PKC Regulates YAP Expression through Alternative Splicing of YAP 3'UTR Pre-mRNA by hnRNP F.

The Yes-associated protein (YAP) is a transcriptional co-activator that plays critical roles in organ development and tumorigenesis, and is verified to be inhibited by the Hippo signaling pathway. In the present study, we show that the YAP 3'UTR is alternatively spliced to generate a novel 950 bp 3'UTR mRNA from the full length 3'UTR region (3483 bp) in human cancer cells. The ratio of full length 3'UTR YAP mRNA to alternatively spliced 3'UTR YAP mRNA is up-regulated by exposure of the cells to PKC inhibitor chelerythrine chloride. Further study using luciferase reporter assay showed that the expression of the alternatively spliced 3'UTR mRNA is much lower compared with the full length 3'UTR mRNA, suggesting that alternatively spliced 3'UTR YAP mRNA may have a shorter half-life than full length 3'UTR mRNA. Interestingly, PKC represses YAP 3'UTR-mediated mRNA stability is dependent on a splicing factor, hnRNP F. Activation of PKC induces nuclear translocation of cytosolic hnRNP F. Ectopic expression of hnRNP F enhances YAP 3'UTR splicing. Our results suggest that hnRNP F regulates YAP 3'UTR-mediated mRNA stability in an alternative splicing-dependent manner, and PKC regulated YAP expression is dependent on nuclear translocation of hnRNP F in human cancer cell lines.

MicroRNA 630 Represses NANOG Expression through Transcriptional and Post-Transcriptional Regulation in Human Embryonal Carcinoma Cells.

The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3'UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the NANOG 3'UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3'UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3'UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3'UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3'UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.

Heterogeneous ribonucleoprotein F regulates YAP expression via a G-tract in 3'UTR.

The Yes-associated protein (YAP) is a transcription coactivator that plays crucial roles in organ size control and tumorigenesis, and was demonstrated to be inhibited by the Hippo signaling pathway. To date, the molecular mechanisms regulating the expression of YAP in human cells remain unknown. In the present study, we found that hnRNP F and hnRNP U negatively regulate YAP expression. We also showed that downregulation of YAP expression by hnRNP F and hnRNP U was not at the transcriptional level. Knockdown of hnRNP F or hnRNP U increased YAP mRNA stability, suggesting the downregulation of YAP expression was by a post-transcriptional mechanism. A putative hnRNP F binding site was identified in the YAP 3'UTR at 685 to 698, and deletion of this putative hnRNP F element abolished the down-regulation effect of YAP mRNA stability by hnRNP F. Binding of the hnRNP F to the YAP 3'UTR was demonstrated by Cross-linked RNA Immunoprecipitation. mRNA stability is a possible secondary effect of alternative splicing or other nuclear process. Understanding the regulation of YAP expression would provide insights into the mechanisms underlying the maintenance of tissue size homeostasis and tumorigenesis.

Dietary Leucine Supplement Ameliorates Hepatic Steatosis and Diabetic Nephropathy in db/db Mice.

Dietary leucine supplementation has been explored for the therapeutic intervention of obesity and obesity-induced metabolic dysfunctions. In this study, we aim to examine the effects of dietary leucine supplementation in db/db mice. Mice were treated with or without leucine (1.5% w/v) in drinking water for 12 weeks. The leucine supplement was found to reduce insulin resistance and hepatic steatosis in db/db mice. Using Nuclear Magnetic Resonance (NMR)-based lipidomics, we found that the reduction of hepatic triglyceride synthesis was correlated with attenuated development of fatty liver. In addition, diabetic nephropathy (DN) was also ameliorated by leucine. Using liquid chromatography⁻time-of-flight mass spectrometry (LC-TOF MS)-based urine metabolomics analysis, we found that the disturbance of the tricarboxylic acid (TCA) cycle was reversed by leucine. The beneficial effects of leucine were probably due to AMP-activated protein kinase (AMPK) activation in the liver and kidneys of db/db mice. Thus, dietary leucine supplementation may potentially be a nutritional intervention to attenuate hepatic steatosis and early DN in type II diabetes.

Novel bifurcation stents coated with bioabsorbable nanofibers with extended and controlled release of rosuvastatin and paclitaxel.

A novel bifurcation stent coated with bioabsorbable nanofibers that deliver the extended and controlled release of rosuvastatin and paclitaxel was developed. Bioabsorbable bifurcation stents, consisting of a double-slit tubular main body and two spiral branches, were manufactured. Bi-layered poly (lactic-co-glycolic acid) nanofibers that contained rosuvastatin and paclitaxel were used for treating the stents. Various properties of the fabricated stents, including compression strengths, collapse pressure, water contact angle and flow properties within a circulation model, were quantified. In vitro nanofibrous elution chromatography assays from the drug-loading bifurcation stents were carried out for the release patterns of pharmaceuticals. The effectiveness of eluted rosuvastatin and paclitaxel in inhibiting the adhesion of platelets as well as the proliferation of smooth muscle cells (SMCs) were studied, respectively. The experimental results suggest that bioabsorbable nanofibrous bifurcation stents released high concentrations of rosuvastatin and paclitaxel for 27 and 70 days, respectively. The eluted drugs of rosuvastatin and paclitaxel effectively reduced adherent platelets and the proliferation of SMCs. The developed bioabsorbable nanofibrous bifurcation stents herein may provide a promising means of treating cardiovascular bifurcation lesions.

Role of dysfunctional adipocytes in cholesterol-induced nonobese metabolic syndrome.

Many studies have reported the causes of obese metabolic syndrome (MS); however, the causes of nonobese MS (NMS) remain unknown. In this study, we demonstrated that inflamed dysfunctional adipose tissue plays a crucial role in cholesterol-induced NMS. Control (C), high cholesterol (HC) and HC with 10% fructose in drinking water (HCF) diets were fed to Sprague-Dawley rats for 12 weeks. After 12 weeks, the body weights of the C- and HC-fed rats were comparable, but the weights of the HCF-fed rats were relatively low. Cholesterol caused metabolic problems such as high blood pressure, hypercholesterolemia and hypoinsulinemia. The HCF-fed rats exhibited whole-body insulin resistance with low circulating high-density lipoprotein levels. Increases in the tumor necrosis factor α level in the plasma, the number of CD68+ macrophages and the free nuclear factor-κB level in gonadal white adipose tissue (gWAT) resulted in local inflammation, which appeared as inflamed dysfunctional gWAT. Reduced superoxide dismutases (SODs) deteriorate natural antioxidant defense systems and induce reactive oxygen species in gWAT. Dysregulation of plasma levels of catecholamine, adipokines (leptin and adiponectin), hormone-sensitive lipase and perilipin in cholesterol-induced inflamed adipose tissue contributed to increased lipolysis and increased circulating nonesterified fatty acids. Cholesterol activated inflammation, lipolysis and cell death in 3T3-L1 adipocytes. Moreover, Chol-3T3-CM reduced the population of M2-type Raw264.7 macrophages, indicating that the macrophage polarization is mediated by cholesterol. Together, our findings indicate that inflamed dysfunctional adipocytes are critical in NMS, supporting the development of anti-inflammatory agents as potential therapeutic drugs for treating NMS.

Over-expression of ΔNp63α facilitates rat corneal wound healing in vivo.

To investigate the roles of ΔNp63α during corneal wound healing and the genes regulated by ΔNp63α in limbal epithelial cells. Adenovirus or shRNA targeting ΔNp63α were pre-injected into the anterior chamber of rat eyeballs and the central corneal epithelium was then wounded with NaOH. The effects of ΔNp63α expression during wound healing were observed by propidium iodide staining. In addition, limbal epithelial cells were cultured and ectopically expressed ΔNp63α by transfecting Ad-ΔNp63α. Total RNA was extracted from transfected epithelial cells and subjected to a gene expression microarray assay. The results showed that over-expression of ΔNp63α accelerated the process of corneal wound healing while knockdown of ΔNp63α impaired the process. ΔNp63α positively up-regulated several cell growth promoter genes and could be referred as a positive regulator of limbal epithelial cell proliferation. It might also inhibit cell differentiation and cell death by differential target gene regulation.

Lidocaine/ketorolac-loaded biodegradable nanofibrous anti-adhesive membranes that offer sustained pain relief for surgical wounds.

The aim of this study was to develop and evaluate the effectiveness of biodegradable nanofibrous lidocaine/ketorolac-loaded anti-adhesion membranes to sustainably release analgesics on abdominal surgical wounds. The analgesic-eluting membranes with two polymer-to-drug ratios (6:1 and 4:1) were produced via an electrospinning technique. A high-performance liquid chromatography (HPLC) assay was employed to characterize the in vivo and in vitro release behaviors of the pharmaceuticals from the membranes. It was found that all biodegradable anti-adhesion nanofibers released effective concentrations of lidocaine and ketorolac for over 20 days post surgery. In addition, a transverse laparotomy was setup in a rat model for an in vivo assessment of activity of postoperative recovery. No tissue adhesion was observed at 2 weeks post surgery, demonstrating the potential anti-adhesion capability of the drug-eluting nanofibrous membrane. The postoperative activities were recorded for two groups of rats as follows: rats that did not have any membrane implanted (group A) and rats that had the analgesic-eluting membrane implanted (group B). Rats in group B exhibited faster recovery times than those in group A with regard to postoperative activities, confirming the pain relief effectiveness of the lidocaine- and ketorolac-loaded nanofibrous membranes. The experimental results suggested that the anti-adhesion nanofibrous membranes with sustainable elution of lidocaine and ketorolac are adequately effective and durable for the purposes of postoperative pain relief in rats.

Fabrication of Multi-Layered Lidocaine and Epinephrine-Eluting PLGA/Collagen Nanofibers: In Vitro and In Vivo Study.

This study developed multi-layered lidocaine- and epinephrine-eluting biodegradable poly[(d,l)-lactide-co-glyco lide] (PLGA)/collagen nanofibers. An electrospinning technique was employed to fabricate the multi-layer biodegradable drug-eluting nanofibers. After fabrication, the nanofibrous membranes were characterized. The drug release characteristics were also investigated. In addition, the in vivo efficacy of nanofibers for pain relief and hemostasis in palatal oral wounds of rabbits were evaluated. Histological examinations were also completed. The experimental results suggested that all nanofibers exhibited good biocompatibility and eluted effective levels of lidocaine and epinephrine at the initial stages of wound recovery.

Docosapentaenoic acid and docosahexaenoic acid are positively associated with insulin sensitivity in rats fed high-fat and high-fructose diets.

BACKGROUND: The aim of the present study was to compare insulin resistance and metabolic changes using a global lipidomic approach. METHODS: Rats were fed a high-fat diet (HFD) or a high-fructose diet (HFrD) for 12 weeks to induce insulin resistance (IR) syndrome. After 12 weeks feeding, physiological and biochemical parameters were examined. Insulin sensitivity and plasma metabolites were evaluated using a euglycemic-hyperinsulinemic clamp and mass spectrometry, respectively. Pearson's correlation coefficient was used to investigate the strength of correlations. RESULTS: Rats on both diets developed IR syndrome, characterized by hypertension, hyperlipidemia, hyperinsulinemia, impaired fasting glucose, and IR. Compared with HFrD-fed rats, non-esterified fatty acids were lower and body weight and plasma insulin levels were markedly higher in HFD-fed rats. Adiposity and plasma leptin levels were increased in both groups. However, the size of adipocytes was greater in HFD- than HFrD-fed rats. Notably, the lipidomic heat map revealed metabolites exhibiting greater differences in HFD- and HFrD-fed rats compared with controls. Plasma adrenic acid levels were higher in HFD- than HFrD-fed rats. Nevertheless, linoleic and arachidonic acid levels decreased in HFrD-fed rats compared with controls. Plasma concentrations of docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) were significantly reduced after feeding of both diets, particularly the HFrD. There was a strong positive correlation between these two fatty acids and the insulin sensitivity index. CONCLUSIONS: The systemic lipidomic analysis indicated that a reduction in DHA and DPA was strongly correlated with IR in rats under long-term overnutrition. These results provide a potential therapeutic target for IR and metabolic syndrome.

Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation.

To avoid xenogeneic infection, we report a novel protocol for producing animal-derived component-free oral mucosal epithelial cells (OMECs) sheet for transplantation, in which collagenase was used to replace dispase II/trypsin-EDTA for digesting oral mucosal tissue, and human platelet-derived PLTMax to replace fetal bovine serum. The resulting epithelial aggregates were expanded on de-epithelialized amniotic membranes without 3T3 feeder cells, and serum-free EpiLife was used to reduce contamination by submucosal mesenchymal cells. The OMEC sheets thus generated showed similar positive keratin 3/76-positive and keratin 8-negative staining patterns compared with those generated by the original protocol. Colony formation efficiency assay, BrdU label retention assay, and p63 and p75NTR immunostaining results indicated that higher proliferative potentials and more progenitor cells were preserved by the modified protocol. TaqMan array analysis revealed that the transcription of integrin-linked kinase (ILK) was up-regulated along with an increase in ß-catenin signaling and its downstream cell cycle modulators, cyclin D1 and p27KIP1. Furthermore, ILK silencing led to the inhibition of nuclear ß-catenin accumulation, suppressed p63 expression, and reduced the expression of cyclin D1 and p27KIP1; these observations suggest that ILK/ß-catenin pathway may be involved in cell proliferation regulation during the ex vivo expansion of OMECs for transplantation purposes.

Enhancement of tendon-bone healing via the combination of biodegradable collagen-loaded nanofibrous membranes and a three-dimensional printed bone-anchoring bolt.

A composite biodegradable polymeric model was developed to enhance tendon graft healing. This model included a biodegradable polylactide (PLA) bolt as the bone anchor and a poly(D,L-lactide-co-glycolide) (PLGA) nanofibrous membrane embedded with collagen as a biomimic patch to promote tendon-bone interface integration. Degradation rate and compressive strength of the PLA bolt were measured after immersion in a buffer solution for 3 months. In vitro biochemical characteristics and the nanofibrous matrix were assessed using a water contact angle analyzer, pH meter, and tetrazolium reduction assay. In vivo efficacies of PLGA/collagen nanofibers and PLA bolts for tendon-bone healing were investigated on a rabbit bone tunnel model with histological and tendon pullout tests. The PLGA/collagen-blended nanofibrous membrane was a hydrophilic, stable, and biocompatible scaffold. The PLA bolt was durable for tendon-bone anchoring. Histology showed adequate biocompatibility of the PLA bolt on a medial cortex with progressive bone ingrowth and without tissue overreaction. PLGA nanofibers within the bone tunnel also decreased the tunnel enlargement phenomenon and enhanced tendon-bone integration. Composite polymers of the PLA bolt and PLGA/collagen nanofibrous membrane can effectively promote outcomes of tendon reconstruction in a rabbit model. The composite biodegradable polymeric system may be useful in humans for tendon reconstruction.

High-fructose and high-fat feeding correspondingly lead to the development of lysoPC-associated apoptotic cardiomyopathy and adrenergic signaling-related cardiac hypertrophy.

BACKGROUND: The heart is a highly adaptive organ that demonstrates remarkable structural, functional, and metabolic remodeling in response to physiological and pathological stimuli. We hypothesize that the heart undergoes differential adaptations in high-fat and high-fructose diet, resulting in a distinct phenotype. METHODS: High-fat and high-fructose diet-induced obese and non-obese insulin resistance (IR) rat models were used to understand how the heart adapts to long-term (12-week) overnutrition. RESULTS: Rats fed the high-fat diet developed obese IR, whereas high-fructose diet developed non-obese IR. Obese IR rats developed fibrotic hypertrophy with impairment of preload-independent contractility. The sympathetic and renin-angiotensin-aldosterone (RAA) systems and myocardial adrenergic signaling were activated in obese IR rats. Non-obese IR rats developed apoptotic cardiomyopathy with severe systolic dysfunction. Myocardial calcium cycling regulatory proteins (CCRPs) were dysregulated in non-obese IR rats; specifically, troponin I protein expression was downregulated. Moreover, compared with the controls, lipidomics analysis revealed substantial differences in lipid metabolites in non-obese IR and obese IR rats. The overproduction of lysophosphatidylcholine (lysoPC) and fatty acids was observed in non-obese IR rat hearts. A strong correlation was observed between the myocardial lysoPC and plasma troponin I levels. Treatment of cardiomyocytes with lysoPC resulted in cell death in a dose- and time-dependent manner. The overproduction of myocardial lysoPCs was associated with circulating sPLA2 levels. CONCLUSION: Obese IR rats developed severe fibrotic hypertrophy with the activation of adrenergic signaling and sympathetic and RAA systems. The sPLA2-lysoPC may play a crucial role in the induction of apoptotic cardiomyopathy in high fructose-induced non-obese IR rats.

Preservation of human limbal epithelial progenitor cells on carbodiimide cross-linked amniotic membrane via integrin-linked kinase-mediated Wnt activation.

The Wnt pathway is a major signaling pathway that regulates corneal epithelial stem cells. However, little is known about how the ultrastructure of the limbal epithelial basement membrane (EBM) affects Wnt activity. Due to its enhanced matrix stability, the cross-linked amniotic membrane (AM) has gained increasing interest in the field of regenerative medicine. For the first time, we used EDC/NHS cross-linked denuded AM (CLDAM) as a simulated EBM substrate to investigate this mechanism. Human limbal epithelial (HLE) cells were cultured on dishes (HLE/dish), denuded AM (HLE/DAM) or CLDAM (HLE/CLDAM). Compared with HLE/dish or HLE/DAM cultures, HLE/CLDAM cultures showed greater BrdU retention and colony formation efficiency and expressed higher levels of p63, ABCG2, integrin ß1, and integrin-linked kinase (ILK). Nuclear ß-catenin and TCF-4 levels were higher in HLE/CLDAM cultures compared with HLE cells cultured on collagen IV, laminin, Matrigel, or DAM. Silencing of ILK in HLE/CLDAM cultures resulted in decreased levels of nuclear ß-catenin, TCF-4 and deltaNp63α, whereas cytokeratin 12 expression increased. Over-expression of ILK in HLE/dish cultures had the opposite effects. Accordingly, we proposed that the CLDAM matrix, with its higher rigidity and rougher ultrastructure, better preserved HLE progenitor cells in vitro, possibly by activating integrin ß1/ILK, which indirectly activated Wnt/ß-catenin and subsequently deltaNp63α. Crosstalk between the integrin ß1/ILK and Wnt/ß-catenin pathways appears to play a crucial role in limbal progenitor cell survival on EBM. STATEMENT OF SIGNIFICANCE: We demonstrated the superior capability of carbodiimide cross-linked denuded amniotic membrane (CLDAM) than natural DAM to preserve limbo-corneal epithelial progenitor cells in vitro, then we used CLDAM as a simulated epithelial basement membrane (EBM) to study how EBM maintains limbal epithelial stem cells (LESCs). We found that integrin-linked kinase (ILK) is an important mediator that transfers survival signals detected by integrin ß1 to the Wnt/ß-catenin pathway, which in turn up-regulates deltaNp63α, a master gene that regulates LESC function. The rougher surface of the limbal EBM suggests that the surface complexity of the LESC niche may be important in regulating LESC function, which is triggered by the recognition of topographic cues by integrin ß1, followed by activation of the ILK/Wnt/ß-catenin/p63 cascade.

Lysophosphatidic acid induces YAP-promoted proliferation of human corneal endothelial cells via PI3K and ROCK pathways.

The first two authors contributed equally to this work.Silence of p120-catenin has shown promise in inducing proliferation in human corneal endothelial cells (HCECs), but there is concern regarding off-target effects in potential clinical applications. We aimed to develop ex vivo expansion of HCECs using natural compounds, and we hypothesized that lysophosphatidic acid (LPA) can unlock the mitotic block in contact-inhibited HCECs via enhancing nuclear translocation of yes-associated protein (YAP). Firstly, we verified that exogenous YAP could induce cell proliferation in contact-inhibited HCEC monolayers and postconfluent B4G12 cells. In B4G12 cells, enhanced cyclin D1 expression, reduced p27(KIP1)/p21(CIP1) levels, and the G1/S transition were detected upon transfection with YAP. Secondly, we confirmed that LPA induced nuclear expression of YAP and promoted cell proliferation. Moreover, PI3K and ROCK, but not ERK or p38, were required for LPA-induced YAP nuclear translocation. Finally, cells treated with LPA or transfected with YAP remained hexagonal in shape, in addition to unchanged expression of ZO-1, Na/K-ATPase, and smooth muscle actin (SMA), suggestive of a preserved phenotype, without endothelial-mesenchymal transition. Collectively, our findings indicate an innovative strategy for ex vivo cultivation of HCECs for transplantation and cell therapy.

Effects of hyperbaric oxygen on the osteogenic differentiation of mesenchymal stem cells.

BACKGROUND: Hyperbaric oxygenation was shown to increase bone healing in a rabbit model. However, little is known about the regulatory factors and molecular mechanism involved.We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is mediated via increases in the osteogenic differentiation of mesenchymal stem cells (MSCs) which are regulated by Wnt signaling. METHODS: The phenotypic characterization of the MSCs was analyzed by flow cytometric analysis. To investigate the effects of HBO on Wnt signaling and osteogenic differentiation of MSCs, mRNA and protein levels of Wnt3a, beta-catenin, GSK-3beta, Runx 2, as well as alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining were analyzed after HBO treatment. To investigate the effects of HBO on Wnt processing and secretion, the expression of Wntless and vacuolar ATPases were quantified after HBO treatment. RESULTS: Cells expressed MSC markers such as CD105, CD146, and STRO-1. The mRNA and protein levels of Wnt3a, ß-catenin, and Runx 2 were up-regulated, while GSK-3ß was down-regulated after HBO treatment. Western blot analysis showed an increased ß-catenin translocation with a subsequent stimulation of the expression of target genes after HBO treatment. The above observation was confirmed by small interfering (si)RNA treatment. HBO significantly increased alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining of osteogenically differentiated MSCs. We further showed that HBO treatment increased the expression of Wntless, a retromer trafficking protein, and vacuolar ATPases to stimulate Wnt processing and secretion, and the effect was confirmed by siRNA treatment. CONCLUSIONS: HBO treatment increased osteogenic differentiation of MSCs via regulating Wnt processing, secretion, and signaling.

Simultaneous quantification of trans-resveratrol and its sulfate and glucuronide metabolites in rat tissues by stable isotope-dilution UPLC-MS/MS analysis.

A rapid, sensitive, and selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in the multiple reaction monitoring (MRM) mode has been developed and validated to investigate the distribution of trans-Resveratrol (Resv) and its metabolites in rats following intravenous (IV) administration at 20mg/kg body weight (BW). Resv and Resv metabolites were analyzed in the negative electrospray ionization mode and eluted with retention times of about 0.69-2.22min with a runtime of 7min. Stable deuterium-labeled Resv and its metabolites were used as the internal standards to correct for matrix effects and to allow for accurate quantification of Resv and its metabolites in a complex biological system. The method was validated with respect to linearity, within- and between-day precision, limit of quantification, recovery, and accuracy for all analytes. Upon examination at 0.5, 1, 2, and 4h post-administration, concentrations of Resv and its metabolites were the highest in the kidney, followed by plasma; specifically, the glucuronidated forms were the most abundant. In the liver and the brain, the predominant forms were the sulfated derivatives. In contrast, heart tissue contained the highest concentration of unmodified Resv at 0.5h post IV administration. The combined use of UPLC-MS/MS and isotope-dilution analysis, proved to be accurate and reliable in identifying and quantifying Resv and its various metabolites in biological samples at the nanomolar range. This technology is potentially applicable for other pharmacokinetic studies.

Hyperbaric oxygen promotes osteogenic differentiation of bone marrow stromal cells by regulating Wnt3a/ß-catenin signaling--an in vitro and in vivo study.

We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is increased via osteogenic differentiation of bone marrow stromal cells (BMSCs), which is regulated by Wnt3a/ß-catenin signaling. Our in vitro data showed that HBO increased cell proliferation, Wnt3a production, LRP6 phosphorylation, and cyclin D1 expression in osteogenically differentiated BMSCs. The mRNA and protein levels of Wnt3a, ß-catenin, and Runx2 were upregulated while those of GSK-3ß were downregulated after HBO treatment. The relative density ratio (phospho-protein/protein) of Akt and GSK-3ß was both up-regulated while that of ß-catenin was down-regulated after HBO treatment. We next investigated whether HBO affects the accumulation of ß-catenin. Our Western blot analysis showed increased levels of translocated ß-catenin that stimulated the expression of target genes after HBO treatment. HBO increased TCF-dependent transcription, Runx2 promoter/Luc gene activity, and the expression of osteogenic markers of BMSCs, such as alkaline phosphatase activity, type I collagen, osteocalcin, calcium, and the intensity of Alizarin Red staining. HBO dose dependently increased the bone morphogenetic protein (BMP2) and osterix production. We further demonstrated that HBO increased the expression of vacuolar-ATPases, which stimulated Wnt3a secretion from BMSCs. Finally, we showed that the beneficial effects of HBO on bone formation were related to Wnt3a/ß-catenin signaling in a rabbit model by histology, mechanical testing, and immunohistochemical assays. Accordingly, we concluded that HBO increased the osteogenic differentiation of BMSCs by regulating Wnt3a secretion and signaling.

Neurodegeneration in streptozotocin-induced diabetic rats is attenuated by treatment with resveratrol.

AIM: Diabetes mellitus-associated hyperglycemia and oxidative stress have been shown to have detrimental effects on the brain and may lead to impairment of cognitive functions. Resveratrol (Rsv), a polyphenolic antioxidant, has been shown to have moderate hypoglycemic and prominent hypolipidemic effects in diabetic rats. In the present study, we examined if Rsv improves the diabetic encephalopathy and explored its possible underlying mechanisms. METHODS: Male SD rats were treated with streptozotocin (65 mg/kg), and the diabetic rats were orally fed with Rsv (0.75 mg/kg, every 8 h) or normal saline for 4 weeks. Animals were then sacrificed and the brain tissues (hippocampus) processed for biochemical and histological studies. RESULTS: Neurodegeneration and astrocytic activation were noted in the hippocampus of the diabetic rats. Tumor necrosis factor-α, IL-6 transcripts and nuclear factor-κB expression were increased in the brain. In addition, neuropathic alterations in the hippocampus were evident in diabetic rats, including increased blood vessel permeability and VEGF expression, decreased mitochondrial number and AMP-activated protein kinase activity. In Rsv-treated diabetic rats, the aforementioned abnormalities were all attenuated. CONCLUSION: These observations suggest that Rsv significantly attenuated neurodegeneration and astrocytic activation in the hippocampus of diabetic rats. Our results suggested that Rsv could potentially be a new therapeutic agent for diabetic encephalopathy and neurodegeneration.

Nanog expression is negatively regulated by protein kinase C activities in human cancer cell lines.

Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.