Assess Protein Expression Research Articles

Endometrial attachment and invasion is decreased with estrogen receptor beta knock down

Endometriosis requires estrogen for development and growth. Levels of estrogen receptor beta (ERβ) in endometriosis are reported to be 100 times higher than normal endometrial tissue and inhibition of ERβ activity by an ERβ selective antagonist suppresses endometriotic lesion growth in mice.1,2 Here, we assessed the role of ERβ in the development of the early endometriotic lesion by examining the effect of ERβ knock down on endometrial stromal cell (ESC) cell attachment and invasion. In vitro study. ERβ was knocked-down (ERβKD) with lipofectamine using shRNA in an immortalized human ESC line (tHESC). Western blot and densitometric analysis were used to assess protein expression. Established in vitro assays were used to assess WT and ERβKD tHESC attachment to peritoneal mesothelial cells (PMCs) and invasion through a monolayer of PMCs. Results were analyzed with unpaired t-tests. tHESCs with ERβKD demonstrated decreased protein expression by Western blot and densitometric analysis. ERβKD attachment to PMCs decreased in comparison to WT (42% vs 67%, respectively; p<0.001). ERβKD invasion through PMCs decreased in ERβKD compared to WT (264 ±16 vs 404 ± 22, respectively; p<0.005). ERβKD decreases tHESCs adhesion to and invasion through PMCs. ERβ may serve as a future therapeutic target to decrease early endometriotic lesion formation.

P53 introduction enhances chemotherapy-induced apoptosis in Hela cells

Background: Cervical cancer remains a global health-related issue with over half a million new cases reported annually. However, despite different therapeutic regimens being utilized, over a million women die of cervical cancer each year. This makes it the most lethal cancer and indicates the urgent need for novel therapeutic strategies. This study was to investigate the functional roles of p53 over-expression in enhancing Lobaplatin (loba)-induced cell growth inhibition and apoptosis in cervical carcinoma cells. Methods: Hela cells were treated with recombinant p53 adenovirus (rAdp53), lobaplatin (loba), or rAdp53 plus loba combination. Subsequently, morphology, Hoechst staining, flow cytometry, and cell proliferation assay were conducted to measure apoptosis and cell growth inhibition in vitro . Western blotting was performed to assess protein expression. Xenograft tumor mouse model was used to detect the combined anti-cancer effects of rAdp53 and loba in vivo . Results: In vitro assays demonstrated that neither sub-lethal rAdp53 nor loba have any apparent apoptotic effect on Hela cells. The combination of rAdp53 and loba induced significant apoptosis and cell growth inhibition. P53 over-expression up-regulated the proapoptotic proteins Bax and Bak. Xenograft cervical tumors derived from Hela cells demonstrated that co-treatment with rAdp53 and loba effectively inhibited tumor growth in vivo . Conclusions: P53 restoration can sensitize cervical cancer cells to loba to induce apoptosis and cell growth inhibition.

Relationship Between ADAMTS8, ADAMTS18, and ADAMTS20 (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) Expressions and Tumor Molecular Classification, Clinical Pathological Parameters, and Prognosis in Breast Invasive Ductal Carcinoma.

BackgroundThe aim of this study was to investigate the correlations between ADAMTSs expression and breast invasive ductal carcinoma (IDC), and to offer a theoretical basis for novel treatment methods for IDC patients.Material/MethodsNon-proliferative catheter of breast fibroadenoma (FA) and IDC were used as the normal control and experimental group, respectively. Immunohistochemical (IHC) staining and Western blot (WB) analysis was used to assess protein expression levels of ADAMTS8, ADAMTS18, and ADAMTS20 in both FA and IDC tissues. The results of IHC, the relationship between the protein expression and the tumor molecular classification, and clinical pathological parameters were all evaluated.ResultsIHC and WB results showed that the expression of ADAMTS8/18 in IDC samples was higher than in FA samples, while the expression of ADAMTS20 in IDC samples was lower than that in FA samples. According to the results of WB, the level of ADAMTS8 was higher in the HER2+ group than in the HER2− group and FA group. The expression of ADAMTS18 in the HR+ (including ER+ and PR+) group was significantly higher than in the HR− group and FA group. The expression of ADAMTS18 protein was also higher in the Ki67+ group than in the Ki67− group. ADAMTS20 was higher in HER2+ IDC compared with the basal subtype of IDC.ConclusionsADAMTS8/18/20 levels were not significantly correlated to the molecular subtype of IDC. ADAMTS18/20 was significantly associated with histological grade of IDC. ADAMTS8 may predict poor prognosis results of IDC patients.

In Vivo Potency Assay for Adeno-Associated Virus-Based Gene Therapy Vectors Using AAVrh.10 as an Example.

The development of a drug product requires rigorous methods of characterization and quality control to assure drug potency. Gene therapy products, a relatively new strategy for drug design with very few licensed examples, represent a unique challenge for the measure of potency. Unlike traditional drugs, potency for a gene therapeutic is a tally of the measures of multiple steps, including infectivity, transcription, translation, protein modifications, proper localization of the protein product, and protein function. This is particularly challenging for products based on the adeno-associated virus (AAV) platform, which has poor in vitro infectivity, limiting the sensitivity and thus the usefulness of cell-based assays. A rigorous in vivo assay has been established that separately evaluates infection, transcription, and resulting protein levels with specifications for each based on real time polymerase chain reaction (DNA and RNA) and standard protein assays. For an acceptance criterion, an administered vector must have vector DNA, transgene mRNA, and transgene expressed protein each concurrently meet individual specifications or the production lot fails. Using the AAVrh.10 serotype as a model vector and three different transgenes as examples, the assay is based on intravenous administration of the vector to male mice. At 2 weeks, the harvested liver is homogenized and assessed for vector genome levels (to assess for vector delivery), mRNA (to assess vector infectivity and transcription), and protein in the liver or serum (to assess protein expression). For all AAV vectors, the assay is robust and reproducible: vector DNA (linearity 102-109 copies, coefficient of variation) intra-assay <0.8%, inter-assay <0.5%; mRNA intra-assay <3.3%, inter-assay <3.4%. The reproducibility of the assay for transgene expressed protein is product specific. This in vivo potency assay is a strategy for characterization and a quantitative lot release test, providing a path forward to meet regulatory drug requirements for any AAV gene therapy vectors.

Clustered intergenic region sequences as predictors of factor H Binding Protein expression patterns and for assessing Neisseria meningitidis strain coverage by meningococcal vaccines.

Factor H binding protein (fHbp) is a major protective antigen in 4C-MenB (Bexsero®) and Trumenba®, two serogroup B meningococcal vaccines, wherein expression level is a determinant of protection. Examination of promoter-containing intergenic region (IGR) sequences indicated that nine fHbp IGR alleles covered 92% of 1,032 invasive meningococcal strains with variant 1 fHbp alleles. Relative expression values for fHbp were determined for 79 meningococcal isolates covering ten IGR alleles by quantitative reverse transcriptase polymerase chain reaction (qRT PCR). Derivation of expression clusters of IGR sequences by linear regression identified five expression clusters with five nucleotides and one insertion showing statistically associations with differences in expression level. Sequence analysis of 273 isolates examined by the Meningococcal Antigen Typing Scheme, a sandwich ELISA, found that coverage depended on the IGR expression cluster and vaccine peptide homology combination. Specific fHbp peptide-IGR expression cluster combinations were designated as ‘at risk’ for coverage by 4C-MenB and were detected in multiple invasive meningococcal disease cases confirmed by PCR alone and occurring in partially-vaccinated infants. We conclude that sequence-based analysis of IGR sequences is informative for assessing protein expression and has utility for culture-independent assessments of strain coverage by protein-based vaccines.

Acute intermittent hypoxia and rehabilitative training following cervical spinal injury alters neuronal hypoxia- and plasticity-associated protein expression.

One of the most promising approaches to improve recovery after spinal cord injury (SCI) is the augmentation of spontaneously occurring plasticity in uninjured neural pathways. Acute intermittent hypoxia (AIH, brief exposures to reduced O2 levels alternating with normal O2 levels) initiates plasticity in respiratory systems and has been shown to improve recovery in respiratory and non-respiratory spinal systems after SCI in experimental animals and humans. Although the mechanism by which AIH elicits its effects after SCI are not well understood, AIH is known to alter protein expression in spinal neurons in uninjured animals. Here, we examine hypoxia- and plasticity-related protein expression using immunofluorescence in spinal neurons in SCI rats that were treated with AIH combined with motor training, a protocol which has been demonstrated to improve recovery of forelimb function in this lesion model. Specifically, we assessed protein expression in spinal neurons from animals with incomplete cervical SCI which were exposed to AIH treatment + motor training either for 1 or 7 days. AIH treatment consisted of 10 episodes of AIH: (5 min 11% O2: 5 min 21% O2) for 7 days beginning at 4 weeks post-SCI. Both 1 or 7 days of AIH treatment + motor training resulted in significantly increased expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) relative to normoxia-treated controls, in neurons both proximal (cervical) and remote (lumbar) to the SCI. All other markers examined were significantly elevated in the 7 day AIH + motor training group only, at both cervical and lumbar levels. These markers included vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and phosphorylated and nonphosphorylated forms of the BDNF receptor tropomyosin-related kinase B (TrkB). In summary, AIH induces plasticity at the cellular level after SCI by altering the expression of major plasticity- and hypoxia-related proteins at spinal regions proximal and remote to the SCI. These changes occur under the same AIH protocol which resulted in recovery of limb function in this animal model. Thus AIH, which induces plasticity in spinal circuitry, could also be an effective therapy to restore motor function after nervous system injury.

High Glucose-Mediated Tyrosine Nitration of PI3-Kinase: A Molecular Switch of Survival and Apoptosis in Endothelial Cells.

Diabetes and hyperglycemia are associated with increased retinal oxidative and nitrative stress and vascular cell death. Paradoxically, high glucose stimulates expression of survival and angiogenic growth factors. Therefore, we examined the hypothesis that high glucose-mediated tyrosine nitration causes inhibition of the survival protein PI3-kinase, and in particular, its regulatory p85 subunit in retinal endothelial cell (EC) cultures. Retinal EC were cultured in high glucose (HG, 25 mM) for 3 days or peroxynitrite (PN, 100 µM) overnight in the presence or absence of a peroxynitrite decomposition catalyst (FeTPPs, 2.5 µM), or the selective nitration inhibitor epicatechin (100 µM). Apoptosis of ECs was assessed using TUNEL assay and caspase-3 activity. Immunoprecipitation and Western blot were used to assess protein expression and tyrosine nitration of p85 subunit and its interaction with the p110 subunit. HG or PN accelerated apoptosis of retinal ECs compared to normal glucose (NG, 5 mM) controls. HG- or PN-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI3-kinase that inhibited its association with the catalytic p110 subunit and impaired PI3-kinase/Akt kinase activity. Decomposing peroxynitrite or blocking tyrosine nitration of p85 restored the activity of PI3-kinase, and prevented apoptosis and activation of p38 MAPK. Inhibiting p38 MAPK or overexpression of the constitutively activated Myr-Akt construct prevented HG- or peroxynitrite-mediated apoptosis. In conclusion, HG impairs pro-survival signals and causes accelerated EC apoptosis, at least in part via tyrosine nitration and inhibition of PI3-kinase. Inhibitors of nitration can be used in adjuvant therapy to delay diabetic retinopathy and microvascular complication.

The net acid extruders NHE1, NBCn1 and MCT4 promote mammary tumor growth through distinct but overlapping mechanisms.

High metabolic and proliferative rates in cancer cells lead to production of large amounts of H+ and CO2 , and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na+ /H+ exchanger NHE1, the Na+ - HCO3- cotransporter NBCn1, and the lactate-H+ cotransporters MCT1 and -4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively. NHE1 and NBCn1 expression both correlated positively with progesterone receptor status, NHE1 correlated negatively and NBCn1 positively with HER2 status, whereas MCT4 expression correlated with lymph node status. Stable shRNA-mediated knockdown (KD) of either NHE1 or NBCn1 in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line significantly reduced steady-state intracellular pH (pHi ) and capacity for pHi recovery after an acid load. Importantly, KD of any of the three transporters reduced in vivo primary tumor growth of MDA-MB-231 xenografts. However, whereas KD of NBCn1 or MCT4 increased tumor-free survival and decreased in vitro proliferation rate and colony growth in soft agar, KD of NHE1 did not have these effects. Moreover, only MCT4 KD reduced Akt kinase activity, PARP and CD147 expression and cell motility. This work reveals that different types of net acid extruding transporters, NHE1, NBCn1 and MCT4, are frequently expressed in patient mammary tumor tissue and demonstrates for the first time that they promote growth of TNBC human mammary tumors in vivo via distinct but overlapping mechanisms.

Protein Kinases Type II (PKG II) Combined with L-Arginine Significantly Ameliorated Xenograft Tumor Development: Is L-Arginine a Potential Alternative in PKG II Activation?

BackgroundThe mammalian cyclic guanosine monophosphate (cGMP)-dependent protein kinases type II (PKG II) plays critical physiological or pathological functions in different tissues. However, the biological effects of PKG II are dependent on cGMP. Published data indicated that L-arginine (L-Arg) promoted NO production, NO can activate soluble guanylate cyclase (sGC), and catalyzes guanosine triphosphate (GTP) into cGMP, which suggested L-Arg could activate PKG II. Therefore, the present work was performed to address: (i) whether L-Arg could be a potential alternative in PKG II activation, and (ii) whether L-Arg also contributes to PKG II against cancer.Material/MethodsNude BALB/c mice were inoculated with human MCF-7, HepG2, and SW480 cell lines via subcutaneous (s.c.) injecting. After 7 days of inoculation, Ad-PKG II was injected into the cancer tissues every 4 days, and the next day 10 μmol/mouse L-Arg was administered. Western blotting and immunohistochemistry were used to assess protein expression.ResultsOur results demonstrated that L-Arg significantly activated PKG II and effectively ameliorated xenograft tumor development through inhibiting cancer growth, angiogenesis, and metastasis, which was partially dependent on blocking of epidermal growth factor receptor (EGFR) activity, as well as downstream signaling pathways such as Erk1/2.ConclusionsOur results provide an exciting new insight: L-Arg is a potential alternative to PKG II activation.

Overcoming Autofluorescence to Assess GFP Expression During Normal Physiology and Aging in Caenorhabditis elegans.

Green fluorescent protein (GFP) is widely used as a molecular tool to assess protein expression and localization. In C. elegans, the signal from weakly expressed GFP fusion proteins is masked by autofluorescence emitted from the intestinal lysosome-related gut granules. For instance, the GFP fluorescence from SKN-1 transcription factor fused to GFP is barely visible with common GFP (FITC) filter setups. Furthermore, this intestinal autofluorescence increases upon heat stress, oxidative stress (sodium azide), and during aging, thereby masking GFP expression even from proximal tissues. Here, we describe a triple band GFP filter setup that separates the GFP signal from autofluorescence, displaying GFP in green and autofluorescence in yellow. In addition, yellow fluorescent protein (YFP) remains distinguishable from both the yellowish autofluorescence and GFP with this triple band filter setup. Although some GFP intensity might be lost with the triple band GFP filter setup, the advantage is that no modification of currently used transgenic GFP lines is needed and these GFP filters are easy to install. Hence, by using this triple band GFP filter setup, the investigators can easily distinguish autofluorescence from GFP and YFP in their favorite transgenic C. elegans lines.

Influence of Tobacco Smoke Exposure on the Protein Expression of α7 and α4 Nicotinic Acetylcholine Receptors in Squamous Cell Carcinoma Tumors of the Upper Aerodigestive Tract (Out of the Larynx).

Purpose:To assess protein expression of α7 and α4 nicotinic acetylcholine receptors (nAChR) subtypes in squamous cell carcinoma of the upper aerodigestive track (out of the larynx) according to tobacco smoke exposure, considering the general characteristics of the patients.Methods:The α7 and α4 nAChR subtypes were assessed by immunohistochemistry in tumor samples from 33 patients with novel diagnosis of squamous cell carcinoma of the upper aerodigestive tract (out of the larynx).Results:Current smokers were middle-age men with alcohol consumption, whereas elderly women with no alcohol consumption prevailed among nonsmokers. Expression of α4 nAChR was high in all groups, with an influence of alcohol use, although expression of α7 nAChR was low in current smokers with alcohol use. Expression of α4 with no expression of α7 nAChR was associated with advanced disease.Conclusions:Squamous cell carcinoma tumors of the upper aerodigestive tract (out of the larynx) may show desensitization of α4 nAChR. Advanced disease at diagnosis might be associated with desensitization of α4 with decrease in α7 nAChR.

One-week exposure to a free-choice high-fat high-sugar diet does not disrupt blood–brain barrier permeability in fed or overnight fasted rats

Objectives: The hypothalamus lies adjacent to the third ventricle and is in close proximity with the median eminence (ME), a circumventricular organ with an incomplete blood–brain barrier (BBB) which controls direct entry of nutrients into the brain. The blood–CSF barrier of the hypothalamus shows dynamic changes upon neuroendocrine events and adjusts permeability with the tight junction (TJ) complex. It has been shown that chronic exposure to a high-fat diet (HFD) affects BBB permeability. HFD also induces leptin resistance and alters neuropeptide expression in the arcuate nucleus (Arc) of the hypothalamus starting early during overnutrition. We hypothesized altered integrity of the BBB to occur after exposing rats to a free-choice high-fat high-sugar (fcHFHS) diet for 1 week.Methods: We measured diffusion of Evans blue dye over the ME and assessed expression of the TJ proteins ZO-1, claudin-5, and occludin in the tanycytic wall of the third ventricle. Furthermore, we assessed protein expression of glucose transporter 1 (GLUT-1), which is highly expressed in the Arc-ME complex and facilitates glucose transport over the BBB.Results: fcHFHS-fed rats increased caloric intake compared to control, however, there was no effect of the fcHFHS diet on permeability of the BBB, nor changes in protein expression of tight TJ proteins or GLUT-1. Fasting acutely affects the BBB and we hypothesized that exposure to the fcHFHS diet affects the BBB differently compared to chow after fasting. We did not, however, find any differences in Evans blue diffusion nor protein expression between chow- and fcHFHS-fed rats when fasted overnight.Conclusions: We conclude that short-term consumption of a fcHFHS diet does not change permeability or diffusion in the hypothalamus barrier in ad libitum fed or fasted rats.

MiR-181a promotes growth of thyroid cancer cells by targeting tumor suppressor RB1.

MicroRNAs (miRs) are critical regulators in cancer development and progression. The current study aimed to investigate the expression and potential function of miR-181a in thyroid cancer. A total of 15 paired thyroid cancer tissues and adjacent normal tissues were subjected to Real-time Polymerase Chain Reaction (PCR) to evaluate miR-181a expression. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, enzyme linked immunosorbent assay (ELISA) or flow cytometry was employed to assess the growth activity, apoptosis and cell cycle, respectively, upon modulation of the miR-181a expression in TPC-1 cells. Western blot was used to assess protein expression. The interaction between miR-181a and RB1 was tested by luciferase activity assay. The expression of miR-181a was significantly upregulated in thyroid cancer tissues compared with the adjacent tissues. Inhibition of miR-181a attenuated cell growth, which could be abrogated by miR-181a co-transfection. MiR-181a overexpression reduced apoptosis and promoted cell cycle progression; inhibition of miR-181a exerted opposite effects on both cell cycle and apoptosis. MiR-181a directly suppressed RB1 expression. RB1 expression in tumor tissues was downregulated and negatively correlated with miR-181a expression. miR-181a plays an oncogenic role in thyroid cancer; by targeting RB1, it promotes cell cycle progression and inhibits apoptosis.

Effect of social interactions on hippocampal protein expression in animal dominant and submissive model of behavioral disorders.

Psychiatric conditions, in many cases, arise from social interactions necessary for optimal mental functioning. Dominance and submissiveness are two opposite poles of behavior, stemming from processes of social interactions between members inside one group or species. Extreme dominance and submissiveness expressions in humans is accompanied by mental impairments, including mania and depression. Here, taking advantage of animals bred selectively for traits of dominance and submissiveness, we assess protein expression profiles in dominant and submissive mice in the context of social interaction. Proteins extracted from hippocampi of naïve and social interaction subjected dominant, submissive and wild type mice (15 mice per each group) are quantified using label-free quantitative LC/MS/MS analysis. Complexity of social interaction-related protein expression is resolved by factor analysis and enriched with GO and protein-protein interaction functional network analyses. In total, 1146 proteins exhibiting expression changes in the wild type mice, as well as dominant and submissive mice are enriched in protein datasets responsible for: 1) socially triggered dominance (90 proteins), 2) inherent submissiveness (75 proteins), 3) socially triggered submissiveness (117 proteins), and 4) social interaction triggered protein expression changes, related to resilience/adaptation to stress (69 proteins). Among the most enriched categories, extensive changes are found in proteins related to presynaptic release, ion channel regulation, circadian rhythm, MAPK, ErbB and NF-kB pathways. Data extracted from this first extensive proteomic study of a social interaction paradigm may facilitate decoding of molecular mechanisms responsible for pathogenesis of psychiatric disorders.

SUMO co-expression modifies KV 11.1 channel activity.

The voltage-gated potassium channel KV 11.1 is the molecular basis for the IKr current, which plays an important role in cardiac physiology. Its malfunction is associated with both inherited and acquired cardiac arrhythmias. Native currents differ from those in experimental models, suggesting additional regulatory mechanisms. We hypothesized that the post-translational modification sumoylation fine-tunes channel activity. The functional effects of sumoylation on KV 11.1 were addressed by employing two-electrode voltage-clamp (TEVC) experiments in Xenopus laevis oocytes. Site-directed mutagenesis enabled a further analysis of the SUMO-target amino acids. We assessed protein expression levels and used confocal imaging for localization studies. Co-expression with Ubc9 and SUMO alters the electrophysiological properties of KV 11.1 leading to a decrease in steady-state current amplitude largely due to faster inactivation and alteration of deactivation kinetics. We identified three lysines (K21, K93 and K116) in the PAS domain as the putative SUMO-targets. This study indicates KV 11.1 as a sumoylation target and offers three main targets: K21, K93, and K116. Furthermore, it proposes an underlying mechanism for the observed kinetic impact of the PAS domain.

Movement disorder in GNAO1 encephalopathy associated with gain-of-function mutations.

To define molecular mechanisms underlying the clinical spectrum of epilepsy and movement disorder in individuals with de novo mutations in the GNAO1 gene. We identified all GNAO1 mutations reported in individuals with epilepsy (early infantile epileptiform encephalopathy 17) or movement disorders through April 2016; 15 de novo mutant alleles from 25 individuals were introduced into the Gαo subunit by site-directed mutagenesis in a mammalian expression plasmid. We assessed protein expression and function in vitro in HEK-293T cells by Western blot and determined functional Gαo-dependent cyclic adenosine monophosphate (cAMP) inhibition with a coexpressed α2A adrenergic receptor. Of the 15 clinical GNAO1 mutations studied, 9 show reduced expression and loss of function (LOF; <90% maximal inhibition). Six other mutations show variable levels of expression but exhibit normal or even gain-of-function (GOF) behavior, as demonstrated by significantly lower EC50 values for α2A adrenergic receptor-mediated inhibition of cAMP. The GNAO1 LOF mutations are associated with epileptic encephalopathy while GOF mutants (such as G42R, G203R, and E246K) or normally functioning mutants (R209) were found in patients with movement disorders with or without seizures. Both LOF and GOF mutations in Gαo (encoded by GNAO1) are associated with neurologic pathophysiology. There appears to be a strong predictive correlation between the in vitro biochemical phenotype and the clinical pattern of epilepsy vs movement disorder.

The Role of MicroRNA-143-5p in the Differentiation of Dental Pulp Stem Cells into Odontoblasts by Targeting Runx2 via the OPG/RANKL Signaling Pathway.

This study aims to elucidate the mechanisms by which microRNA-143-5p (miR-143-5p) targets runt-related transcription factor 2 (Runx2) in the differentiation of dental pulp stem cells (DPSCs) into odontoblasts, through regulating the osteoprotegerin receptor activator of the nuclear factor-κB ligand (OPG/RANKL) signaling pathway. Following transfection, DPSCs were divided into blank, control, miR-143-5p mimics, miR-143-5p inhibitors, miR-143-5p inhibitors + siRunx2 and siRunx2 groups. Alkaline phosphatase (ALP) activity and mineralized nodules were detected using ALP kit and alizarin red staining. Quantitative reverse transcriptase real time PCR (qRT-PCR) was conducted to measure mRNA expressions of miR-143-5p, Runx2, OPG, and RANKL. Western blotting was used to assess protein expression of odontoblast differentiation-related proteins. Transwell assay and an extracellular matrix (ECM) adhesion cell assay were employed to examine cell migration and cell adhesion. Compared with the blank group, the miR-143-5p mimics and siRunx2 groups showed decreased ALP activity, decreased mineralized nodules and displays of calcium. Fewer migrated cells, weakened cell adhesion, decreased protein expression of dentin phosphoprotein (DPP), dentin sialoprotein (DSP), dentin matrix protein 1 (DMP1), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), OPG and Runx2, and increased RANKL protein expressions were observed. Additionally, opposite results were observed in the miR-143-5p inhibitors group, demonstrating that down-regulated miR-143-5p promotes the differentiation of DPSCs into odontoblasts by enhancing Runx2 expression via the OPG/RANKL signaling pathway. Based on findings in this study, it is postulated that the enhancement of Runx2 expression via the regulation of the OPG/RANKL signaling pathway could be a beneficial approach for dental pulp regeneration. J. Cell. Biochem. 119: 536-546, 2018. © 2017 Wiley Periodicals, Inc.

198 Heart failure increases mitochondrial s-nitrosylation

Heart failure affects over 5 50 000 people living in the United Kingdom (1) and roughly half of those present with reduced cardiac ejection fraction (2). The electron transport chain...

FBP1 is highly expressed in human hypertrophic scars and increases fibroblast proliferation, apoptosis, and collagen expression

ABSTRACTPurpose: FBP1, one of the far-upstream element binding proteins(FBPs), is a distal upstream binding protein of c-myc, which is highly expressed in tumor tissues. This study aimed to investigate FBP1 expression in human hypertrophic scars and to determine the effects of FBP1 on fibroblasts. Materials and methods: Human normal skin and scar specimens were collected during clinical surgery. One portion of each tissue specimen was embedded in paraffin and sliced to observe differences in histological features and FBP1 expression by immunohistochemistry and western blotting. The other portion of each tissue specimen was cultured to obtain fibroblasts. Fibroblasts from the second to the sixth passage were used for the experiments, which were divided into the following two groups: an experimental group, whose cells were transfected with an siRNA targeting FBP1, and a control group, whose cells where not transfected. MTT and TUNEL assays were performed, respectively, to assess fibroblast proliferation and apoptosis, and western blotting was performed to assess protein expression. Results: We obtained fibroblasts by primary tissue culture and found that FBP1 was highly expressed in hypertrophic scars. MTT assay showed that an siRNA targeting FBP1 significantly reduced fibroblast proliferation in siRNA-treated cells compared to control cells. TUNEL assay showed that there was no difference in apoptosis between the two groups; however, western blotting showed that collagen I, collagen III, c-myc, caspase-3, and caspase-9 expression levels were all decreased in the experimental group. Conclusion: FBP1 is highly expressed in human hypertrophic scars and increases fibroblast proliferation, apoptosis and collagen expression.

Abstract 150: Hypoxia and Ischemia Promote a Maladaptive Platelet Phenotype

Background: An understudied area of platelet biology is that ischemic diseases such as peripheral vascular disease (PVD) can alter the platelet phenotype, and this may lead to unpredictable effects of anti-platelet agents. Methods: Isolated murine and human platelets were exposed to a reduced oxygen environment (hypoxia chamber, 5% O 2 ). Platelet activation was assessed as mean fluorescence intensity (MFI) of surface p-selectin by flow cytometry, and morphologic changes by confocal microscopy. A murine model of critical limb ischemia (CLI) was used to assess changes in platelet activity in wild-type (WT) and in platelet ERK5 -/- mice. Limb blood flow was assessed by laser Doppler imaging. Hypoxia was additionally examined in mice by unilateral pneumonectomy. Western blotting was used to assess protein expression. Results: In human platelets ex vivo at 5% O 2 , platelet activation increased to 5076 MFI±409 from 3548 MFI±187 at 20% O 2 (P=0.005). With the ERK5 inhibitor XMD892, platelet activation at 5% O 2 decreased from 5677 MFI±312 to 3175 MFI±247 (p=0.001). In a murine model of PVD, enhanced platelet activation was sustained for weeks vs. sham, and limb blood flow in platelet ERK5 -/- mice one week after ischemia was augmented by 26%: 0.61±0.03 vs 0.87±0.03 vs, WT mice (p=0.0002), with 50% less platelet activity. In mice with unilateral pneumonectomy vs. sham surgery, observed hypoxia was accompanied by increased platelet ERK5 activation coincident with enhanced platelet activation. In patients with CLI, augmented platelet activation in spite of aspirin treatment was observed via the thromboxane and the PAR1 receptors, and especially via P2Y 12 receptor. Conclusions: Hypoxia and ischemic tissue injury, such as with critical limb ischemia, change the phenotype of the platelet, promoting a pro-thrombotic state that is partly dependent on platelet ERK5. Platelet phenotype and function should be better characterized in ischemic diseases.