rate. This article reviews the various aspects of rationally designing AAV capsids like by site-directed mutagenesis, directed evolution and combinatorial libraries which can create vectors having not only immune evasive property but also enhanced gene expression and transduction capability. One or more combinations of these strategies have strong potential to create novel vectors which will have suitable clinical efficiency even at a low dose.
PMCID: PMC4251935  PMID: 25425174
Adeno associated virus; Capsid bioengineering; AAV; Site directed mutagenesis
18.  N-3 polyunsaturated fatty acids decrease levels of doxorubicin-induced reactive oxygen species in cardiomyocytes -- involvement of uncoupling protein UCP2 
Use of the chemotherapeutic drug doxorubicin (DOX) is associated with serious cardiotoxicity, as it increases levels of reactive oxygen species (ROS). N-3 polyunsaturated fatty acid dietary supplements can be of benefit to patients undergoing cancer therapy. The aims of this study were to determine whether DOX-induced cardiotoxicity is related to mitochondrial uncoupling proteins and whether eicosapentaenoic acid (EPA, C20:5 n-3) or docosahexaenoic acid (DHA, C22:6 n-3) affects DOX-induced cardiomyocyte toxicity.
Treatment of H9C2 cells with DOX resulted in decreased cell viability and UCP2 expression. Treatment with 100 μM EPA or 50 μM DHA for 24 h resulted in a maximal mitochondria concentration of these fatty acids and increased UCP2 expression. Pretreatment with 100 μM EPA or 50 μM DHA prevented the DOX-induced decrease in UCP2 mRNA and protein levels, but these effects were not seen with EPA or DHA and DOX cotreatment. In addition, the DOX-induced increase in ROS production and subsequent mitochondrial membrane potential change (∆ψ) were significantly attenuated by pretreatment with EPA or DHA.
EPA or DHA pre-treatment inhibits the DOX-induced decrease in UCP2 expression, increase in ROS production, and subsequent mitochondrial membrane potential change that contribute to the cardiotoxicity of DOX.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-014-0101-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4237738  PMID: 25407516
EPA; DHA; Doxorubicin; ROS; UCP2
19.  The pathological effects of CCR2+ inflammatory monocytes are amplified by an IFNAR1-triggered chemokine feedback loop in highly pathogenic influenza infection 
Highly pathogenic influenza viruses cause high levels of morbidity, including excessive infiltration of leukocytes into the lungs, high viral loads and a cytokine storm. However, the details of how these pathological features unfold in severe influenza infections remain unclear. Accumulation of Gr1 + CD11b + myeloid cells has been observed in highly pathogenic influenza infections but it is not clear how and why they accumulate in the severely inflamed lung. In this study, we selected this cell population as a target to investigate the extreme inflammatory response during severe influenza infection.
We established H1N1 IAV-infected mouse models using three viruses of varying pathogenicity and noted the accumulation of a defined Gr1 + CD11b + myeloid population correlating with the pathogenicity. Herein, we reported that CCR2+ inflammatory monocytes are the major cell compartments in this population. Of note, impaired clearance of the high pathogenicity virus prolonged IFN expression, leading to CCR2+ inflammatory monocytes amplifying their own recruitment via an interferon-α/β receptor 1 (IFNAR1)-triggered chemokine loop. Blockage of IFNAR1-triggered signaling or inhibition of viral replication by Oseltamivir significantly suppresses the expression of CCR2 ligands and reduced the influx of CCR2+ inflammatory monocytes. Furthermore, trafficking of CCR2+ inflammatory monocytes from the bone marrow to the lung was evidenced by a CCR2-dependent chemotaxis. Importantly, leukocyte infiltration, cytokine storm and expression of iNOS were significantly reduced in CCR2−/− mice lacking infiltrating CCR2+ inflammatory monocytes, enhancing the survival of the infected mice.
Our results indicated that uncontrolled viral replication leads to excessive production of inflammatory innate immune responses by accumulating CCR2+ inflammatory monocytes, which contribute to the fatal outcomes of high pathogenicity virus infections.
PMCID: PMC4243311  PMID: 25407417
Influenza A virus; CCR2+ inflammatory monocytes; IFNAR1; CCL2; CCL7 and CCL12
20.  Hyaluronan and cardiac regeneration 
Hyaluronan (HA) is abundantly expressed in several human tissues and a variety of roles for HA has been highlighted. Particularly relevant for tissue repair, HA is actively produced during tissue injury, as widely evidenced in wound healing investigations. In the heart HA is involved in physiological functions, such as cardiac development during embryogenesis, and in pathological conditions including atherosclerosis and myocardial infarction. Moreover, owing to its relevant biological properties, HA has been widely used as a biomaterial for heart regeneration after a myocardial infarction. Indeed, HA and its derivatives are biodegradable and biocompatible, promote faster healing of injured tissues, and support cells in relevant processes including survival, proliferation, and differentiation. Injectable HA-based therapies for cardiovascular disease are gaining growing attention because of the benefits obtained in preclinical models of myocardial infarction. HA-based hydrogels, especially as a vehicle for stem cells, have been demonstrated to improve the process of cardiac repair by stimulating angiogenesis, reducing inflammation, and supporting local and grafted cells in their reparative functions. Solid-state HA-based scaffolds have been also investigated to produce constructs hosting mesenchymal stem cells or endothelial progenitor cells to be transplanted onto the infarcted surface of the heart. Finally, applying an ex-vivo mechanical stretching, stem cells grown in HA-based 3D scaffolds can further increase extracellular matrix production and proneness to differentiate into muscle phenotypes, thus suggesting a potential strategy to create a suitable engineered myocardial tissue for cardiac regeneration.
PMCID: PMC4226915  PMID: 25358954
Hyaluronan; Myocardial infarction; Cardiac regeneration; Adult stem cells
21.  The emerging role of hepatitis B virus Pre-S2 deletion mutant proteins in HBV tumorigenesis 
Chronic hepatitis B virus (HBV) infection can cause hepatocellular carcinoma (HCC). Several hypotheses have been proposed to explain the mechanisms of HBV tumorigenesis, including inflammation and liver regeneration associated with cytotoxic immune injuries and transcriptional activators of mutant HBV gene products. The mutant viral oncoprotein-driven tumorigenesis is prevailed at the advanced stage or anti-HBe-positive phase of chronic HBV infection. Besides HBx, the pre-S2 (deletion) mutant protein represents a newly recognized oncoprotein that is accumulated in the endoplasmic reticulum (ER) and manifests as type II ground glass hepatocytes (GGH). The retention of pre-S2 mutant protein in ER can induce ER stress and initiate an ER stress-dependent VEGF/Akt/mTOR and NFκB/COX-2 signal pathway. Additionally, the pre-S2 mutant large surface protein can induce an ER stress-independent pathway to transactivate JAB-1/p27/RB/cyclin A,D pathway, leading to growth advantage of type II GGH. The pre-S2 mutant protein-induced ER stress can also cause DNA damage, centrosome overduplication, and genomic instability. In 5-10% of type II GGHs, there is co-expression of pre-S2 mutant protein and HBx antigen which exhibited enhanced oncogenic effects in transgenic mice. The mTOR signal cascade is consistently activated throughout the course of pre-S2 mutant transgenic livers and in human HCC tissues, leading to metabolic disorders and HCC tumorigenesis. Clinically, the presence of pre-S2 deletion mutants in sera frequently develop resistance to nucleoside analogues anti-virals and predict HCC development. The pre-S2 deletion mutants and type II GGHs therefore represent novel biomarkers of HBV-related HCCs. A versatile DNA array chip has been developed to detect pre-S2 mutants in serum. Overall, the presence of pre-S2 mutants in serum has implications for anti-viral treatment and can predict HCC development. Targeting at pre-S2 mutant protein-induced, ER stress-dependent, mTOR signal cascade and metabolic disorders may offer potential strategy for chemoprevention or therapy in high risk chronic HBV carriers.
PMCID: PMC4200140  PMID: 25316153
Ground glass hepatocytes; Pre-S mutants; Endoplasmic reticulum stress; Chronic HBV infection; Hepatocellular carcinoma
22.  Higher glucose level can enhance the H. pylori adhesion and virulence related with type IV secretion system in AGS cells 
Hyperglycemia increases the risk of gastric cancer in H. pylori-infected patients. High glucose could increase endothelial permeability and cancer-associated signaling. These suggest high glucose may affect H. pylori or its infected status.
We used two strains to investigate whether H. pylori growth, viability, adhesion and CagA-phosphorylation level in the infected-AGS cells were influenced by glucose concentration (100, 150, and 200 mg/dL).
The growth curves of both strains in 200 mg/dL of glucose were maintained at the highest optimal density after 48 h and the best viability of both strains were retained in the same glucose condition at 72 h. Furthermore, adhesion enhancement of H. pylori was significantly higher in 200 mg/dL of glucose as compared to that in 100 and 150 mg/dL (p < 0.05). CagA protein also increased in higher glucose condition. The cell-associated CagA and phosphorylated-CagA was significantly increased in 150 and 200 mg/dL of glucose concentrations as compared to that of 100 mg/dL (p < 0.05), which were found to be dose-dependent.
Higher glucose could maintain H. pylori growth and viability after 48 h. H. pylori adhesion and CagA increased to further facilitate the enhancement of cell-associated CagA and phosphorylated CagA in higher glucose conditions.
PMCID: PMC4196111  PMID: 25296847
Glucose; Helicobacter pylori; CagA
23.  Study of the association of adrenomedullin and basic-fibroblast growth factors with the peripheral arterial blood flow and endothelial dysfunction biomarkers in type 2 diabetic patients with peripheral vascular insufficiency 
Progressive micro-vascular vaso-degeneration is the major factor in progression of diabetic complications. Adrenomedullin (AM) and basic-Fibroblast growth factor (b-FGF) are strongly correlated with angiogenesis in vascular diseases. This study aims to provide base line data regarding the vascular effects and correlation of AM, and b-FGF with the peripheral blood flow in diabetic patients with peripheral vascular disease (PVD), and their effect on endothelial dysfunction markers. Ninety age- and sex matched females were enrolled in the study: 30 were controls, 30 had diabetes without complications (group II) and 30 had diabetes with PVD (group III) diagnosed by ankle/ brachial index (A/BI). Plasma levels of AM, b-FGF, intercellular adhesion molecule −1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were measured by indirect enzyme immunoassay (ELISA).
There was a significant increase in plasma AM, VCAM-1and ICAM-1, while a significant decrease in plasma b-FGF in diabetic patients with PVD (p < 0.05). A positive correlation was observed between plasma AM, b-FGF and A/BI and a negative correlation with VCAM −1 and ICAM in diabetic PVD. AM was not a predictor, while b-FG, VCAM-1 and ICAM-1 could be predictors for peripheral blood flow in diabetic PVD.
This study elucidates for the first time that AM and b-FGF are correlated and have a direct impact on the peripheral blood flow, the rise of AM in diabetic PVD may be a consecutive and compensatory vasculo-protective effect as its angiogenic and anti-inflammatory properties act to relief the endothelial insult. Down expression of b-FGF may be a predisposing factor for micro-vascular derangement. It is not clear if the rise of AM and the decline of b- FGF levels may be consequences or predisposing factors for VCAM-1 and ICAM-1 elevation as these endothelial dysfunction biomarkers could reduce peripheral blood flow and vascular integrity. It is optimistic to believe that drug intervention through AM and b-FGF administration together with reversing the endothelial inflammatory process by targeting VCAM and ICAM could reduce the prevalence of diabetic vascular complications, reduce the risk of cerebrovascular and cardiovascular morbidity in diabetes through normalizing vascular endothelium function and peripheral blood flow.
PMCID: PMC4195904  PMID: 25287126
Diabetic vasculopathy; Adrenomedullin; Basic-Fibroblast growth factor
24.  Up-regulation of S100A16 expression promotes epithelial-mesenchymal transition via Notch1 pathway in breast cancer 
Our previous studies demonstrated that S100A16 promotes adipogenesis and is involved in weight gain attenuation induced by dietary calcium. Till now, the function of S100A16 in the breast cancer remains to be elucidated.
In this study, we observed that S100A16 was expressed in higher levels in human breast cancer tissues compared with paired adjacent non-cancerous tissues. Further examination showed that overexpression of S100A16 in MCF-7 cells could increase cell proliferation and colony formation. One major mechanistic change was that S100A16 was able to up-regulate the transcription factors Notch1, ZEB1, and ZEB2, which had the capacities to directly repress the expression of epithelial markers E-cadherin and β-catenin but increase mesenchymal markers N-cadherin and vimentin, a characterized phenotype of epithelial-mensenchymal transition (EMT). In addition to display with morphologic change, migration and invasion were increased in S100A16 over-expressed MCF-7 cells. Importantly, knockdown of Notch1 by specific siRNA could reverse the EMT induced by S100A16 overexpression, which confirmed that Notch1 played a critical role in the process of EMT induced by S100A16.
All together, our data indicated that S100A16 had a potential function to regulate some embryonic transcription factors to promote EMT in breast cancer cells which may be an important target site for the therapy of breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-014-0097-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4197258  PMID: 25287362
Breast cancer; S100A16; EMT; Notch1; MCF-7
25.  MicroRNA-5p and -3p co-expression and cross-targeting in colon cancer cells 
Two mature miRNA species may be generated from the 5’ and 3’ arms of a pre-miRNA precursor. In most cases, only one species remains while the complementary species is degraded. However, co-existence of miRNA-5p and -3p species is increasingly being reported. In this work, we aimed to systematically investigate co-expression of miRNA-5p/3p in colon cancer cells in a genome-wide analysis, and to examine cross-targeting of the dysregulated miRNAs and 5p/3p species.
Four colon cancer cell lines were examined relative to two normal colon tissues. Of the 1,190 miRNAs analyzed, 92 and 36 were found to be up- or down-regulated, respectively, in cancer cells. Nineteen co-expressed miRNA-5p/3p pairs were further identified suggesting frequent 5p/3p co-accumulation in colon cancer cells. Of these, 14 pairs were co-up-regulated and 3 pairs were co-down-regulated indicating concerted 5p/3p dysregulation. Nine dysregulated miRNA pairs fell into three miRNA gene families, namely let-7, mir-8/200 and mir-17, which showed frequent cross-targeting in the metastasis process. Focusing on the let-7d-5p/3p pair, the respectively targeted IGF1R and KRAS were shown to be in a reverse relationship with expression of the respective miRNA, which was confirmed in transient transfection assays using let-7d mimic or inhibitor. Targeting of KRAS by let-7d was previous reported; targeting of IGF1R by let-7d-5p was confirmed in luciferase assays in this study. The findings of let-7d-5p/3p and multiple other miRNAs targeting IGF1R, KRAS and other metastasis-related factors suggest that 5p/3p miRNAs contribute to cross-targeting of multiple cancer-associated factors and processes possibly to evade functional abolishment when any one of the crucial factors are inactivated.
miRNA-5p/3p species are frequently co-expressed and are coordinately regulated in colon cancer cells. In cancer cells, multiple cross-targeting by the miRNAs, including the co-existing 5p/3p species, frequently occurs in an apparent safe-proof scheme of miRNA regulation of important tumorigenesis processes. Further systematic analysis of co-existing miRNA-5p/3p pairs in clinical tissues is important in elucidating 5p/3p contributions to cancer pathogenesis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12929-014-0095-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4195866  PMID: 25287248
microRNA-5p and -3p pairs; Colon cancer; miRNA cross targeting; Safe-proof regulation

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