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International Journal of Biological Sciences



International Journal of Biological Sciences RSS feed -- Volume 12



Published: Thu, 22 Dec 2016 04:00:00 GMT

Last Build Date: Thu, 22 Dec 2016 04:00:00 GMT

 



Evaluation of Breast Cancer Stem Cells and Intratumor Stemness Heterogeneity in Triple-negative Breast Cancer as Prognostic Factors

2016

Triple-negative breast cancer (TNBC) is a tumor subtype with aggressive behavior and poor clinical outcome for lacking effective therapies. Breast cancer stem cells (BCSCs) have been suggested to have tumor-initiating properties, but it remains unclear whether their presence contributes to the increased aggressiveness and poor prognosis of TNBC. Also, the breast cancers display frequent inter- and intra-tumor heterogeneity, which adds the complexity in diagnosis and predicting prognosis. Here we investigated the clinical relevance and prognostic value of the BCSC markers, CD44+/CD24-, aldehyde dehydrogenase family 1 member A1 (ALDH1A1) and CD133 in 88 TNBC cases. We found that a few patients displayed spatial heterogeneity of the BCSC markers in expression, which was defined as intratumor stemness heterogeneity (ITSH) below. There was no significant correlation between any BCSC marker alone or ITSH and progression-free survival (PFS). Interestingly, the combined BCSC phenotype by CD44+/CD24- and ALDH1A1 was significantly associated with worse PFS (P = 0.009). Further stratification analysis revealed that this combined BCSC phenotype was an independent prognostic factor for PFS in some subgroups. In conclusion, we demonstrated the existence of ITSH in TNBC and found that the ITSH as well as a single BCSC marker was not significantly associated with survival, whereas combing the analysis of BCSC markers could improve prognostic value. Our findings may lead to an improvement of prognostic indicators in TNBC.




Anticancer Effects of a New SIRT Inhibitor, MHY2256, against Human Breast Cancer MCF-7 Cells via Regulation of MDM2-p53 Binding

2016

The sirtuins (SIRTs), a family of NAD+-dependent class III histone deacetylase, are involved in various biological processes including cell survival, division, senescence, and metabolism via activation of the stress-response pathway. Recently, inhibition of SIRTs has been considered a promising anticancer strategy, but their precise mechanisms of action are not well understood. In particular, the relevance of p53 to SIRT-induced effects has not been fully elucidated. We investigated the anticancer effects of a novel SIRT inhibitor, MHY2256, and its efficacy was compared to that of salermide in MCF-7 (wild-type p53) and SKOV-3 (null-type p53) cells. Cell viability, SIRT1 enzyme activity, cell cycle regulation, apoptosis, and autophagic cell death were measured. We compared sensitivity to cytotoxicity in MCF-7 and SKOV-3 cells. MHY2256 significantly decreased the viability of MCF-7 (IC50, 4.8 μM) and SKOV-3 (IC50, 5.6 μM) cells after a 48 h treatment period. MHY2256 showed potent inhibition (IC50, 0.27 mM) against SIRT1 enzyme activity compared with nicotinamide (IC50, >1 mM). Moreover, expression of SIRT (1, 2, or 3) protein levels was significantly reduced by MHY2256 treatment in both MCF-7 and SKOV-3 cells. Flow cytometry analysis revealed that MHY2256 significantly induced cell cycle arrest in the G1 phase, leading to an effective increase in apoptotic cell death in MCF-7 and SKOV-3 cells. A significant increase in acetylated p53, a target protein of SIRT, was observed in MCF-7 cells after MHY2256 treatment. MHY2256 up-regulated LC3-II and induced autophagic cell death in MCF-7 cells. Furthermore, MHY2256 markedly inhibited tumor growth in a tumor xenograft model of MCF-7 cells. These results suggest that a new SIRT inhibitor, MHY2256, has anticancer activity through p53 acetylation in MCF-7 human breast cancer cells.




Signal transduction mechanism for glucagon-induced leptin gene expression in goldfish liver

2016

Leptin is a peripheral satiety hormone that also plays important roles in energy homeostasis in vertebrates ranging from fish to mammals. In teleost fish, however, the regulatory mechanism for leptin gene expression still remains unclear. In this study, we found that glucagon, a key hormone in glucose homeostasis, was effective at elevating the leptin-AI and leptin-AII transcript levels in goldfish liver via both in vivo intraperitoneal injection and in vitro cells incubation approaches. The responses of leptin-AI and leptin-AII mRNA to glucagon treatment were highly comparable. In contrast, blockade of local glucagon action could reduce the basal and induced leptin-AI and leptin-AII mRNA expression. The stimulation of leptin levels by glucagon was caused by the activation of adenylate cyclase (AC)/cyclic-AMP (cAMP)/ protein kinase A (PKA), and probably cAMP response element-binding protein (CREB) cascades. Our study described the effect and signal transduction mechanism of glucagon on leptin gene expression in goldfish liver, and may also provide new insight into leptin as a mediator in the regulatory network of energy metabolism in the fish model.




Cyclooxygenase-2 in tumor-associated macrophages promotes metastatic potential of breast cancer cells through Akt pathway

2016

Tumor-associated macrophages (TAMs) promote cancer development and progression by releasing various cytokines and chemokines. Previously, we have found that the number of COX-2+ TAMs was associated with lymph node metastasis in breast cancer. However, the mechanism remains enigmatic. In this study, we show that COX-2 in breast TAMs enhances the metastatic potential of breast cancer cells. COX-2 in TAMs induces MMP-9 expression and promotes epithelial-mesenchymal transition (EMT) in breast cancer cells. In addition, COX-2/PGE2 induces IL-6 release in macrophages. Furthermore, we find that the activation of Akt pathway in cancer cells is crucial for the pro-metastatic effect of COX-2+ TAMs by regulating MMP-9 and EMT. These findings indicate that TAMs facilitate breast cancer cell metastasis through COX-2-mediated intercellular communication.




Genes involved in angiogenesis and mTOR pathways are frequently mutated in Asian patients with pancreatic neuroendocrine tumors

2016

Introduction: To address the issue of limited data on and inconsistent findings for genetic alterations in pancreatic neuroendocrine tumors (pNETs), we analyzed sequences of known pNET-associated genes for their impact on clinical outcomes in a Taiwanese cohort. Methods: Tissue samples from 40 patients with sporadic pNETs were sequenced using a customized sequencing panel that analyzed 43 genes with either an established or potential association with pNETs. Genetic mutations and clinical outcomes were analyzed for potential associations. Results: Thirty-three patients (82.5%) survived for a median 5.9 years (range, 0.3-18.4) of follow up. The median number of mutations per patient was 3 (range, 0-16). The most frequent mutations were in ATRX (28%), MEN1 (28%), ASCL1 (28%), TP53 (20%), mTOR (20%), ARID1A (20%), and VHL (20%). The mutation frequencies in the MEN1 (including MEN1/PSIP1/ARID1A), mTOR (including mTOR/PIK3CA/AKT1/PTEN /TS1/TSC2/ATM), DAXX/ATRX, and angiogenesis (including VHL/ANGPT1/ANGPT2 /HIF1A) pathways were 48%, 48%, 38%, and 45%, respectively. Mutations in ATRX were associated with WHO grade I pNET (vs. grade II or III, p = 0.043), and so were those in genes involved in angiogenesis (p = 0.002). Patients with mutated MEN1 and DAXX/ATRX pathways showed a trend toward better survival, compared to patients with the wild-type genes (p = 0.08 and 0.12, respectively). Conclusion: Genetic profiles of Asian patients with pNETs were distinct from Caucasian patient profiles. Asian patients with pNETs were more frequently mutated for the mTOR and angiogenesis pathways. This could partially explain the better outcome observed for targeted therapy in Asian patients with pNETs.




Dose-dependent Effects of Strontium Ranelate on Ovariectomy Rat Bone Marrow Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells

2016

In clinic, strontium ranelate (SrR) is a useful drug to treat osteoporosis by orally taken method, but some side effect appeared in recent years. The aim of this study is to evaluate the effectiveness and safety of SrR on cells by direct application, to study the possibility of local application of this drug. Qualitative ALP staining, quantitative ALP activity assay, alizarin red staining, realtime PCR and westernblot assay were used to evaluate the osteogenesis ability of SrR under normal or osteogenic induction environment of ovariectomy bone marrow mesenchymal stem cells (OVX-BMSCs). The angiogenesis ability of SrR was studied by immunofluorescence staining of CD31 and vWF of OVX-BMSCs under angiogenesis induction environment, transwell, tubeformation and realtime PCR assay of HUVECs. Signaling pathway of PI3K/AKT/mTOR was also studied. The result demonstrated that SrR could enhance proliferation and osteogenic differentiation of OVX-BMSCs. The osteogenesis effect of SrR has been proved by the better performed of ALP activity, alizarin red staining and the remarkable up-regulation of ALP, Col-I, Runx2, OCN, BMP-2, BSP, OPG of the OVX-BMSCs, and reduction of RANKL. In addition, SrR promotes angiogenesis differentiation of both OVX-BMSCs and HUVECs. Higher intensity of immunostaining of CD31 and vWF, better result of transwell and tubeformation assay could be observed in SrR treated group, and increasing mRNA levels of VEGF and Ang-1 in the OVX-BMSCs, VEGF in HUVECs were learnt. Signaling pathway assay showed that PI3K/AKT/mTOR signaling pathway was involved in this SrR triggered angiogenesis procedure. The thrombosis marker ET-1, PAI-1 and t-PA were up-regulated, but no significant differences for low concentration (<0.5mM). The concentration between 0.25-0.5mM may be more appropriate for local application, and locally application of SrR could be considered as a promising way for bone regeneration.




The Correlation Between PARP1 and BRCA1 in AR Positive Triple-negative Breast Cancer

2016

Triple-negative breast cancer (TNBC) lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression and thus cannot benefit from conventional hormonal or anti-HER2 targeted therapies. Anti-androgen therapy has shown a certain effect on androgen receptor (AR) positive TNBC. The emerging researches have proved that poly (ADP-ribose) polymerase (PARP) inhibitor is effective in BRCA1-deficient breast cancers. We demonstrated that combination of AR antagonist (bicalutamide) and PARP inhibitor (ABT-888) could inhibit cell viability and induce cell apoptosis significantly whatever in vitro or in vivo setting in AR-positive TNBC. Previous studies have proved that both BRCA1 and PARP1 have close connections with AR in prostate cancer. We explored the correlation among AR, PARP1 and BRCA1 in TNBC for the first time. After BRCA1 overexpression, the expression of AR and PARP1 were decreased in mRNA and protein levels. Additionally, AR positively regulated PARP1 while PARP1 also up-regulated AR expression in vitro. We also confirmed BRCA1 expression was negatively correlated with AR and PARP1 in TNBC patients using a tissue microarray with TNBC patient samples. These results suggest that the combination of bicalutamide and PARP inhibitor may be a potential strategy for TNBC patients and merits further evaluation.




LIS1 Regulates Osteoclastogenesis through Modulation of M-SCF and RANKL Signaling Pathways and CDC42

2016

We have previously reported that depletion of LIS1, a key regulator of microtubules and cytoplasmic dynein motor complex, in osteoclast precursor cells by shRNAs attenuates osteoclastogenesis in vitro. However, the underlying mechanisms remain unclear. In this study, we show that conditional deletion of LIS1 in osteoclast progenitors in mice led to increased bone mass and decreased osteoclast number on trabecular bone. In vitro mechanistic studies revealed that loss of LIS1 had little effects on cell cycle progression but accelerated apoptosis of osteoclast precursor cells. Furthermore, deletion of LIS1 prevented prolonged activation of ERK by M-CSF and aberrantly enhanced prolonged JNK activation stimulated by RANKL. Finally, lack of LIS1 abrogated M-CSF and RANKL induced CDC42 activation and retroviral transduction of a constitutively active form of CDC42 partially rescued osteoclastogenesis in LIS1-deficient macrophages. Therefore, these data identify a key role of LIS1 in regulation of cell survival of osteoclast progenitors by modulating M-CSF and RANKL induced signaling pathways and CDC42 activation.




Exosomes Derived from Human Endothelial Progenitor Cells Accelerate Cutaneous Wound Healing by Promoting Angiogenesis Through Erk1/2 Signaling

2016

Chronic skin wounds represent one of the most common and disabling complications of diabetes. Endothelial progenitor cells (EPCs) are precursors of endothelial cells and can enhance diabetic wound repair by facilitating neovascularization. Recent studies indicate that the transplanted cells exert therapeutic effects primarily via a paracrine mechanism and exosomes are an important paracrine factor that can be directly used as therapeutic agents for regenerative medicine. However, application of exosomes in diabetic wound repair has been rarely reported. In this study, we demonstrated that the exosomes derived from human umbilical cord blood-derived EPCs (EPC-Exos) possessed robust pro-angiogenic and wound healing effects in streptozotocin-induced diabetic rats. By using a series of in vitro functional assays, we found that EPC-Exos could be incorporated into endothelial cells and significantly enhance endothelial cells' proliferation, migration, and angiogenic tubule formation. Moreover, microarray analyses indicated that exosomes treatment markedly altered the expression of a class of genes involved in Erk1/2 signaling pathway. It was further confirmed with functional study that this signaling process was the critical mediator during the exosomes-induced angiogenic responses of endothelial cells. Therefore, EPC-Exos are able to stimulate angiogenic activities of endothelial cells by activating Erk1/2 signaling, which finally facilitates cutaneous wound repair and regeneration.




Autophagy activation protects shock wave induced renal tubular epithelial cell apoptosis may through modulation of Akt/ GSK-3β pathway

2016

Purpose: Extracorporeal shock wave lithotripsy (ESWL) is well documented to exert destructive effect to renal cells and its mechanism is not clear. Autophagy is one of cell basic response for stressful conditions and it is important to determine cell's fate. The aim of this study is to elucidate the role of autophagy in the process of shock wave-induced renal cells injury.

Methods: NRK-52E cell, a rat renal tubular epithelial cell, was exposed to shock wave at the voltage of 14KV. GFP-LC3 puncta was used to monitor Autophagy flux in the process of shock wave injury. Autophagic relative proteins, such as light chain 3 (LC3), beclin-1 and p62, were also examined. Cell variability and apoptosis were detected when inhibition autophagy with 3-methyladenine (3MA) or stimulating its activity with rapamycin during the process of shock wave injury. The role of Akt/ GSK-3β and its connection with autophagy in the process of shock wave injury were also investigated.

Results: Shock wave was confirmed to activate autophagy in renal cells, which was manifested in LC3-II turnover, beclin-1 induction and degradation of p62. Inhibition autophagy enhanced cell damage or apoptosis, whereas its stimulating was able to exert protection from shock wave injury. Akt/ GSK-3β, a cell-survival signaling pathway, can also be activated during the process. And its activation could be suppressed by blockade autophagy.

Conclusion: Autophagy is a self-protective response for renal cells from shock wave injury. The cyto-protection of autophagy may be connected with modulation Akt/ GSK-3β pathway.




Pathological Characterization Of IFNAR(-/-) Mice Infected With Bluetongue Virus Serotype 4

2016

Bluetongue virus (BTV) replicates in lymphoid tissues where infected mononuclear leukocytes secrete proinflammatory and vasoactive mediators that can contribute to bluetongue (BT) pathogenesis. Using the well-characterized IFNAR(-/-) mice animal model, we have now studied the histopathology and dynamics of leukocyte populations in different target tissues (spleen, thymus, and lung) during BTV-4 infection by histological and immunohistochemical techniques. The spleen and thymus of BTV-4 infected mice showed severe lymphoid depletion on H&E stained sections. This finding was confirmed by IHC, showing moderate decreased immunopositivity against CD3 in the thymus, and scarce immunoreactivity against CD3 and CD79 in the rest of the white pulp in the spleen, together with an increase in MAC387 immunostaining. BTV-4 infection also induced the expression of active caspase-3 in the spleen, where apoptotic debris was observed by H&E. A dramatic increase in iNOS immunoreactivity associated to necrotic areas of the white pulp was observed, being less noticeable in the thymus and the lung. The induction of pro-inflammatory cytokines in tissues where BTV replicates was evaluated by measuring transcript levels by RT-qPCR. BTV-4 infection led to enhance transcription of IFN-γ, TNF, IL-6, IL-12-p40, and IL-1β mRNA in the thymus, spleen and lung, correlating with the level of virus replication in these tissues. Disease progression and pathogenesis in IFNAR(-/-) mice closely mimics hallmarks of bluetongue disease in ruminants. IFNAR(-/-) mice are a good choice to facilitate a faster advance in the field of orbiviruses.




MiR-99a and MiR-491 Regulate Cisplatin Resistance in Human Gastric Cancer Cells by Targeting CAPNS1

2016

Cisplatin is the first-line agent utilized for the clinical treatment of a wide variety of solid tumors including gastric cancer. However, the intrinsic or acquired cisplatin resistance is often occurred in patients with gastric cancer and resulted in failure of cisplatin therapy. In order to investigate if miRNA involves in cisplatin resistance of human gastric cancer, we first screened and compared the expression of miRNAs between cisplatin resistant gastric cancer cell lines SGC-7901/DDP and BGC-823/DDP and their sensitive parental cells by miRNAs microarray and followed by analysis of 2D-GE/MS to identify their target proteins. We found both miR-99a and miR-491 were upregulated while their target gene calpain small subunit 1 (CAPNS1) was downregulated in resistant gastric cancer cells. Dual-luciferase- reporter assays with wild-type and mutated CAPNS1 3'-UTR confirmed their specificity of targeting. Inhibition of miR-99a and miR-491, or overexpress CAPNS1 can enhance cisplatin sensitivity of the resistant cells while transfection of two miRNAs' mimics or si-CAPNS1 in the sensitive cells can induce their resistance. Moreover, our results demonstrated CAPNS1 positively regulated calpain1 and calpain2, the catalytic subunits of CAPNS1, and cleaved caspase3 which further cleaved PARP1 and directly induced apoptosis. Therefore, miR-99a and miR-491 might be work as novel molecules regulate cisplatin resistance by directly targeting CAPNS1 associated pathway in human gastric cancer cells.




CRISPR-Cas9: from Genome Editing to Cancer Research

2016

Cancer development is a multistep process triggered by innate and acquired mutations, which cause the functional abnormality and determine the initiation and progression of tumorigenesis. Gene editing is a widely used engineering tool for generating mutations that enhance tumorigenesis. The recent developed clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) system renews the genome editing approach into a more convenient and efficient way. By rapidly introducing genetic modifications in cell lines, organs and animals, CRISPR-Cas9 system extends the gene editing into whole genome screening, both in loss-of-function and gain-of-function manners. Meanwhile, the system accelerates the establishment of animal cancer models, promoting in vivo studies for cancer research. Furthermore, CRISPR-Cas9 system is modified into diverse innovative tools for observing the dynamic bioprocesses in cancer studies, such as image tracing for targeted DNA, regulation of transcription activation or repression. Here, we view recent technical advances in the application of CRISPR-Cas9 system in cancer genetics, large-scale cancer driver gene hunting, animal cancer modeling and functional studies.




Catalpol Protects Pre-Myelinating Oligodendrocytes against Ischemia-induced Oxidative Injury through ERK1/2 Signaling Pathway

2016

The vulnerability of pre-myelinating oligodendrocytes (PreOLs) to ischemic injury plays an important role in the pathogenesis and progression of perinatal white matter injury. Although oxidative stress is thought to be a major pathogenic mechanism predisposing the PreOLs to injury, no effective therapies have been identified to date. The present study aimed to investigate the direct protective effects of catalpol, a potent antioxidant and free radical scavenger, on ischemia-induced oxidative damage in PreOLs and to explore whether the ERK1/2 signaling pathway contributed to the protection provided by catalpol. Primary cultures of PreOLs exposed to oxygen-glucose deprivation (OGD) followed by reperfusion were used as an in vitro model of ischemia. Pretreatment with 0.5 mM catalpol for 1 h prior to OGD treatment significantly reversed ischemia-induced apoptosis in PreOLs and myelination deficits by inhibiting intracellular Ca2+ increase, reducing mitochondrial damage, and ameliorating overproduction of reactive oxygen species (ROS). The expression levels of phosphorylated ERK1/2 (p-ERK1/2) and activated poly-ADP-ribose polymerase-1 (PARP-1) were also markedly decreased by catalpol treatment. Blocking the ERK1/2 signaling pathway with the MEK inhibitor U0126 and catalpol significantly protected PreOLs from ROS-mediated apoptosis under OGD. Taken together, these results suggest that catalpol protects PreOLs against ischemia-induced oxidative injury through ERK1/2 signaling pathway. Catalpol may be a candidate for treating ischemic white matter damage.




Distributed Features of Vimentin-Containing Neural Precursor Cells in Olfactory Bulb of SOD1G93A Transgenic Mice: a Study about Resource of Endogenous Neural Stem Cells

2016

No any effective treatments can prevent from the motor neuron degeneration in amyotrophic lateral sclerosis (ALS) at present. In order to modulating the endogenous neural precursor cells (NPCs) to repairing the degenerative motor neurons in ALS, we studied the alteration of endogenous vimentin-containing NPCs in olfactory bulb (OB) at the different stages of SOD1 wlid-type and G93A transgenic mice. The results showed that the vimentin-containing cells (VCCs) were mainly distributed in the glomerular layer (Gl), the accessory OB (AOB), the OB core, the granular cell layer (GRO) and the mitral cell layer (MI)+the internal plexiform layer (IPL) of the OB of adult mice. Almost all VCCs in Gl, OB core and GRO were the GFAP positive cells. Almost all VCCs in AOB were the Oligo-2 positive cells. Fewer VCCs in MI+IPL were the NeuN positive cells. VCCs significantly increased in the OB core and Gl of adult OB at the pre-onset, onset and progression stages of ALS-like G93A transgenic disease, particularly in OB core. All increased VCCs were the GFAP positive cells. Our data suggested that there extensively existed the endogenous vimentin-containing NPCs in the OB of adult mice, which was a potential resource of neural stem cells, they could differentiate into astrocyte, oligodendrocyte and neuron cells, were a potential astrocyte neuroregenerative response in adult OB in the ALS-like disease, were a potential pathway to repair the degenerated motor neurons.




Conserved chemosensory proteins in the proboscis and eyes of Lepidoptera

2016

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are endowed with several different functions besides being carriers for pheromones and odorants. Based on a previous report of a CSP acting as surfactant in the proboscis of the moth Helicoverpa armigera, we revealed the presence of orthologue proteins in two other moths Plutella xylostella and Chilo suppressalis, as well as two butterflies Papilio machaon and Pieris rapae, using immunodetection and proteomic analysis. The unusual conservation of these proteins across large phylogenetic distances indicated a common specific function for these CSPs. This fact prompted us to search for other functions of these proteins and discovered that CSPs are abundantly expressed in the eyes of H. armigera and possibly involved as carriers for carotenoids and visual pigments. This hypothesis is supported by ligand-binding experiments and docking simulations with retinol and β-carotene. This last orange pigment, occurring in many fruits and vegetables, is an antioxidant and the precursor of visual pigments. We propose that structurally related CSPs solubilise nutritionally important carotenoids in the proboscis, while they act as carriers of both β-carotene and its derived products 3-hydroxyretinol and 3-hydroxyretinal in the eye. The use of soluble olfactory proteins, such as CSPs, as carriers for visual pigments in insects, here reported for the first time, parallels the function of retinol-binding protein in vertebrates, a lipocalin structurally related to vertebrate odorant-binding proteins.




Genetic Variant in Flavin-Containing Monooxygenase 3 Alters Lipid Metabolism in Laying Hens in a Diet-Specific Manner

2016

Genetic variant T329S in flavin-containing monooxygenase 3 (FMO3) impairs trimethylamine (TMA) metabolism in birds. The TMA metabolism that under complex genetic and dietary regulation, closely linked to cardiovascular disease risk. We determined whether the genetic defects in TMA metabolism may change other metabolic traits in birds, determined whether the genetic effects depend on diets, and to identify genes or gene pathways that underlie the metabolic alteration induced by genetic and diet factors. We used hens genotyped as FMO3 c.984 A>T as well as those with the homozygous normal genotype. For each genotype, hens were provided with either a corn-soybean meal basal diets (SM), which contains lower levels of TMA precursor, or the basal diets supplemented with 21% of rapeseed meal (RM), which contains higher levels of TMA precursor. An integrative analysis of metabolomic and transcriptomic was used to explore the metabolic patterns of FMO3 genetic variant in hens that were fed the two defined diets. In birds that consumed SM diets, the T329S mutation increased levels of plasma TMA and lipids, FMO3 mRNA levels, and the expression of genes involved in long chain polyunsaturated fatty acid biosynthesis. In birds that consumed RM diets, the T329S mutation induced fishy odor syndrome, a repression in LXR pathway and a reciprocal change in lipid metabolism. Variations in TMA and lipid metabolism were linked to the genetic variant in FMO3 in a diet-specific manner, which suggest FMO3 functions in TMA metabolism and lipid homeostasis. LXR pathway and polyunsaturated fatty acid metabolism are two possible mechanisms of FMO3 action in response to dietary TMA precursor.




Insights into the Nanog gene: A propeller for stemness in primitive stem cells

2016

Self-renewal and pluripotency are two major characteristics of embryonic stem cells (ESCs) that allow ESCs to maintain stem cell population, and differentiate into multiple types of adult tissues. Nanog is the key transcription factor that controls both self-renewal and pluripotency of ESCs. Similarly, cancer stem cells (CSCs) are capable of preserving population and initiating new tumor development by self-renewal. Expression of Nanog family proteins can be increased in many types of cancer which is correlated with tumor outcomes. In this review we summarized the recent understanding of the roles and mechanisms of Nanog in ESC regulation under physiological conditions. In addition, we describe the function of Nanog family proteins in different types of cancer, and the association of Nanog with clinical outcomes. Taken together, Nanog proteins are central regulators controlling both ESCs and CSCs, and are considered as a prognostic marker in many types of cancer. These findings supported the possibility of novel therapeutic potentials of Nanog against cancers.




Aloe-emodin suppresses hypoxia-induced retinal angiogenesis via inhibition of HIF-1α/VEGF pathway

2016

Background: Aloe-emodin (AE) has been reported to possess the antiangiogenic effect on laser induced choroidal neovascularization. AE inhibits the vessel formation in the zebrafish embryos. However, it is still unclear whether AE can alleviate neovascularization. Here, we investigated the inhibitory effect of AE on the hypoxia-induced retinal neovascularization and the possible mechanisms.

Methods: We established a vascular endothelial growth factor (VEGF) secretion model under chemical induced hypoxia by exposure of 150 µM CoCl2 to the ARPE-19 cells, then treated the cells with different concentrations of AE (0.2, 1.0 and 5.0 µg/mL) or a special hypoxia-inducible factor 1α (HIF-1α) inhibitor [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, YC-1, 1.0 µg/mL]. The cellular supernatants were collected 48 h later to measure the VEGFA concentrations by human VEGFA enzyme-linked immunosorbent assay (ELISA) kits, the mRNA expressions of VEGFA, HIF-1α and prolyl hydroxylase-2 (PHD-2) by quantitative reverse transcription-PCR (qRT-PCR) and the protein expressions of HIF-1α and PHD-2 by Western blots. For in vivo study, the rat pups with oxygen-induced retinopathy were treated with Conbercept ophthalmic injection (1.0 mg/kg) or AE (5.0 and 10.0 mg/kg) for five days, then the retinal avascular areas were assessed via visualization of the retinal vasculature with ADPase and hematoxylin & eosin (H&E) stains.

Results: AE inhibits the VEGFA secretion of ARPE-19 cells under hypoxia condition, decreases the mRNA expressions of VEGFA and PHD-2 and the protein expressions of VEGFA, HIF-1α and PHD-2 in vitro and prevents hypoxia-induced retinal neovascularization in vivo.

Conclusions: AE ameliorates retinal neovascularization throuth inhibition of the HIF-1α/VEGF signaling pathway. AE may be developed as a potential drug for the prevention and treatment of diabetic retinopathy.




IL28B SNP rs12979860 is the Critical Predictor for Sustained Viral Response in Chinese Children Aged 1 to 6 Years with Chronic Hepatitis C

2016

Clinical data on children with chronic hepatitis C (CHC) remain extremely limited. This study investigated sustained virologic response (SVR) to alfa-interferon 2b plus RBV treatment in children aged 1-6 years with unsafe injection-acquired CHC. 154 children with CHC aged 1 to 6 years were enrolled, 101 of them were male (65.6%) and 53 were female (34.4%), and they were treated with alfa-interferon at a dose of 1-5 MIU/m2 3 times weekly plus oral RBV (15 mg/kg/day) for 48 weeks. 57(39.3 %) of them were genotype 1b, 73(50.3%) were genotypes 2a, 15(10.3%) were undecided type. SVR was achieved in 53 of 57(93.0%) patients with genotype 1b, in 72 (98.6%) of 73 with genotype 2a, 15(100.0%) of 15 with undecided type. There was no significant statistical difference in SVR between male and female (98.0% vs 94.3%, p=0.340), genotype 2a and those with genotype 1b(98.6% vs 93.0%, p=0.160), ALT>40U/L group and ALT≤40U/L group(96.7% vs 96.8%, p=1.000), AST>40U/L group and AST≤40U/L group(95.9% vs 98.2%, p=0.654) as well as lower baseline viral load group (<6×105 IU/ml) and higher baseline viral load group(≥6×105 IU/ml)(97.3% vs 95.3%, p=0.916). Leucopenia, neutropenia, hemoglobin concentration decrease, fever, platelet count decrease and rash were 8.4%, 69.5%, 24.0%, 50.6%, 1.9% and 4.5%, respectively. And only 12(7.8%) individuals developed thyroid autoantibodies. The SVR was higher in patients with IL-28B genotype C/C than C/T (99.0% vs 80%, p=0.002). Compared with HCV viral genotype, ALT level and baseline viral load, IL-28B rs12979860 is more suitable for predicting antiviral efficacy in children with CHC. It is inappropriate to take the increase of ALT level as an essential indicator for antiviral treatment in children aged 1-6 years.




Ginsenoside Rg1 Protects against Oxidative Stress-induced Neuronal Apoptosis through Myosin IIA-actin Related Cytoskeletal Reorganization

2016

Oxidative stress-induced cytoskeletal dysfunction of neurons has been implicated as a crucial cause of cell apoptosis or death in the central nervous system (CNS) diseases, such as neurodegenerative and psychiatric diseases. The application of neuroprotectants rescuing the neurons from cytoskeletal damage and apoptosis can be a potential treatment for these CNS diseases. Ginsenoside Rg1 (Rg1), one of the major active components of ginseng, has been reported possessing notable neuroprotective activities. However, there is rare report about its effect on cytoskeleton and its undergoing mechanism. The current study is to reveal the regulatory effects of Rg1 on cytoskeletal and morphological lesion in oxidative stress-induced neuronal apoptosis. The results demonstrated that pre-treatment with Rg1 (0.1-10 μM) attenuated hydrogen peroxide (H2O2)-induced neuronal apoptosis and oxidative stress through reducing the intracellular reactive oxygen species (ROS) production and methane dicarboxylic aldehyde (MDA) level. The Rg1 treatment also abolished H2O2-induced morphological changes, including cell rounding, membrane blebbing, neurite retraction and nuclei condensation, which were generated by myosin IIA-actin interaction. These effects were mediated via the down-regulation of caspase-3, ROCK1 (Rho-associated kinase1) activation and myosin light chain (MLC, Ser-19) phosphorylation. Furthermore, inhibiting myosin II activity with blebbistatin partly blocked the neuroprotective effects of Rg1. The computer-aided homology modelling revealed that Rg1 preferentially positioned in the actin binding cleft of myosin IIA and might block the binding of myosin IIA to actin filaments. Accordingly, the neuroprotective mechanism of Rg1 is related to the activity that inhibits myosin IIA-actin interaction and the caspase-3/ROCK1/MLC signaling pathway. These findings put some insights into the unique neuroprotective properties of Rg1 associated with the regulation of myosin IIA-actin cytoskeletal structure under oxidative stress and provide experimental evidence for Rg1 in CNS diseases.




The Evolving Functions of Autophagy in Ocular Health: A Double-edged Sword

2016

Autophagy plays an adaptive role in cell survival, development, differentiation and intracellular homeostasis. Autophagy is recognized as a 'self-cannibalizing' process that is active during stresses such as starvation, chemotherapy, infection, ageing, and oxygen shortage to protect organisms from various irritants and to regenerate materials and energy. However, autophagy can also lead to a form of programmed cell death distinct from apoptosis.

Components of the autophagic pathway are constitutively expressed at a high level in the eye, including in the cornea, lens, retina, and orbit. In addition, the activation of autophagy is directly linked to the development of eye diseases such as age-related macular degeneration (ARMD), cataracts, diabetic retinopathy (DR), glaucoma, photoreceptor degeneration, ocular tumours, ocular infections and thyroid-associated ophthalmopathy (TAO). A high level of autophagy defends against external stress; however, excessive autophagy can result in deterioration, as observed in ocular diseases such as ARMD and DR.

This review summarizes recent developments elucidating the relationship between autophagy and ocular diseases and the potential roles of autophagy in the pathogenesis and treatment of these diseases.




RNA interference of chitin synthase genes inhibits chitin biosynthesis and affects larval performance in Leptinotarsa decemlineata (Say)

2016

Dietary introduction of bacterially expressed double-stranded RNA (dsRNA) has great potential for management of Leptinotarsa decemlineata. Identification of the most attractive candidate genes for RNA interference (RNAi) is the first step. In the present paper, three complete chitin synthase cDNA sequences (LdChSAa, LdChSAb and LdChSB) were cloned. LdChSAa and LdChSAb, two splicing variants of LdChSA gene, were highly expressed in ectodermally-derived epidermal cells forming epidermis, trachea, foregut and hindgut, whereas LdChSB was mainly transcribed in midgut cells. Feeding bacterially expressed dsChSA (derived from a common fragment of LdChSAa and LdChSAb), dsChSAa, dsChSAb and dsChSB in the second- and fourth-instar larvae specifically knocked down their target mRNAs. RNAi of LdChSAa+LdChSAb and LdChSAa lowered chitin contents in whole body and integument samples, and thinned tracheal taenidia. The resulting larvae failed to ecdyse, pupate, or emerge as adults. Comparably, knockdown of LdChSAb mainly affected pupal-adult molting. The LdChSAb RNAi pupae did not completely shed the old larval exuviae, which caused failure of adult emergence. In contrast, silencing of LdChSB significantly reduced foliage consumption, decreased chitin content in midgut sample, damaged midgut peritrophic matrix, and retarded larval growth. As a result, the development of the LdChSB RNAi hypomorphs was arrested. Our data reveal that these LdChSs are among the effective candidate genes for an RNAi-based control strategy against L. decemlineata.




Epstein-Barr virus lytic reactivation regulation and its pathogenic role in carcinogenesis

2016

Epstein-Barr virus (EBV) has been associated with several types of human cancers. In the host, EBV can establish two alternative modes of life cycle, known as latent or lytic and the switch from latency to the lytic cycle is known as EBV reactivation. Although EBV in cancer cells is found mostly in latency, a small number of lytically-infected cells promote carcinogenesis through the release of growth factors and oncogenic cytokines. This review focuses on the mechanisms by which EBV reactivation is controlled by cellular and viral factors, and discusses how EBV lytic infection contributes to human malignancies.




Histone Deacetylase Inhibitor Trichostatin a Promotes the Apoptosis of Osteosarcoma Cells through p53 Signaling Pathway Activation

2016

Purpose: The purpose of this study was to investigate the profile of histone deacetylase (HDAC) activity and expression in osteosarcoma cells and tissues from osteosarcoma patients and to examine the mechanism by which a histone deacetylase (HDAC) inhibitor, Trichostatin A (TSA), promotes the apoptosis of osteosarcoma cells.

Methods: HDAC activity and histone acetyltransferase (HAT) activity were determined in nuclear extracts of MG63 cells, hFOB 1.19 cells and tissues from 6 patients with primary osteosarcoma. The protein expression of Class I HDACs (1, 2, 3 and 8) and the activation of the p53 signaling pathway were examined by Western blot. Cell growth and apoptosis were determined by 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) assay and flow cytometry, respectively.

Results: Nuclear HDAC activity and class I HDAC expression were significantly higher in MG63 cells than in hFOB 1.19 cells, and a similar trend was observed in the human osteosarcoma tissues compared with the paired adjacent non-cancerous tissues. TSA significantly inhibited the growth of MG63 cells and promoted apoptosis in a dose-dependent manner through p53 signaling pathway activation.

Conclusion: Class I HDACs play a central role in the pathogenesis of osteosarcoma, and HDAC inhibitors may thus have promise as new therapeutic agents against osteosarcoma.




P-selectin-mediated LOX expression promotes insulinoma growth in Rip1-Tag2 mice by increasing tissue stiffness

2016

P-selectin, a cell adhesion molecule, is an important member of the selectin family. Recent studies have shown that P-selectin deletion inhibits tumor growth in Rip1-Tag2 mice by suppressing platelet accumulation in tumor tissues. This study aimed to evaluate whether and how P-selectin affects tumor stiffness in Rip1-Tag2 mice. To explore the role of P-selectin in tissue stiffness, we demonstrated that tumor progression in Rip1-Tag2 mice was correlated with tissue stiffness using immunofluorescence and histological staining. Furthermore, we showed that P-selectin deficiency significantly decreased tissue stiffness by inhibiting lysyl oxidase (LOX) expression. Our experiments involving Rip1-Tag2 mice treated with the LOX inhibitor BAPN showed that BAPN significantly abolished collagen deposition to decrease tumor stiffness and thus inhibit tumor growth. These results indicate that P-selectin deletion significantly decreases tumor stiffness in Rip1-Tag2 mice by inhibiting LOX expression. Further study demonstrated that P-selectin-mediated platelet accumulation increases tissue stiffness mainly by increasing LOX expression and thus promotes tumor growth. Therefore, P-selectin may be an effective therapeutic targeting for treating human insulinomas.




Blockage of epithelial to mesenchymal transition and upregulation of let 7b are critically involved in ursolic acid induced apoptosis in malignant mesothelioma cell

2016

Malignant pleural mesothelioma (MPN), which is caused by asbestos exposure, is one of aggressive lung tumors. In the present study, we elucidated the anti-tumor mechanism of ursolic acid in malignant mesotheliomas. Ursolic acid significantly exerted cytotoxicity in a time and dose dependent manner in H28, H2452 and MSTO-211H mesothelioma cells and inhibited cell proliferation by colony formation assay in a dose-dependent fashion. Also, ursolic acid treatment accumulated the sub-G1 population, attenuated the expression of procapase 9, cyclin D1, pAKT, p-glycogen synthase kinase 3-alpha/beta (pGSK3α/β), β-catenin and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and also cleaved caspase 3 and poly (ADP-ribose) polymerase (PARP) in mesothelioma cells. Furthermore, ursolic acid treatment blocked epithelial and mesenchymal transition (EMT) molecules by activating E-cadherin as an epithelial marker and attenuating Vimentin, and Twist as mesenchymal molecules. Interestingly, miRNA array revealed that 23 miRNAs (>2 folds) including let-7b and miRNA3613-5p, miRNA134 and miRNA196b were significantly upregulated while 33 miRNAs were downregulated in ursolic acid treated H2452 cells. Furthermore, overexpression of let 7b using let-7b mimics enhanced the antitumor effect of ursolic acid to attenuate the expression of procaspases 3, pro-PARP, pAKT, β-catenin and Twist and increase sub-G1 accumulation in H2452 mesothelioma cells. Overall, our findings suggest that ursolic acid induces apoptosis via inhibition of EMT and activation of let7b in mesothelioma cells as a potent chemotherapeutic agent for treatment of malignant mesotheliomas.




An Efficient Light-Inducible P53 Expression System for Inhibiting Proliferation of Bladder Cancer Cell

2016

Optogenetic gene expression systems enable spatial-temporal modulation of gene transcription and cell behavior. Although applications in biomedicine are emerging, the utility of optogenetic gene switches remains elusive in cancer research due to the relative low gene activation efficiency. Here, we present an optimized CRISPR-Cas9-based light-inducible gene expression device that controls gene transcription in a dose-dependent manner. To prove the potential utility of this device, P53 was tested as a functional target in the bladder cancer cell models. It was illustrated that the light-induced P53 inhibited proliferation of 5637 and UMUC-3 cell effectively. The “light-on” gene expression system may demonstrate a novel therapeutic strategy for bladder cancer intervention.




Exosomes from Human Synovial-Derived Mesenchymal Stem Cells Prevent Glucocorticoid-Induced Osteonecrosis of the Femoral Head in the Rat

2016

Osteonecrosis of the femoral head (ONFH) represents a debilitating complication following glucocorticoid (GC)-based therapy. Synovial-derived mesenchymal stem cells (SMSCs) can exert protective effect in the animal model of GC-induced ONFH by inducing cell proliferation and preventing cell apoptosis. Recent studies indicate the transplanted cells exert therapeutic effects primarily via a paracrine mechanism and exosomes are an important paracrine factor that can be directly used as therapeutic agents for tissue engineering. Herein, we provided the first demonstration that the early treatment of exosomes secreted by human synovial-derived mesenchymal stem cells (SMSC-Exos) could prevent GC-induced ONFH in the rat model. Using a series of in vitro functional assays, we found that SMSC-Exos could be internalized into bone marrow derived stromal cells (BMSCs) and enhance their proliferation and have anti-apoptotic abilities. Finally, SMSC-Exos may be promising for preventing GC-induced ONFH.




From Nutrient to MicroRNA: a Novel Insight into Cell Signaling Involved in Skeletal Muscle Development and Disease

2016

Skeletal muscle is a remarkably complicated organ comprising many different cell types, and it plays an important role in lifelong metabolic health. Nutrients, as an external regulator, potently regulate skeletal muscle development through various internal regulatory factors, such as mammalian target of rapamycin (mTOR) and microRNAs (miRNAs). As a nutrient sensor, mTOR, integrates nutrient availability to regulate myogenesis and directly or indirectly influences microRNA expression. MiRNAs, a class of small non-coding RNAs mediating gene silencing, are implicated in myogenesis and muscle-related diseases. Meanwhile, growing evidence has emerged supporting the notion that the expression of myogenic miRNAs could be regulated by nutrients in an epigenetic mechanism. Therefore, this review presents a novel insight into the cell signaling network underlying nutrient-mTOR-miRNA pathway regulation of skeletal myogenesis and summarizes the epigenetic modifications in myogenic differentiation, which will provide valuable information for potential therapeutic intervention.




Indoxyl Sulfate Enhance the Hypermethylation of Klotho and Promote the Process of Vascular Calcification in Chronic Kidney Disease

2016

Chronic kidney disease (CKD) is a state of Klotho deficiency. The Klotho expression may be suppressed due to DNA hypermethylation in cancer cells so we have investigated the effects and possible mechanisms by which Klotho expression is regulated in human aortic smooth muscle cells (HASMCs). The vascular Klotho hypermethylation in radial arteries of patients with end-stage renal disease was described. Cultured HASMCs and 5/6-nephrectomized Sprague Dawley (SD) rats treated with indoxyl sulfate (IS) were used as in vitro and in vivo models, respectively. IS increased CpG hypermethylation of the Klotho gene and decreased Klotho expression in HASMCs, and potentiated HASMCs calcification. The expression of DNA methyltransferase (DNMT) 1 and 3a in HASMCs treated with IS was significantly increased and specific inhibition of DNA methyltransferase 1 by 5-aza-2'-deoxycytidine(5Aza-2dc) caused demethylation of the Klotho gene and increased Klotho expression. In rats, injection of IS potentiated vascular calcification, increased CpG hypermethylation of the Klotho gene and decreased Klotho expression in the aortic medial layer and all of these changes could be reverted by 5Aza-2dc treatment. Transcriptional suppression of vascular Klotho gene expression by IS and epigenetic modification of Klotho by IS may be an important pathological mechanism of vascular calcification in CKD.




Deficiency of DJ-1 Ameliorates Liver Fibrosis through Inhibition of Hepatic ROS Production and Inflammation

2016

Liver fibrosis is a global health problem and previous studies have demonstrated that reactive oxygen species (ROS) play important roles in fibrogenesis. Parkinson disease (autosomal recessive, early onset) 7 (Park7) also called DJ-1 has an essential role in modulating cellular ROS levels. DJ-1 therefore may play functions in liver fibrogenesis and modulation of DJ-1 may be a promising therapeutic approach. Here, wild-type (WT) and DJ-1 knockout (DJ-1 KO) mice were administrated with carbon tetrachloride (CCl4) to induce liver fibrosis or acute liver injury. Results showed that DJ-1 depletion significantly blunted liver fibrosis, accompanied by marked reductions in liver injury and ROS production. In the acute CCl4 model, deficiency of DJ-1 showed hepatic protective functions as evidenced by decreased hepatic damage, reduced ROS levels, diminished hepatic inflammation and hepatocyte proliferation compared to WT mice. In vitro hepatic stellate cells (HSCs) activation assays indicated that DJ-1 has no direct effect on the activation of HSCs in the context of with or without TGFβ treatment. Thus our present study demonstrates that in CCl4-induced liver fibrosis, DJ-1 deficiency attenuates mice fibrosis by inhibiting ROS production and liver injury, and further indirectly affecting the activation of HSCs. These results are in line with previous studies that ROS promote HSC activation and fibrosis development, and suggest the therapeutic value of DJ-1 in treatment of liver fibrosis.




miR-628 Promotes Burn-Induced Skeletal Muscle Atrophy via Targeting IRS1

2016

Skeletal muscle atrophy is a common clinical feature among patients with severe burns. Previous studies have shown that miRNAs play critical roles in the regulation of stress-induced skeletal muscle atrophy. Our previous study showed that burn-induced skeletal muscle atrophy is mediated by miR-628. In this study, compared with sham rats, rats subjected to burn injury exhibited skeletal muscle atrophy, as well as significantly decreased insulin receptor substrate 1 (IRS1) protein expression and significantly increased skeletal muscle cell apoptosis. An miRNA array showed that the levels of miR-628, a potential regulator of IRS1 protein translation, were also clearly elevated. Second, L6 myocyte cell apoptosis increased after induction of miR-628 expression, and IRS1 and p-Akt protein expression decreased significantly. Expression of the cell apoptosis-related proteins FoxO3a and cleaved caspase 3 also increased after induction of miR-628 expression. Finally, forced miR-628 expression in normal rats resulted in increased cell apoptosis and skeletal muscle atrophy, as well as changes in IRS1/Akt/FoxO3a signaling pathway activity consistent with the changes in protein expression described above. Inhibiting cell apoptosis with Z-VAD-FMK resulted in alleviation of burn-induced skeletal muscle atrophy. In general, our results indicate that miR-628 mediates burn-induced skeletal muscle atrophy by regulating the IRS1/Akt/FoxO3a signaling pathway.




Dmp1 Null Mice Develop a Unique Osteoarthritis-like Phenotype

2016

Patients with hypophosphatemia rickets (including DMP1 mutations) develop severe osteoarthritis (OA), although the mechanism is largely unknown. In this study, we first identified the expression of DMP1 in hypertrophic chondrocytes using immunohistochemistry (IHC) and X-gal analysis of Dmp1-knockout-lacZ-knockin heterozygous mice. Next, we characterized the OA-like phenotype in Dmp1 null mice from 7-week-old to one-year-old using multiple techniques, including X-ray, micro-CT, H&E staining, Goldner staining, scanning electronic microscopy, IHC assays, etc. We found a classical OA-like phenotype in Dmp1 null mice such as articular cartilage degradation, osteophyte formation, and subchondral osteosclerosis. These Dmp1 null mice also developed unique pathological changes, including a biphasic change in their articular cartilage from the initial expansion of hypertrophic chondrocytes at the age of 1-month to a quick diminished articular cartilage layer at the age of 3-months. Further, these null mice displayed severe enlarged knees and poorly formed bone with an expanded osteoid area. To address whether DMP1 plays a direct role in the articular cartilage, we deleted Dmp1 specifically in hypertrophic chondrocytes by crossing the Dmp1-loxP mice with Col X Cre mice. Interestingly, these conditional knockout mice didn't display notable defects in either the articular cartilage or the growth plate. Because of the hypophosphatemia remained in the entire life span of the Dmp1 null mice, we also investigated whether a high phosphate diet would improve the OA-like phenotype. A 8-week treatment of a high phosphate diet significantly rescued the OA-like defect in Dmp1 null mice, supporting the critical role of phosphate homeostasis in maintaining the healthy joint morphology and function. Taken together, this study demonstrates a unique OA-like phenotype in Dmp1 null mice, but a lack of the direct impact of DMP1 on chondrogenesis. Instead, the regulation of phosphate homeostasis by DMP1 via the axis of “FGF23-renal phosphorus reabsorption” is vital for maintaining a healthy joint.




Expression of Iron Regulatory Protein 1 Is Regulated not only by HIF-1 but also pCREB under Hypoxia

2016

The inconsistent of responses of IRP1 and HIF-1 alpha to hypoxia and the similar tendencies in the changes of IRP1 and pCREB contents led us to hypothesize that pCREB might be involved in the regulation of IRP1 under hypoxia. Here, we investigated the role of pCREB in IRP1 expression in HepG2 cells under hypoxia using quantitative PCR, western blot, immunofluorescence, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). We demonstrated that 1) Hypoxia increased pCREB levels inside of the nucleus; 2) Putative CREs were found in the IRP1 gene; 3) Nuclear extracts of HepG2 cells treated with hypoxia could bind to CRE1 and CRE3, and 100-fold competitor of putative CREs could abolish the binding activity to varying degrees; 4) pCREB was found in the CRE1 and CRE3 DNA-protein complexes of EMSA; 5) CRE1 and CRE3 binding activity of IRP1 depended on CREB activation but not on HIF-1; 6) Increased IRP1 expression under hypoxia could be prevented by LY294002; 7) ChIP assays demonstrated that pCREB binds to IRP1 promoter; and 8) HIF-1 and/or HIF-2 siRNA had no effect on the expression of pCREB and IRP1 proteins in cells treated with hypoxia for 8 hours. Our findings evidenced for the involvement of pCREB in IRP1 expression and revealed a dominant role of PI3K/Akt pathway in CREB activation under hypoxia and also suggested that dual-regulation of IRP1 expression by HIF-1 and pCERB or other transcription factor(s) under hypoxia might be a common mechanism in most if not all of hypoxia-inducible genes.




Changes in the Expression of FUS/TLS in Spinal Cords of SOD1 G93A Transgenic Mice and Correlation with Motor-Neuron Degeneration

2016

In order to searching the possible pathogenesis of amyotrophic lateral sclerosis (ALS), we examined the expression and distribution of FUS/TLS protein in the different anatomic regions, segments and neural cells of adult spinal cord at the different stages of the SOD1 wild-type and G93A transgenic mice using the fluorescent immunohistochemistry. Result revealed that, in the SOD1 wild-type mice, the FUS/TLS expression almost wasn't detected. However, in the SOD1 G93A mice, the FUS/TLS expression in the white matter was significantly more than that in the gray matter. In the white matter, the FUS/TLS expression in the anterior funiculus was more than that in the lateral funiculus more than that in the posterior funiculus. In the gray matter, the FUS/TLS expression in the ventral horn was more than that surrounding the central canal more than that in the dorsal horn. The FUS/TLS expression in the thoracic segment was more than that in the cervical segment more than that in the lumbar segment. Almost all FUS/TLS expressed in the nuclear of the GFAP positive cell at the onset stage, but it expressed in both the nuclear and the cytoplasm of the GFAP positive cell at the progression stage, almost didn't detected FUS/TLS expression in the NeuN and Oligo positive cells. The FUS/TLS expression was positively correlated with the neuron death. Our data suggested that the expressive increase and mislocalization of FUS/TLS in the astrocyte cell might cause the motor neuron degenerative death in the SOD1 G93A transgenic mice.




A Metabolomic Approach (1H HRMAS NMR Spectroscopy) Supported by Histology to Study Early Post-transplantation Responses in Islet-transplanted Livers

2016

Intrahepatic transplantation of islets requires a lot of islets because more than 50% of the graft is lost during the 24 hours following transplantation. We analyzed, in a rat model, early post-transplantation inflammation using systemic inflammatory markers, or directly in islet-transplanted livers by immunohistochemistry. 1H HRMAS NMR was employed to investigate metabolic responses associated with the transplantation. Inflammatory markers (Interleukin-6, α2-macroglobulin) are not suitable to follow islet reactions as they are not islet specific. To study islet specific inflammatory events, immunohistochemistry was performed on sections of islet transplanted livers for thrombin (indicator of the instant blood-mediated inflammatory reaction (IBMIR)) and granulocytes and macrophages. We observed a specific correlation between IBMIR and granulocyte and macrophage infiltration after 12 h. In parallel, we identified a metabolic response associated with transplantation: after 12 h, glucose, alanine, aspartate, glutamate and glutathione were significantly increased. An increase of glucose is a marker of tissue degradation, and could be explained by immune cell infiltration. Alanine, aspartate and glutamate are inter-connected in a common metabolic pathway known to be activated during hypoxia. An increase of glutathione revealed the presence of antioxidant protection. In this study, IBMIR visualization combined with 1H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation.




Class A Scavenger Receptor Exacerbates Osteoclastogenesis by an Interleukin-6-Mediated Mechanism through ERK and JNK Signaling Pathways

2016

Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, which are important for bone health. Class A scavenger receptor (SR-A) is a multifunctional molecule that functions during differentiation of monocyte into macrophages and osteoclasts. To further characterize the role of SR-A in osteoclasts, we used the murine tooth movement model (TM) and the murine anterior cruciate ligament transection model of osteoarthritis (ACLT OA). In these two models the bones involved are of different origin and have different properties. Bone resorption was decreased in SR-A-/- mice compared to SR-A+/+ mice. Further evaluation showed that the number of multinucleated osteoclasts in SR-A-/- mice, compared to SR-A+/+ mice, was significantly decreased both in vivo and in vitro. The levels of interleukin-6 (IL-6) produced by osteoclasts were reduced in SR-A-/- mice compared to SR-A+/+ mice. In the in vitro marrow-derived osteoclast formation assay and in both mouse models, osteoclastogenesis was restored to normal in SR-A-/- mice by administration of recombinant murine IL-6. Moreover, neutralization of IL-6 reduced the number of osteoclasts formed in SR-A+/+ mice of TM model. Both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38, signaling pathways were downregulated in receptor activator of nuclear factor-κB ligand (RANKL)-stimulated SR-A-/- osteoclasts. Importantly, when treated with either ERK or JNK inhibitor, the numbers of osteoclasts generated from RANKL-induced bone marrow derived-macrophages of SR-A+/+ mice, and their IL-6 production, were significantly decreased. This suggests that SR-A activates the ERK and JNK signaling pathways, and promotes production of IL-6 by osteoclasts to further stimulate osteoclast formation.




Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells

2016

Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na+ channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium.




The Overexpression of TDP-43 Protein in the Neuron and Oligodendrocyte Cells Causes the Progressive Motor Neuron Degeneration in the SOD1 G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

2016

The recent investigation suggested that the TDP-43 protein was closely related to the motor neuron degeneration in amyotrophic lateral sclerosis (ALS), but the pathogenesis contributed to motor neuron degeneration largely remained unknown. Therefore, we detected the alteration of TDP-43 expression and distribution in the adult spinal cord of the SOD1 G93A transgenic mouse model for searching the possible pathogenesis of ALS. We examined the TDP-43 expression and distribution in the different anatomic regions, segments and neural cells in the adult spinal cord at the different stages of the SOD1 wild-type and G93A transgenic model by the fluorescent immunohistochemical technology. We revealed that the amount of TDP-43 positive cell was cervical>lumbar>thoracic segment, that in the ventral horn was more than that in the dorsal horn, a few of TDP-43 protein sparsely expressed and distributed in the other regions, the TDP-43 protein weren't detected in the white matter and the central canal. The TDP-43 protein was mostly expressed and distributed in the nuclear of neuron cells and the cytoplasm of oligodendrocyte cells of the gray matter surrounding the central canal of spinal cord by the granular shape in the SOD1 wild-type and G93A transgenic mice. The amount of TDP-43 positive cell significantly increased at the onset and progression stages of ALS following with the increase of neuron death in spinal cord, particularly in the ventral horn of cervical segment at the progression stage. Our results suggested that the overexpression of TDP-43 protein in the neuron and oligodendrocyte cell causes the progressive motor neuron degeneration in the ALS-like mouse model.




Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae

2016

Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture. We chose eight commonly used housekeeping genes as candidates. The qRT-PCR expression data for these genes were evaluated from seven populations: a susceptible and three acaricide resistant populations feeding on lima beans, and three other susceptible populations which had been shifted host from lima beans to three other plant species. The stability of the candidate reference genes was then assessed using four different algorithms (comparative ΔCt method, geNorm, NormFinder, and BestKeeper). Additionally, we used an online web-based tool (RefFinder) to assign an overall final rank for each candidate gene. Our study found that CycA and Rp49 are best for investigating gene expression in acaricide susceptible and resistant populations. GAPDH, Rp49, and Rpl18 are best for host plant shift studies. And GAPDH and Rp49 were the most stable reference genes when investigating gene expression under changes in both experimental conditions. These results will facilitate research in revealing molecular mechanisms underlying the xenobiotic adaptation of this notorious agricultural pest.




Role of Osteopontin in Liver Diseases

2016

Osteopontin (OPN), a multifunctional protein, is involved in numerous pathological conditions including inflammation, immunity, angiogenesis, fibrogenesis and carcinogenesis in various tissues. Extensive studies have elucidated the critical role of OPN in cell signaling such as regulation of cell proliferation, migration, inflammation, fibrosis and tumor progression. In the liver, OPN interacts with integrins, CD44, vimentin and MyD88 signaling, thereby induces infiltration, migration, invasion and metastasis of cells. OPN is highlighted as a chemoattractant for macrophages and neutrophils during injury in inflammatory liver diseases. OPN activates hepatic stellate cells (HSCs) to exert an enhancer in fibrogenesis. The role of OPN in hepatocellular carcinoma (HCC) has also generated significant interests, especially with regards to its role as a diagnostic and prognostic factor. Interestingly, OPN acts an opposing role in liver repair under different pathological conditions. This review summarizes the current understanding of OPN in liver diseases. Further understanding of the pathophysiological role of OPN in cellular interactions and molecular mechanisms associated with hepatic inflammation, fibrosis and cancer may contribute to the development of novel strategies for clinical diagnosis, monitoring and therapy of liver diseases.




Novel FOXC2 Mutation in Hereditary Distichiasis Impairs DNA-Binding Activity and Transcriptional Activation

2016

Distichiasis presents as double rows of eyelashes arising from aberrant differentiation of the meibomian glands of the eyelids, and it may be sporadic or hereditary. FOXC2 gene mutations in hereditary distichiasis are rarely reported. Here, we examined two generations of a Chinese family with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was amplified and sequenced in all family members. Subcellular localization and luciferase assays were performed to assess the activity of the mutant FOXC2 protein. Clinical examinations showed distichiasis, lower eyelid ectropion, congenital ptosis and photophobia in all affected individuals. Sequence analysis revealed a novel frameshift mutation, c.964_965insG, in the coding region of the FOXC2 gene. This mutation caused protein truncation due to the presence of a premature stop codon. A fluorescence assay showed that this mutation did not change the nuclear localization of the protein. However, it impaired DNA-binding activity and decreased transcriptional activation. This is the first report of a FOXC2 mutation in hereditary distichiasis in the Chinese population. The findings of our study expand the FOXC2 mutation spectrum and contribute to the understanding of the genotype-phenotype correlation of this disease.




An Effective Molecular Target Site in Hepatitis B Virus S Gene for Cas9 Cleavage and Mutational Inactivation

2016

Chronic hepatitis B infection remains incurable because HBV cccDNA can persist indefinitely in patients recovering from acute HBV infection. Given the incidence of HBV infection and the shortcomings of current therapeutic options, a novel antiviral strategy is urgently needed. To inactivate HBV replication and destroy the HBV genome, we employed genome editing tool CRISPR/Cas9. Specifically, we found a CRISPR/Cas9 system (gRNA-S4) that effectively targeted the HBsAg region and could suppress efficiently viral replication with minimal off-target effects and impact on cell viability. The mutation mediated by CRISPR/Cas9 in HBV DNA both in a stable HBV-producing cell line and in HBV transgenic mice had been confirmed and evaluated using deep sequencing. In addition, we demonstrated the reduction of HBV replication was caused by the mutation of S4 site through three S4 region-mutated monoclonal cells. Besides, the gRNA-S4 system could also reduce serum surface-antigen levels by 99.91 ± 0.05% and lowered serum HBV DNA level below the negative threshold in the HBV hydrodynamics mouse model. Together, these findings indicate that the S4 region may be an ideal target for the development of innovative therapies against HBV infection using CRISPR/Cas9.




Cooperation of Doxycycline with Phytochemicals and Micronutrients Against Active and Persistent Forms of Borrelia sp

2016

Phytochemicals and micronutrients represent a growing theme in antimicrobial defense; however, little is known about their anti-borreliae effects of reciprocal cooperation with antibiotics. A better understanding of this aspect could advance our knowledge and help improve the efficacy of current approaches towards Borrelia sp. In this study, phytochemicals and micronutrients such as baicalein, luteolin, 10-HAD, iodine, rosmarinic acid, and monolaurin, as well as, vitamins D3 and C were tested in a combinations with doxycycline for their in vitro effectiveness against vegetative (spirochetes) and latent (rounded bodies, biofilm) forms of Borrelia burgdorferi and Borrelia garinii. Anti-borreliae effects were evaluated according to checkerboard assays and supported by statistical analysis. The results showed that combination of doxycycline with flavones such as baicalein and luteolin exhibited additive effects against all morphological forms of studied Borrelia sp. Doxycycline combined with iodine demonstrated additive effects against spirochetes and biofilm, whereas with fatty acids such as monolaurin and 10-HAD it produced FICIs of indifference. Additive anti-spirochetal effects were also observed when doxycycline was used with rosmarinic acid and both vitamins D3 and C. Antagonism was not observed in any of the cases. This data revealed the intrinsic anti-borreliae activity of doxycycline with tested phytochemicals and micronutrients indicating that their addition may enhance efficacy of this antibiotic in combating Borrelia sp. Especially the addition of flavones balcalein and luteolin to a doxycycline regimen could be explored further in defining more effective treatments against these bacteria.




Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron

2016

Cells make use of molecular motors to transport small molecules, macromolecules and cellular organelles to target region to execute biological functions, which is utmost important for polarized cells, such as neurons. In particular, cytoskeleton motors play fundamental roles in neuron polarization, extension, shape and neurotransmission. Cytoskeleton motors comprise of myosin, kinesin and cytoplasmic dynein. F-actin filaments act as myosin track, while kinesin and cytoplasmic dynein move on microtubules. Cytoskeleton motors work together to build a highly polarized and regulated system in neuronal cells via different molecular mechanisms and functional regulations. This review discusses the structures and working mechanisms of the cytoskeleton motors in neurons.




Analysis of PBase Binding Profile Indicates an Insertion Target Selection Mechanism Dependent on TTAA, But Not Transcriptional Activity

2016

Transposons and retroviruses are important pathogenic agents and tools for mutagenesis and transgenesis. Insertion target selection is a key feature for a given transposon or retrovirus. The piggyBac (PB) transposon is highly active in mice and human cells, which has a much better genome-wide distribution compared to the retrovirus and P-element. However, the underlying reason is not clear. Utilizing a tagged functional PB transposase (PBase), we were able to conduct genome-wide profiling for PBase binding sites in the mouse genome. We have shown that PBase binding mainly depends on the distribution of the tetranucleotide TTAA, which is not affected by the presence of PB DNA. Furthermore, PBase binding is negatively influenced by the methylation of CG sites in the genome. Analysis of a large collection of PB insertions in mice has revealed an insertion profile similar to the PBase binding profile. Interestingly, this profile is not correlated with transcriptional active genes in the genome or transcriptionally active regions within a transcriptional unit. This differs from what has been previously shown for P-element and retroviruses insertions. Our study provides an explanation for PB's genome-wide insertion distribution and also suggests that PB target selection relies on a new mechanism independent of active transcription and open chromatin structure.




Gamma-secretase Inhibitor Prevents Proliferation and Migration of Ductus Arteriosus Smooth Muscle Cells through the Notch3-HES1/2/5 Pathway

2016

Patent ductus arteriosus (PDA) can cause morbidity and mortality in neonates. Vascular remodeling, characterized by proliferation and migration of smooth muscle cells (SMCs), is an essential process for postnatal DA closure. Notch signaling is an important mediator of vascular remodelling but its role in DA is unkonwn. We investigated the effects and underlying mechanisms of γ-secretase inhibitor DAPT, a Notch signaling inhibitor on angiotensin II (Ang II)-induced proliferation and migration of DASMCs. Proliferation and migration of DASMCs cultured from neonatal Wistar rats were induced by Ang II, with or without DAPT pre-treatment. In addition, potential underlying mechanisms including cell cycle progression, Ca2+ influx, reactive oxygen species (ROS) production, signal transduction of MAPK and Akt, and Notch receptor with its target gene pathway were examined. We found that DAPT inhibited Ang II-induced DASMCs proliferation and migration dose dependently. DAPT also arrested the cell cycle progression in the G0/G1-phase, and attenuated calcium overload and ROS production caused by Ang II. Moreover, DAPT inhibited nuclear translocation of Notch3 receptor intracellular domain, with decreased expression of its down-stream genes including HES1, HES2 and HES5. Finally, Ang II-activated ERK1/2, JNK and Akt were also counteracted by DAPT. In conclusion, DAPT inhibits Ang II-induced DASMCs proliferation and migration. These effects are potentially mediated by decreased calcium influx, reduced ROS production, and down-regulation of ERK1/2, JNK and Akt, through the Notch3-HES1/2/5 pathway. Therefore, Notch signaling has a role in DA remodeling and may provide a target pathway for therapeutic intervention of PDA.




Alcohol Increases Liver Progenitor Populations and Induces Disease Phenotypes in Human IPSC-Derived Mature Stage Hepatic Cells

2016

Alcohol consumption has long been a global problem affecting human health, and has been found to influence both fetal and adult liver functions. However, how alcohol affects human liver development and liver progenitor cells remains largely unknown. Here, we used human induced pluripotent stem cells (iPSCs) as a model to examine the effects of alcohol, on multi-stage hepatic cells including hepatic progenitors, early and mature hepatocyte-like cells derived from human iPSCs. While alcohol has little effect on endoderm development from iPSCs, it reduces formation of hepatic progenitor cells during early hepatic specification. The proliferative activities of early and mature hepatocyte-like cells are significantly decreased after alcohol exposure. Importantly, at a mature stage of hepatocyte-like cells, alcohol treatment increases two liver progenitor subsets, causes oxidative mitochondrial injury and results in liver disease phenotypes (i.e., steatosis and hepatocellular carcinoma associated markers) in a dose dependent manner. Some of the phenotypes were significantly improved by antioxidant treatment. This report suggests that fetal alcohol exposure may impair generation of hepatic progenitors at early stage of hepatic specification and decrease proliferation of fetal hepatocytes; meanwhile alcohol injury in post-natal or mature stage human liver may contribute to disease phenotypes. This human iPSC model of alcohol-induced liver injury can be highly valuable for investigating alcoholic injury in the fetus as well as understanding the pathogenesis and ultimately developing effective treatment for alcoholic liver disease in adults.




Multiple Growth Factors, But Not VEGF, Stimulate Glycosaminoglycan Hyperelongation in Retinal Choroidal Endothelial Cells

2016

A major feature of early age-related macular degeneration (AMD) is the thickening of Bruch's membrane in the retina and an alteration in its composition with increased lipid deposition. In certain pathological conditions proteoglycans are responsible for lipid retention in tissues. Growth factors are known to increase the length of glycosaminoglycan chains and this can lead to a large increase in the interaction between proteoglycans and lipids. Using choroidal endothelial cells, we investigated the effects of a number of AMD relevant growth factors TGFβ, thrombin, PDGF, IGF and VEGF on proteoglycan synthesis. Cells were characterized as of endothelial origin using the specific cell markers endothelial nitric oxide synthesis and von Willebrand factor and imaged using confocal microscopy. Cells were treated with growth factors in the presence and absence of the appropriate inhibitors and were radiolabeled with [35S]-SO4. Proteoglycans were isolated by ion exchange chromatography and sized using SDS-PAGE. Radiosulfate incorporation was determined by the cetylpyridinium chloride (CPC) precipitation technique. To measure cellular glycosaminoglycan synthesizing capacity we added xyloside and assessed the xyloside-GAGs by SDS-PAGE. TGFβ, thrombin, PDGF & IGF dose-dependently stimulated radiosulfate incorporation and GAG elongation as well as xyloside-GAG synthesis, however VEGF treatment did not stimulate any changes in proteoglycan synthesis. VEGF did not increase pAKT but caused a large increase in pERK relative to the response to PDGF. Thus, AMD relevant agonists cause glycosaminoglycan hyperelongation of proteoglycans synthesised and secreted by retinal choroidal endothelial cells. The absence of a response to VEGF is intriguing and identifies proteoglycans as a novel potential target in AMD. Future studies will examine the relevance of these changes to enhanced lipid binding and the development of AMD.




Rapamycin Reduced Ischemic Brain Damage in Diabetic Animals Is Associated with Suppressions of mTOR and ERK1/2 Signaling

2016

The objectives of the present study are to investigate the activation of mTOR and ERK1/2 signaling after cerebral ischemia in diabetic rats and to examine the neuroprotective effects of rapamycin. Ten minutes transient global cerebral ischemia was induced in straptozotocin-induced diabetic hyperglycemic rats and non-diabetic, euglycemic rats. Brain samples were harvested after 16 h of reperfusion. Rapamycin or vehicle was injected 1 month prior to the induction of ischemia. The results showed that diabetes increased ischemic neuronal cell death and associated with elevations of p-P70S6K and Ras/ERK1/2 and suppression of p-AMPKα. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and suppressed phosphorylation of P70S6K and ERK1/2. It is concluded that diabetes activates mTOR and ERK1/2 signaling pathways in rats subjected to transient cerebral ischemia and inhibition of mTOR by rapamycin reduces ischemic brain damage and suppresses the mTOR and ERK1/2 signaling in diabetic settings.




A Review of Neoadjuvant Chemoradiotherapy for Locally Advanced Rectal Cancer

2016

Neoadjuvant chemoradiotherapy has become the standard treatment for locally advanced rectal cancer. Neoadjuvant chemoradiotherapy not only can reduce tumor size and recurrence, but also increase the tumor resection rate and anus retention rate with very slight side effect. Comparing with preoperative chemotherapy, preoperative chemoradiotherapy can further reduce the local recurrence rate and downstage. Middle and low rectal cancers can benefit more from neoadjuvant chemradiotherapy than high rectal cancer. It needs to refine the selection of appropriate patients and irradiation modes for neoadjuvant chemoradiotherapy. Different therapeutic reactions to neoadjuvant chemoradiotherapy affect the type of surgical techniques, hence calling for the need of much attention. Furthermore, many problems such as accurate staging before surgery, selection of suitable neoadjuvant chemoradiotherapy method, and sensitivity prediction to preoperative radiotherapy need to be well settled.




Cis-Natural Antisense Transcripts Are Mainly Co-expressed with Their Sense Transcripts and Primarily Related to Energy Metabolic Pathways during Muscle Development

2016

Cis-natural antisense transcripts (cis-NATs) are a new class of RNAs identified in various species. However, the biological functions of cis-NATs are largely unknown. In this study, we investigated the transcriptional characteristics and functions of cis-NATs in the muscle tissue of lean Landrace and indigenous fatty Lantang pigs. In total, 3,306 cis-NATs of 2,469 annotated genes were identified in the muscle tissue of pigs. More than 1,300 cis-NATs correlated with their sense genes at the transcriptional level, and approximately 80% of them were co-expressed in the two breeds. Furthermore, over 1,200 differentially expressed cis-NATs were identified during muscle development. Function annotation showed that the cis-NATs participated in muscle development mainly by co-expressing with genes involved in energy metabolic pathways, including citrate cycle (TCA cycle), glycolysis or gluconeogenesis, mitochondrial activation and so on. Moreover, these cis-NATs and their sense genes abruptly increased at the transition from the late fetal stages to the early postnatal stages and then decreased along with muscle development. In conclusion, the cis-NATs in the muscle tissue of pigs were identified and determined to be mainly co-expressed with their sense genes. The co-expressed cis-NATs and their sense gene were primarily related to energy metabolic pathways during muscle development in pigs. Our results offered novel evidence on the roles of cis-NATs during the muscle development of pigs.




Carbon Monoxide Improves Neurologic Outcomes by Mitochondrial Biogenesis after Global Cerebral Ischemia Induced by Cardiac Arrest in Rats

2016

Mitochondrial dysfunction contributes to brain injury following global cerebral ischemia after cardiac arrest. Carbon monoxide treatment has shown potent cytoprotective effects in ischemia/reperfusion injury. This study aimed to investigate the effects of carbon monoxide-releasing molecules on brain mitochondrial dysfunction and brain injury following resuscitation after cardiac arrest in rats. A rat model of cardiac arrest was established by asphyxia. The animals were randomly divided into the following 3 groups: cardiac arrest and resuscitation group, cardiac arrest and resuscitation plus carbon monoxide intervention group, and sham control group (no cardiac arrest). After the return of spontaneous circulation, neurologic deficit scores (NDS) and S-100B levels were significantly decreased at 24, 48, and 72 h, but carbon monoxide treatment improved the NDS and S-100B levels at 24 h and the 3-day survival rates of the rats. This treatment also decreased the number of damaged neurons in the hippocampus CA1 area and increased the brain mitochondrial activity. In addition, it increased mitochondrial biogenesis by increasing the expression of biogenesis factors including peroxisome proliferator-activated receptor-γ coactivator-1α, nuclear respiratory factor-1, nuclear respiratory factor-2 and mitochondrial transcription factor A. Thus, this study showed that carbon monoxide treatment alleviated brain injury after cardiac arrest in rats by increased brain mitochondrial biogenesis.




Fibroblast Growth Factor Receptor 3 Deficiency Does Not Impair the Osteoanabolic Action of Parathyroid Hormone on Mice

2016

Summary: PTH stimulates bone formation in Fgfr3 knockout mice through promotion of proliferation and differentiation in osteoblasts.

Introduction: Previous studies showed that endogenous fibroblast growth factor 2 (FGF-2) is required for parathyroid hormone (PTH)-stimulated bone anabolic effects, however, the exact mechanisms by which PTH stimulate bone formation and the function of FGF receptors in mediating these actions are not fully defined. FGF receptor 3 (FGFR3) has been characterized as an important regulator of bone metabolism and is confirmed to cross-talk with PTH/PTHrP signal in cartilage and bone development.

Methods: Fgfr3 knockout and wild-type mice at 2-month-old and 4-month-old were intraperitoneally injected with PTH intermittently for 4 weeks and then the skeletal responses to PTH were assessed by dual energy X-ray absorptiometry (DEXA), micro-computed tomography (μCT) and bone histomorphometry.

Results: Intermittent PTH treatment improved bone mineral density (BMD) and femoral mechanical properties in both Fgfr3-/- and wild-type mice. Histomorphometric analysis showed that bone formation and bone resorption were increased in both genotypes following PTH treatment. PTH treatment increased trabecular bone volume (BV/TV) in WT and Fgfr3-deficient mice. The anabolic response in Fgfr3-deficient and wild-type bone is characterized by an increase of both bone formation and resorption-related genes following PTH treatment. In addition, we found that Fgfr3 null osteoblasts (compared to wild-type controls) maintained normal abilities to response to PTH-stimulated increase of proliferation, differentiation, expression of osteoblastic marker genes (Cbfa1, Osteopontin and Osteocalcin), and phosphorylation of Erk1/2.

Conclusions: Bone anabolic effects of PTH were not impaired by the absence of FGFR3, suggesting that the FGFR3 signaling may not be required for osteoanabolic effects of PTH activities.




BDNF-mediates Down-regulation of MicroRNA-195 Inhibits Ischemic Cardiac Apoptosis in Rats

2016

Background: Our previous studies suggested that brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) axis inhibited cardiomyocyte apoptosis in myocardial infarction (MI). However, the relationship between BDNF and microRNA (miRNA) in cardiomyocytes are unclear. The present study was performed to investigate the role of miR-195 and the interplay between BDNF and miR-195 in ischemic cardiomyocyte apoptosis.

Methods: Male Wistar rats were subjected to coronary artery ligation, and primary neonatal rat ventricular myocytes were treated with hypoxia or hydrogen peroxide (H2O2). BDNF level in rat ventricles was measured by enzyme linked immunosorbent assay (ELISA). miR-195 mimic, inhibitor or negative control was transfected into the cardiomyocytes. Cell viability and apoptosis were detected by MTT assay and TdT-mediated dUTP nick end labeling (TUNEL) staining, respectively. Cardiac function and apoptosis were detected in MI rats intravenously injected with antagomiR-195. Luciferase assay, Western blot and Real-time RT-PCR were employed to clarify the interplay between miR-195 and BDNF.

Results: miR-195 level was dynamically regulated in response to MI and significantly increased in ischemic regions 24 h post-MI as well as in hypoxic or H2O2-treated cardiomyocytes. Meanwhile, BDNF protein level was rapidly increased in MI rats and H2O2-treated cardiomyocytes. Apoptosis in both hypoxic and H2O2-treated cardiomyocytes were markedly reduced and cell viability was increased by miR-195 inhibitor. Moreover, inhibition of miR-195 significantly improved cardiac function of MI rats. Bcl-2 but not BDNF was validated as the direct target of miR-195. Furthermore, BDNF abolished the pro-apoptotic role of miR-195, which was reversed by its scavenger TrkB-Fc.

Conclusion: Up-regulation of miR-195 in ischemic cardiomyocytes promotes ischemic apoptosis by targeting Bcl-2. BDNF mitigated the pro-apoptotic effect of miR-195 in rat cardiomyocytes. These findings may provide better understanding of the pro-apoptotic role of miR-195 in MI and suggest that BDNF/miR-195/Bcl-2 axis may be beneficial for limiting myocardial ischemic injury.




Selecting Cells for Bioartificial Liver Devices and the Importance of a 3D Culture Environment: A Functional Comparison between the HepaRG and C3A Cell Lines

2016

Recently, the first clinical trials on Bioartificial Livers (BALs) loaded with a proliferative human hepatocyte cell source have started. There are two cell lines that are currently in an advanced state of BAL development; HepaRG and HepG2/C3A. In this study we aimed to compare both cell lines on applicability in BALs and to identify possible strategies for further improvement. We tested both cell lines in monolayer- and BAL cultures on growth characteristics, hepatic differentiation, nitrogen-, carbohydrate-, amino acid- and xenobiotic metabolism. Interestingly, both cell lines adapted the hepatocyte phenotype more closely when cultured in BALs; e.g. monolayer cultures produced lactate, while BAL cultures showed diminished lactate production (C3A) or conversion to elimination (HepaRG), and urea cycle activity increased upon BAL culturing in both cell lines. HepaRG-BALs outperformed C3A-BALs on xenobiotic metabolism, ammonia elimination and lactate elimination, while protein synthesis was comparable. In BAL cultures of both cell lines ammonia elimination correlated positively with glutamine production and glutamate consumption, suggesting ammonia elimination was mainly driven by the balance between glutaminase and glutamine synthetase activity. Both cell lines lacked significant urea cycle activity and both required multiple culture weeks before reaching optimal differentiation in BALs.

In conclusion, culturing in BALs enhanced hepatic functionality of both cell lines and from these, the HepaRG cells are the most promising proliferative cell source for BAL application.




The Involving Roles of Intrahepatic and Extrahepatic Stem/Progenitor Cells (SPCs) to Liver Regeneration

2016

Liver regeneration is usually attributed to mature hepatocytes, which possess a remarkable potential to proliferate under mild to moderate injury. However, when the liver is severely damaged or hepatocyte proliferation is greatly inhibited, liver stem/progenitor cells (LSPCs) will contribute to the liver regeneration process. LSPCs in the developing liver have been extensively characterized, however, their contributing role to liver regeneration has not been completely understood. In addition to the restoration of the liver parenchymal tissue by hepatocytes or/and LSPCs, or in some cases bone marrow (BM) derived cells, such as hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), the wound healing after injury in terms of angiopoiesis by liver sinusoidal endothelial cells (LSECs) or/and sinusoidal endothelial progenitor cells (SEPCs) is another important aspect taking place during regeneration. To conclude, liver regeneration can be mainly divided into three distinct restoring levels according to the cause and severity of injury: hepatocyte dominant regeneration, LSPCs mediated regeneration, extrahepatic stem cells participative regeneration. In this review, we focus on the recent findings of liver regeneration, especially on those related to stem/progenitor cells (SPCs)-mediated regeneration and their potential clinical applications and challenges.




Protective Effect of Thymoquinone against Cyclophosphamide-Induced Hemorrhagic Cystitis through Inhibiting DNA Damage and Upregulation of Nrf2 Expression

2016

Cyclophosphamide (CYP) induced hemorrhagic cystitis is a dose-limiting side effect involving increased oxidative stress, inflammatory cytokines and suppressed activity of nuclear factor related erythroid 2-related factor (Nrf2). Thymoquinone (TQ), an active constituent of Nigella sativa seeds, is reported to increase the expression of Nrf2, exert antioxidant action, and anti-inflammatory effects in the experimental animals. The present study was designed to explore the effects of TQ on CYP-induced hemorrhagic cystitis in Balb/c mice. Cystitis was induced by a single intraperitoneal injection of CYP (200 mg/kg). TQ was administered intraperitoneally at 5, 10 and 20 mg/kg doses twice a day, for three days before and three days after the CYP administration. The efficacy of TQ was determined in terms of the protection against the CYP-induced histological perturbations in the bladder tissue, reduction in the oxidative stress, and inhibition of the DNA fragmentation. Immunohistochemistry was performed to examine the expression of Nrf2. TQ protected against CYP-induced oxidative stress was evident from significant reduction in the lipid peroxidation, restoration of the levels of reduced glutathione, catalase and superoxide dismutase activities. TQ treatment significantly reduced the DNA damage evident as reduced DNA fragmentation. A significant decrease in the cellular infiltration, edema, epithelial denudation and hemorrhage were observed in the histological observations. There was restoration and rise in the Nrf2 expression in the bladder tissues of mice treated with TQ. These results confirm that, TQ ameliorates the CYP-induced hemorrhagic cystitis in mice through reduction in the oxidative stress, inhibition of the DNA damage and through increased expression of Nrf2 in the bladder tissues.




The Protein Elicitor PevD1 Enhances Resistance to Pathogens and Promotes Growth in Arabidopsis

2016

The protein elicitor PevD1, isolated from Verticillium dahlia, could enhance resistance to TMV in tobacco and Verticillium wilt in cotton. Here, the pevd1 gene was over-expressed in wild type (WT) Arabidopsis, and its biological functions were investigated. Our results showed that the transgenic lines were more resistant to Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 than the WT line was. In transgenic plants, both the germination time and bolting time required were significantly shorter and fresh weights and plant heights were significantly higher than those in the WT line. A transcriptomics study using digital gene expression profiling (DGE) was performed in transgenic and WT Arabidopsis. One hundred and thirty-six differentially expressed genes were identified. In transgenic Arabidopsis, three critical regulators of JA biosynthesis were up-regulated and JA levels were slightly increased. Three important repressors of the ABA-responsive pathway were up-regulated, indicating that ABA signal transduction may be suppressed. One CML and two WRKY TFs involved in Ca2+-responsive pathways were up-regulated, indicating that this pathway may have been triggered. In conclusion, we show that PevD1 is involved in regulating several plant endogenous signal transduction pathways and regulatory networks to enhance resistance and promote growth and development in Arabidopsis.




Yorkie Facilitates Organ Growth and Metamorphosis in Bombyx

2016

The Hippo pathway, which was identified from genetic screens in the fruit fly, Drosophila melanogaster, has a major size-control function in animals. All key components of the Hippo pathway, including the transcriptional coactivator Yorkie that is the most critical substrate and downstream effector of the Hippo kinase cassette, are found in the silkworm, Bombyx mori. As revealed by microarray and quantitative real-time PCR, expression of Hippo pathway genes is particularly enriched in several mitotic tissues, including the ovary, testis, and wing disc. Developmental profiles of Hippo pathway genes are generally similar (with the exception of Yorkie) within each organ, but vary greatly in different tissues showing nearly opposing expression patterns in the wing disc and the posterior silk gland (PSG) on day 2 of the prepupal stage. Importantly, the reduction of Yorkie expression by RNAi downregulated Yorkie target genes in the ovary, decreased egg number, and delayed larval-pupal-adult metamorphosis. In contrast, baculovirus-mediated YorkieCA overexpression upregulated Yorkie target genes in the PSG, increased PSG size, and accelerated larval-pupal metamorphosis. Together the results show that Yorkie potentially facilitates organ growth and metamorphosis, and suggest that the evolutionarily conserved Hippo pathway is critical for size control, particularly for PSG growth, in the silkworm.




Generation of a Mouse Full-length Balancer with Versatile Cassette-shuttling Selection Strategy

2016

Balancer chromosomes are important tools for a variety of genetic manipulations in lower model organisms, owing to their ability to suppress recombination. In mouse, however, such effort has not been accomplished, mostly due to the size of the chromosomes and the complexity of multiple step chromosomal engineering. We developed an effective and versatile cassette-shuttling selection (CASS) strategy involving only two selection markers to achieve the sequential production of multiple large inversions along the chromosome. Using this strategy, we successfully generated the first full-length balancer in mice and showed that Balancer 17M-GFP can efficiently suppress recombination. Our study has not only generated a useful genetic resource, but also provided a strategy for constructing mammalian balancer chromosomes.




Long Noncoding RNA: Recent Updates in Atherosclerosis

2016

Long noncoding RNAs belong to a class of noncoding RNAs longer than 200 nucleotides with the epigenetic regulation potential. As a novel molecular regulator, lncRNAs are often dysregulated in various pathological conditions and display multiple functions in a wide range of biological processes. Given that recent studies have indicated that lncRNAs are involved in atherosclerosis-related smooth muscle cell, endothelial cell, macrophage and lipid metabolism regulation, it is pertinent to understand the potential function of lncRNAs in atherosclerosis development. This review will highlight the recent updates of lncRNAs in atherogenesis and also discuss their potential roles as novel therapeutic targets.




Interactions between Autophagy and Inhibitory Cytokines

2016

Autophagy is a degradative pathway that plays an essential role in maintaining cellular homeostasis. Most early studies of autophagy focused on its involvement in age-associated degeneration and nutrient deprivation. However, the immunological functions of autophagy have become more widely studied in recent years. Autophagy has been shown to be an intrinsic cellular defense mechanism in the innate and adaptive immune responses. Cytokines belong to a broad and loose category of proteins and are crucial for innate and adaptive immunity. Inhibitory cytokines have evolved to permit tolerance to self while also contributing to the eradication of invading pathogens. Interactions between inhibitory cytokines and autophagy have recently been reported, revealing a novel mechanism by which autophagy controls the immune response. In this review, we discuss interactions between autophagy and the regulatory cytokines IL-10, transforming growth factor-β, and IL-27. We also mention possible interactions between two newly discovered cytokines, IL-35 and IL-37, and autophagy.




Glycosphingolipid GM3 is Indispensable for Dengue Virus Genome Replication

2016

Dengue virus (DENV) causes the most prevalent arthropod-borne viral disease of humans worldwide. Glycosphingolipids (GSLs) are involved in virus infection by regulating various steps of viral-host interaction. However, the distinct role of GSLs during DENV infection remains unclear. In this study, we used mouse melanoma B16 cells and their GSL-deficient mutant counterpart GM95 cells to study the influence of GSLs on DENV infection. Surprisingly, GM95 cells were highly resistant to DENV infection compared with B16 cells. Pretreatment of B16 cells with synthetase inhibitor of GM3, the most abundant GSLs in B16 cells, or silencing GM3 synthetase T3GAL5, significantly inhibited DENV infection. DENV attachment and endocytosis were not impaired in GM95 cells, but DENV genome replication was obviously inhibited in GM95 cells compared to B16 cells. Furthermore, GM3 was colocalized with DENV viral replication complex on endoplasmic reticulum (ER) inside the B16 cells. Finally, GM3 synthetase inhibitor significantly reduced the mortality rate of suckling mice that challenged with DENV by impairing the viral replication in mouse brain. Taken together, these data indicated that GM3 was not required for DENV attachment and endocytosis, however, essential for viral genome replication. Targeting GM3 could be a novel strategy to inhibit DENV infection.




Asiatic Acid Protects against Cardiac Hypertrophy through Activating AMPKα Signalling Pathway

2016

Background: AMPactivated protein kinase α (AMPKα) is closely involved in the process of cardiac hypertrophy. Asiatic acid (AA), a pentacyclic triterpene, was found to activate AMPKα in our preliminary experiment. However, its effects on the development of cardiac hypertrophy remain unclear. The present study was to determine whether AA could protect against cardiac hypertrophy.

Methods: Mice subjected to aortic banding were orally given AA (10 or 30mg/kg) for 7 weeks. In the inhibitory experiment, Compound C was intraperitoneally injected for 3 weeks after surgery.

Results: Our results showed that AA markedly inhibited hypertrophic responses induced by pressure overload or angiotensin II. AA also suppressed cardiac fibrosis in vivo and accumulation of collagen in vitro. The protective effects of AA were mediated by activation of AMPKα and inhibition of the mammalian target of rapamycin (mTOR) pathway and extracellular signal-regulated kinase (ERK) in vivo and in vitro. However, AA lost the protective effects after AMPKα inhibition or gene deficiency.

Conclusions: AA protects against cardiac hypertrophy by activating AMPKα, and has the potential to be used for the treatment of heart failure.




Deguelin Induces the Apoptosis of Lung Squamous Cell Carcinoma Cells through Regulating the Expression of Galectin-1

2016

Lung cancer is the leading cause of cancer mortality around the world. Despite advances in the targeted therapy, patients with lung squamous cell carcinoma(SCC) still benefit few from it, and the search for potential effective therapies is imperative. Here, we demonstrated that deguelin induced significant apoptosis of lung SCC cells in vitro. Importantly, we found deguelin down-regulated the expression of galectin-1, which was involved in a wide range of tumorous physiologic process. Thus, we both over-expressed and down-regulated galectin-1 to perform its role in deguelin-induced apoptosis. We found that increased galectin-1 attenuated apoptosis of SCC cells exposed to deguelin, while galectin-1 knockdown sensitized lung cancer cells to deguelin treatment. Additionally, we observed that down-regulation of galectin-1 resulted in suppression of Ras/Raf/ERK pathway which was involved in deguelin-induced cell apoptosis. We also found that deguelin had a significant anti-tumor ability with decline of galectin-1 in vivo. In conclusion, these findings confirm that deguelin may act as a new chemo-preventive agent through inducing apoptosis of lung SCC cells in a galectin-1 dependent manner.




Exosomes Secreted by Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Repair Critical-Sized Bone Defects through Enhanced Angiogenesis and Osteogenesis in Osteoporotic Rats

2016

Bone defects caused by trauma, severe infection, tumor resection and skeletal abnormalities are common osteoporotic conditions and major challenges in orthopedic surgery, and there is still no effective solution to this problem. Consequently, new treatments are needed to develop regeneration procedures without side effects. Exosomes secreted by mesenchymal stem cells (MSCs) derived from human induced pluripotent stem cells (hiPSCs, hiPSC-MSC-Exos) incorporate the advantages of both MSCs and iPSCs with no immunogenicity. However, there are no reports on the application of hiPSC-MSC-Exos to enhance angiogenesis and osteogenesis under osteoporotic conditions. HiPSC-MSC-Exos were isolated and identified before use. The effect of hiPSC-MSC-Exos on the proliferation and osteogenic differentiation of bone marrow MSCs derived from ovariectomized (OVX) rats (rBMSCs-OVX) in vitro were investigated. In vivo, hiPSC-MSC-Exos were implanted into critical size bone defects in ovariectomized rats, and bone regeneration and angiogenesis were examined by microcomputed tomography (micro-CT), sequential fluorescent labeling analysis, microfil perfusion and histological and immunohistochemical analysis. The results in vitro showed that hiPSC-MSC-Exos enhanced cell proliferation and alkaline phosphatase (ALP) activity, and up-regulated mRNA and protein expression of osteoblast-related genes in rBMSCs-OVX. In vivo experiments revealed that hiPSC-MSC-Exos dramatically stimulated bone regeneration and angiogenesis in critical-sized calvarial defects in ovariectomized rats. The effect of hiPSC-MSC-Exos increased with increasing concentration. In this study, we showed that hiPSC-MSC-Exos effectively stimulate the proliferation and osteogenic differentiation of rBMSCs-OVX, with the effect increasing with increasing exosome concentration. Further analysis demonstrated that the application of hiPSC-MSC-Exos+β-TCP scaffolds promoted bone regeneration in critical-sized calvarial defects by enhancing angiogenesis and osteogenesis in an ovariectomized rat model.




A Novel Malaria Pf/Pv Ab Rapid Diagnostic Test Using a Differential Diagnostic Marker Identified by Network Biology

2016

Rapid diagnostic tests (RDTs) can detect anti-malaria antibodies in human blood. As they can detect parasite infection at the low parasite density, they are useful in endemic areas where light infection and/or re-infection of parasites are common. Thus, malaria antibody tests can be used for screening bloods in blood banks to prevent transfusion-transmitted malaria (TTM), an emerging problem in malaria endemic areas. However, only a few malaria antibody tests are available in the microwell-based assay format and these are not suitable for field application. A novel malaria antibody (Ab)-based RDT using a differential diagnostic marker for falciparum and vivax malaria was developed as a suitable high-throughput assay that is sensitive and practical for blood screening. The marker, merozoite surface protein 1 (MSP1) was discovered by generation of a Plasmodium-specific network and the hierarchical organization of modularity in the network. Clinical evaluation revealed that the novel Malaria Pf/Pv Ab RDT shows improved sensitivity (98%) and specificity (99.7%) compared with the performance of a commercial kit, SD BioLine Malaria P.f/P.v (95.1% sensitivity and 99.1% specificity). The novel Malaria Pf/Pv Ab RDT has potential for use as a cost-effective blood-screening tool for malaria and in turn, reduces TTM risk in endemic areas.




Transient Receptor Potential Ankyrin 1 Channel Involved in Atherosclerosis and Macrophage-Foam Cell Formation

2016

Transient receptor potential ankyrin 1 channel (TRPA1) plays an important role in the pathogenesis of inflammatory diseases, yet its role and the underlying mechanism in atherosclerosis remain unclear. We aimed to investigate the role of TRPA1 in atherosclerosis and foam-cell formation in vivo in mice and in vitro in mouse macrophages. Histopathology was examined by hematoxylin and eosin staining, levels of cytokines and lipid profile were evaluated by assay kits, and protein expression was determined by western blot analysis. TRPA1 expression was increased in macrophage foam cells in atherosclerotic aortas of apolipoprotein E-deficient (apoE-/-) mice. Atherosclerotic lesions, hyperlipidemia and systemic inflammation were worsened with chronic administration of the TRPA1 channel antagonist HC030031 or genetic ablation of TRPA1 (TRPA1-/-) in apoE-/- mice. Treatment with allyl isothiocyanate (AITC, a TRPA1 agonist) retarded the progression of atherosclerosis in apoE-/- mice but not apoE-/-TRPA1-/- mice. Mouse macrophages showed oxidized low-density lipoprotein (oxLDL) activated TRPA1 channels. OxLDL-induced lipid accumulation of macrophages was exacerbated by HC030031 or loss of function of TRPA1. Inhibition of TRPA1 activity did not alter oxLDL internalization but impaired cholesterol efflux by downregulating the ATP-binding cassette transporters. Furthermore, tumor necrosis factor-α-induced inflammatory response was attenuated in AITC-activated macrophages. TRPA1 may be a pivotal regulator in the pathogenesis of atherosclerosis and cholesterol metabolism of macrophage foam cells.




Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells

2016

Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles.




Fenofibrate Suppresses Oral Tumorigenesis via Reprogramming Metabolic Processes: Potential Drug Repurposing for Oral Cancer

2016

One anticancer strategy suggests targeting mitochondrial metabolism to trigger cell death through slowing down energy production from the Warburg effect. Fenofibrate is a clinical lipid-lowering agent and an effective anticancer drug. In the present study, we demonstrate that fenofibrate provided novel mechanisms for delaying oral tumor development via the reprogramming of metabolic processes. Fenofibrate induced cytotoxicity by decreasing oxygen consumption rate (OCR) that was accompanied with increasing extracellular acidification rate (ECAR) and reducing ATP content. Moreover, fenofibrate caused changes in the protein expressions of hexokinase II (HK II), pyruvate kinase, pyruvate dehydrogenase, and voltage-dependent anion channel (VDAC), which are associated with the Warburg effect. In addition, fenofibrate reprogrammed the metabolic pathway by interrupting the binding of HK II to VDAC. In an oral cancer mouse model, fenofibrate exhibited both preventive and therapeutic efficacy on oral tumorigenesis. Fenofibrate administration suppressed the incidence rate of tongue lesions, reduced the tumor sizes, decreased the tumor multiplicity, and decreased the immunoreactivities of VDAC and mTOR. The molecular mechanisms involved in fenofibrate's ability to delay tumor development included the down-regulation of mTOR activity via TSC1/2-dependent signaling through activation of AMPK and inactivation of Akt, or via a TSC1/2-independent pathway through direct suppression of raptor. Our findings provide a molecular rationale whereby fenofibrate exerts anticancer and additional beneficial effects for the treatment of oral cancer patients.




Vitamin K2 Ameliorates Damage of Blood Vessels by Glucocorticoid: a Potential Mechanism for Its Protective Effects in Glucocorticoid-induced Osteonecrosis of the Femoral Head in a Rat Model

2016

Glucocorticoid has been reported to decrease blood vessel number and harm the blood supply in the femoral head, which is recognized to be an important mechanism of glucocorticoid-induced osteonecrosis of the femoral head (ONFH). To prevent glucocorticoid-induced ONFH, medication that promotes both bone formation and angiogenesis would be ideal. Vitamin K2 has been revealed to play an important role in bone metabolism; however, few studies have focused on the effect of Vitamin K2 on new vascular formation. Thus, this study aimed to investigate whether Vitamin K2 promoted new blood vessel formation in the presence of glucocorticoids, both in vitro and in vivo. The effect of Vitamin K2 on viability, migration, in vitro tube formation, and VEGF, vWF, CD31, KDR, Flt and PDGFB in EAhy926 incubated with or without dexamethasone were elucidated. VEGF, TGF-β and BMP-2, angiogenesis-related proteins secreted by osteoblasts, were also detected in the osteoblast-like cell line of MG63. In addition, blood vessels of the femoral head in rats administered with or without methylprednisolone and Vitamin K2 were evaluated using angiography and CD31 staining. In vitro studies showed that Vitamin K2 significantly protected endothelial cells from dexamethasone-induced apoptosis, promoted endothelial cell migration and in vitro tube formation. Angiogenesis-related proteins both in EAhy926 and MG63 were also upregulated by Vitamin K2 when cotreated with dexamethasone. In vivo studies showed enhanced blood vessel volume and CD31-positive staining cells in rats cotreated with VK2 and methylprednisolone compared to rats treated with methylprednisolone only. Collectively, Vitamin K2 has the ability to promote angiogenesis in vitro and to ameliorate vessels of the femoral head in glucocorticoid-treated rats in vivo, indicating that Vitamin K2 is a promising drug that may be used to prevent steroid-induced ONFH.




Inhibition of wnt/β-catenin Signaling in Hepatocellular Carcinoma by an Antipsychotic Drug Pimozide

2016

Hepatocellular carcinoma (HCC) is one of the most common forms of malignant cancers in the world, yet very few effective systemic treatments for HCC patients exist. Thus, the development of new treatment modalities presents a great need. The wnt/β-catenin signaling pathway is highly activated in stem cell-like aggressive HCC, which is associated with chemoresistance and poor survival in HCC patients. In a previous study, we found that an FDA-approved psychiatric drug, pimozide (PMZ), has anti-cancer properties in HCC cell lines that express epithelial cell adhesion molecule (EpCAM), a hepatic stem cell marker that is a functional down-stream target of the wnt/β-catenin pathway. In this study, we demonstrate that PMZ effectively inhibits cell growth of HCC cells by disrupting the wnt/β-catenin signaling pathway and reducing EpCAM expression. Thus, PMZ may be a useful molecular entity that could be repurposed as an anti-cancer therapy for treatment of HCC.




A Novel Protein Elicitor (PeBA1) from Bacillus amyloliquefaciens NC6 Induces Systemic Resistance in Tobacco

2016

Here we reported a novel protein elicitor from Bacillus amyloliquefaciens NC6 induced systemic resistance (ISR) in tobacco. The purification was executed by ion-exchange chromatography, native-page extraction and HPLC, and the amino acid sequence was identified by mass spectrometry. This recombinant elicitor protein, expressed in Escherichia coli by an E1 expression vector, had good thermal stability, and the elicitor caused a clearly defined hypersensitive response (HR) necrosis in tobacco leaves. It could also trigger early defence events, including generation of reactive oxygen species (H2O2 and O2-) and phenolic-compound accumulation. Quantitative real-time PCR (Q-RT-PCR) results indicated that several plant defence genes, including the salicylic acid (SA)-responsive PR1a, PR1b, PR5, and phenylalanine ammonia lyase (PAL), as well as the jasmonic acid (JA)-responsive PDF1.2 and CORONATINE INSENSITIVE 1 (COI1), were all up-regulated. Moreover, infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus (TMV) and the fungal pathogen Botrytis cinerea.




Chemoprevention of Low-Molecular-Weight Citrus Pectin (LCP) in Gastrointestinal Cancer Cells

2016

Background & Aims: Low-molecular-weight citrus pectin (LCP) is a complex polysaccharide that displays abundant galactosyl (i.e., sugar carbohydrate) residues. In this study, we evaluated the anti-tumor properties of LCP that lead to Bcl-xL -mediated dampening of apoptosis in gastrointestinal cancer cells.

Methods: We used AGS gastric cancer and SW-480 colorectal cancer cells to elucidate the effects of LCP on cell viability, cell cycle and apoptosis in cultured cells and tumor xenografts.

Results: Significantly decreased cell viabilities were observed in LCP treated AGS and SW-480 cells (P<0.05). Cell cycle-related protein expression, such as Cyclin B1, was also decreased in LCP treated groups as compared to the untreated group. The AGS or SW-480 cell-line tumor xenografts were significantly smaller in the LCP treated group as compared the untreated group (P<0.05). LCP treatment decreased Galectin-3 (GAL-3) expression levels, which is an important gene in cancer metastasis that results in reversion of the epithelial-mesenchymal transition (EMT), and increased suppression of Bcl-xL and Survivin to promote apoptosis. Moreover, results demonstrated synergistic tumor suppressor activity of LCP and 5-FU against gastrointestinal cancer cells both in vivo and in vitro.

Conclusions: LCP effectively inhibits the growth and metastasis of gastrointestinal cancer cells, and does so in part by down-regulating Bcl-xL and Cyclin B to promote apoptosis, and suppress EMT. Thus, LCP alone or in combination with other treatments has a high potential as a novel therapeutic strategy to improve the clinical therapy of gastrointestinal cancer.




Different Modulatory Mechanisms of Renal FXYD12 for Na+-K+-ATPase between Two Closely Related Medakas upon Salinity Challenge

2016

Upon salinity challenge, the Na+-K+-ATPase (NKA) of fish kidney plays a crucial role in maintaining ion and water balance. Moreover, the FXYD protein family was found to be a regulator of NKA. Our preliminary results revealed that fxyd12 was highly expressed in the kidneys of the two closely related euryhaline medaka species (Oryzias dancena and O. latipes) from different natural habitats (brackish water and fresh water). In this study, we investigated the expression and association of renal FXYD12 and NKA α-subunit as well as potential functions of FXYD12 in the two medakas. These findings illustrated and compared the regulatory roles of FXYD12 for NKA in kidneys of the two medakas in response to salinity changes. In this study, at the mRNA and/or protein level, the expression patterns were similar for renal FXYD12 and NKA in the two medakas. However, different patterns of NKA activities and different interaction levels between FXYD12 and NKA were found in the kidneys of these two medakas. The results revealed that different strategies were used in the kidneys of the two medaka species upon salinity challenge. On the other hand, gene knockdown experiments demonstrated that the function of O. dancena FXYD12 allowed maintenance of a high level of NKA activity. The results of the present study indicated that the kidneys of the examined euryhaline medakas originating from brackish water and fresh water exhibited different modulatory mechanisms through which renal FXYD12 enhanced NKA activity to maintain internal homeostasis. Our findings broadened the knowledge of expression and functions of FXYD proteins, the modulators of NKA, in vertebrates.




New Strategies for the Treatment of Solid Tumors with CAR-T Cells

2016

Recent years, we have witnessed significant progresses in both basic and clinical studies regarding novel therapeutic strategies with genetically engineered T cells. Modification with chimeric antigen receptors (CARs) endows T cells with tumor specific cytotoxicity and thus induce anti-tumor immunity against malignancies. However, targeting solid tumors is more challenging than targeting B-cell malignancies with CAR-T cells because of the histopathological structure features, specific antigens shortage and strong immunosuppressive environment of solid tumors. Meanwhile, the on-target/off-tumor toxicity caused by relative expression of target on normal tissues is another issue that should be reckoned. Optimization of the design of CAR vectors, exploration of new targets, addition of safe switches and combination with other treatments bring new vitality to the CAR-T cell based immunotherapy against solid tumors. In this review, we focus on the major obstacles limiting the application of CAR-T cell therapy toward solid tumors and summarize the measures to refine this new cancer therapeutic modality.




Interleukin-10 Family and Tuberculosis: An Old Story Renewed

2016

The interleukin-10 (IL-10) family of cytokines consists of six immune mediators, namely IL-10, IL-19, IL-20, IL-22, IL-24 and IL-26. IL-10, IL-22, IL-24 and IL-26 are critical for the regulation of host defense against Mycobacterium tuberculosis infections. Specifically, IL-10 and IL-26 can suppress the antimycobacterial immunity and promote the survival of pathogen, while IL-22 and IL-24 can generate protective responses and inhibit the intracellular growth of pathogen. Knowledge about the new players in tuberculosis immunology, namely IL-10 family, can inform novel immunity-based countermeasures and host directed therapies against tuberculosis.




Ribose Accelerates Gut Motility and Suppresses Mouse Body Weight Gaining

2016

The increasing prevalence of obesity is closely related to excessive energy consumption. Clinical intervention of energy intake is an attractive strategy to fight obesity. However, the current FDA-approved weight-loss drugs all have significant side effects. Here we show that ribose upregulates gut motility and suppresses mice body weight gain. Ribokinase, which is encoded by Rbks gene, is the first enzyme for ribose metabolism in vivo. Rbks mutation resulted in ribose accumulation in the small intestine, which accelerated gut movement. Ribose oral treatment in wild type mice also enhanced bowel motility and rendered mice resistance to high fat diets. The suppressed weight gain was resulted from enhanced ingested food excretion. In addition, the effective dose of ribose didn't cause any known side effects (i.e. diarrhea and hypoglycemia). Overall, our results show that ribose can regulate gut motility and energy homeostasis in mice, and suggest that administration of ribose and its analogs could regulate gastrointestinal motility, providing a novel therapeutic approach for gastrointestinal dysfunction and weight control.




Ubisol-Q10 Prevents Glutamate-Induced Cell Death by Blocking Mitochondrial Fragmentation and Permeability Transition Pore Opening

2016

Mitochondrial dysfunction and oxidative stress are the major events that lead to the formation of mitochondrial permeability transition pore (mPTP) during glutamate-induced cytotoxicity and cell death. Coenzyme Q10 (CoQ10) has widely been used for the treatment of mitochondrial disorders and neurodegenerative diseases. Comparing to traditional lipid-soluble CoQ10, water soluble CoQ10 (Ubisol-Q10) has high intracellular and intra-mitochondrial distribution. The aims of the present study are to determine the neuroprotective effects of Ubisol-Q10 on glutamate-induced cell death and to explore its functional mechanisms. HT22 neuronal cells were exposed to glutamate. Cell viability was measured and mitochondrial fragmentation was assessed by mitochondrial imaging. The mPTP opening was determined by mitochondrial membrane potential and calcium retention capacity. The results revealed that the anti-glutamate toxicity effects of Ubisol-Q10 was associated with its ability to block mitochondrial fragmentation, to maintain calcium retention capacity and mitochondrial membrane potential, and to prevent mPTP formation, AIF release, and DNA fragmentation. We concluded that Ubisol-Q10 protects cells from glutamate toxicity by preserving the integrity of mitochondrial structure and function. Therefore, adequate CoQ10 supplementation may be beneficial in preventing cerebral stroke and other disorders that involve mitochondrial dysfunction.




A Novel Role of Cab45-G in Mediating Cell Migration in Cancer Cells

2016

Ca2+-binding protein of 45 kDa (Cab45), a CREC family member, is reported to be associated with Ca2+-dependent secretory pathways and involved in multiple diseases including cancers. Cab45-G, a Cab45 isoform protein, plays an important role in protein sorting and secretion at Golgi complex. However, its role in cancer cell migration remains elusive. In this study, we demonstrate that Cab45-G exhibited an increased expression in cell lines with higher metastatic potential and promoted cell migration in multiple types of cancer cells. Overexpression of Cab45-G resulted in an altered expression of the molecular mediators of epithelial-mesenchymal transition (EMT), which is a critical step in the tumor metastasis. Quantitative real-time PCR showed that overexpression of Cab45-G increased the expression of matrix metalloproteinase-2 and -7 (MMP-2 and MMP-7). Conversely, knock-down of Cab45-G reduced the expression of the above MMPs. Moreover, forced expression of Cab45-G upregulated the level of phosphorylated ERK and modulated the secretion of extracellular proteins fibronectin and fibulin. Furthermore, in human cervical and esophageal cancer tissues, the expression of Cab45-G was found to be significantly correlated with that of MMP-2, further supporting the importance of Cab45-G on regulating cancer metastasis. Taken together, these results suggest that Cab45-G could regulate cancer cell migration through various molecular mechanisms, which may serve as a therapeutic target for the treatment of cancers.




Partial Müllerian Duct Retention in Smad4 Conditional Mutant Male Mice

2016

Müllerian duct regression is a complex process which involves the AMH signalling pathway. We have previously demonstrated that besides AMH and its specific type II receptor (AMHRII), BMPR-IA and Smad5 are two essential factors implicated in this mechanism.

Mothers against decapentaplegic homolog 4 (Smad4) is a transcription factor and the common Smad (co-Smad) involved in transforming growth factor beta (TGF-β) signalling pathway superfamily. Since Smad4 null mutants die early during gastrulation, we have inactivated Smad4 in the Müllerian duct mesenchyme. Specific inactivation of Smad4 in the urogenital ridge leads to the partial persistence of the Müllerian duct in adult male mice. Careful examination of the urogenital tract reveals that the Müllerian duct retention is randomly distributed either on one side or both sides. Histological analysis shows a uterus-like structure, which is confirmed by the expression of estrogen receptor α. As previously described in a β-catenin conditional mutant mouse model, β-catenin contributes to Müllerian duct regression. In our mutant male embryos, it appears that β-catenin expression is locally reduced along the urogenital ridge as compared to control mice. Moreover, the expression pattern is similar to those observed in control female mice. This study shows that reduced Smad4 expression disrupts the Wnt/β-catenin signalling leading to the partial persistence of Müllerian duct.




RNA-Seq and iTRAQ Reveal the Dwarfing Mechanism of Dwarf Polish Wheat (Triticum polonicum L.)

2016

The dwarfing mechanism of Rht-dp in dwarf Polish wheat (DPW) is unknown. Each internode of DPW was significantly shorter than it in high Polish wheat (HPW), and the dwarfism was insensitive to photoperiod, abscisic acid (ABA), gibberellin (GA), cytokinin (CK), auxin and brassinolide (BR). To understand the mechanism, three sets of transcripts, DPW, HPW, and a chimeric set (a combination of DPW and HPW), were constructed using RNA sequencing (RNA-Seq). Based on the chimeric transcripts, 2,446 proteins were identified using isobaric tags for relative and absolute quantification (iTRAQ). A total of 108 unigenes and 12 proteins were considered as dwarfism-related differentially expressed genes (DEGs) and differentially expressed proteins (DEPs), respectively. Among of these DEGs and DEPs, 6 DEGs and 6 DEPs were found to be involved in flavonoid and S-adenosyl-methionine (SAM) metabolisms; 5 DEGs and 3 DEPs were involved in cellulose metabolism, cell wall plasticity and cell expansion; 2 DEGs were auxin transporters; 2 DEPs were histones; 1 DEP was a peroxidase. These DEGs and DEPs reduced lignin and cellulose contents, increased flavonoid content, possibly decreased S-adenosyl-methionine (SAM) and polyamine contents and increased S-adenosyl-L-homocysteine hydrolase (SAHH) content in DPW stems, which could limit auxin transport and reduce extensibility of the cell wall, finally limited cell expansion (the cell size of DPW was significantly smaller than HPW cells) and caused dwarfism in DPW.




Dimethyloxaloylglycine Promotes the Angiogenic Activity of Mesenchymal Stem Cells Derived from iPSCs via Activation of the PI3K/Akt Pathway for Bone Regeneration

2016

The vascularization of tissue-engineered bone is a prerequisite step for the successful repair of bone defects. Hypoxia inducible factor-1α (HIF-1α) plays an essential role in angiogenesis-osteogenesis coupling during bone regeneration and can activate the expression of angiogenic factors in mesenchymal stem cells (MSCs). Dimethyloxaloylglycine (DMOG) is an angiogenic small molecule that can inhibit prolyl hydroxylase (PHD) enzymes and thus regulate the stability of HIF-1α in cells at normal oxygen tension. Human induced pluripotent stem cell-derived MSCs (hiPSC-MSCs) are promising alternatives for stem cell therapy. In this study, we evaluated the effect of DMOG on promoting hiPSC-MSCs angiogenesis in tissue-engineered bone and simultaneously explored the underlying mechanisms in vitro. The effectiveness of DMOG in improving the expression of HIF-1α and its downstream angiogenic genes in hiPSC-MSCs demonstrated that DMOG significantly enhanced the gene and protein expression profiles of angiogenic-related factors in hiPSC-MSCs by sustaining the expression of HIF-1α. Further analysis showed that DMOG-stimulated hiPSC-MSCs angiogenesis was associated with the phosphorylation of protein kinase B (Akt) and with an increase in VEGF production. The effects could be blocked by the addition of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In a critical-sized calvarial defect model in rats, DMOG-treated hiPSC-MSCs showed markedly improved angiogenic capacity in the tissue-engineered bone, leading to bone regeneration. Collectively, the results indicate that DMOG, via activation of the PI3K/Akt pathway, promotes the angiogenesis of hiPSC-MSCs in tissue-engineered bone for bone defect repair and that DMOG-treated hiPSC-MSCs can be exploited [...]



In Vitro Antileukemia Activity of ZSTK474 on K562 and Multidrug Resistant K562/A02 Cells

2016

Chronic myelogenous leukemia (CML) is a malignant hematological disorder mainly caused by the Bcr-Abl tyrosine kinase. While Bcr-Abl inhibitors including Imatinib showed antitumor efficacy on many CML patients, resistance was frequently reported in recent years. Therefore, novel drugs for CML are still expected. ZSTK474 is a specific phosphatidylinositol 3-kinase (PI3K) inhibitor that we identified. In the present study, the efficacy of ZSTK474, alone or in combination with Imatinib, on K562 CML cells as well as on its multidrug resistance counterpart K562/A02 cells, was investigated. ZSTK474 inhibited the cell proliferation with an IC50 of 4.69 μM for K562 and 7.57 μM for K562/A02 cells, respectively. Treatment by ZSTK474 resulted in cell cycle arrest in G1 phase, which might be associated with upregulation of p27, and downregulation of cyclin D1. ZSTK474 also inhibited phosphorylation of Akt and GSK-3β, which might be involved in the effect on the above cell cycle-related proteins. Moreover, combination of ZSTK474 and Imatinib indicated synergistic effect on both cell lines. In conclusion, ZSTK474 exhibited antileukemia activity alone, and showed synergistic effect when combined with Imatinib, on CML K562 cells as well as the multidrug resistant ones, providing a potential therapeutic approach for CML patients.




Modeling Energy Dynamics in Mice with Skeletal Muscle Hypertrophy Fed High Calorie Diets

2016

Retrospective and prospective studies show that lean mass or strength is positively associated with metabolic health. Mice deficient in myostatin, a growth factor that negatively regulates skeletal muscle mass, have increased muscle and body weights and are resistant to diet-induced obesity. Their leanness is often attributed to higher energy expenditure in the face of normal food intake. However, even obese animals have an increase in energy expenditure compared to normal weight animals suggesting this is an incomplete explanation. We have previously developed a computational model to estimate energy output, fat oxidation and respiratory quotient from food intake and body composition measurements to more accurately account for changes in body composition in rodents over time. Here we use this approach to understand the dynamic changes in energy output, intake, fat oxidation and respiratory quotient in muscular mice carrying a dominant negative activin receptor IIB expressed specifically in muscle. We found that muscular mice had higher food intake and higher energy output when fed either chow or a high-fat diet for 15 weeks compared to WT mice. Transgenic mice also matched their rate of fat oxidation to the rate of fat consumed better than WT mice. Surprisingly, when given a choice between high-fat diet and Ensure® drink, transgenic mice consumed relatively more calories from Ensure® than from the high-fat diet despite similar caloric intake to WT mice. When switching back and forth between diets, transgenic mice adjusted their intake more rapidly than WT to restore normal caloric intake. Our results show that mice with myostatin inhibition in muscle are better at adjusting energy intake and output on diet[...]



Ecological Trade-offs between Migration and Reproduction Are Mediated by the Nutrition-Sensitive Insulin-Signaling Pathway

2016

Crowding and changes in food availability are two critical environmental conditions that impact an animal's trajectory toward either migration or reproduction. Many insects facing this challenge have evolved wing polyphenisms. When conditions favor reproduction, wing polyphenic species produce adults that either have no wings or short, non-functional wings. Facultative wing growth reflects a physiological and evolutionary trade-off between migration and reproduction, triggered by environmental conditions. How environmental cues are transduced to produce these alternative forms, and their associated ecological shift from migration to reproduction, remains an important unsolved problem in evolutionary ecology. The brown planthopper, a wing polymorphic insect exhibiting strong trade-offs in investment between migration and reproduction, is one of the most serious rice pests in Asia. In this study, we investigated the function of four genes in the insulin-signaling pathway known to couple nutrition with growth, PI3 Kinase (PI3K), PDK1, Akt (Protein Kinase B), and the forkhead gene FOXO. Using a combination of RNA interference and pharmacological inhibitor treatment, we show that all four genes contribute to tissue level regulation of wing polymorphic development in this insect. As predicted, silencing of the NlPI3K, NlAkt and NlPDK1 through dsRNA and with the pharmacological inhibitor Perifosine resulted in short-winged brown planthoppers, whereas knockdown of NlFOXO resulted in long-winged planthoppers. Morphometric analyses confirm that phenotypes from our manipulations mimic what would be found in nature, i.e., major parameters such as bristle number, wing area and body weight are not signif[...]



Revealing the Effects of the Herbal Pair of Euphorbia kansui and Glycyrrhiza on Hepatocellular Carcinoma Ascites with Integrating Network Target Analysis and Experimental Validation

2016

Although the herbal pair of Euphorbia kansui (GS) and Glycyrrhiza (GC) is one of the so-called "eighteen antagonistic medicaments" in Chinese medicinal literature, it is prescribed in a classic Traditional Chinese Medicine (TCM) formula Gansui-Banxia-Tang for cancerous ascites, suggesting that GS and GC may exhibit synergistic or antagonistic effects in different combination designs. Here, we modeled the effects of GS/GC combination with a target interaction network and clarified the associations between the network topologies involving the drug targets and the drug combination effects. Moreover, the "edge-betweenness" values, which is defined as the frequency with which edges are placed on the shortest paths between all pairs of modules in network, were calculated, and the ADRB1-PIK3CG interaction exhibited the greatest edge-betweenness value, suggesting its crucial role in connecting the other edges in the network. Because ADRB1 and PIK3CG were putative targets of GS and GC, respectively, and both had functional interactions with AVPR2 approved as known therapeutic target for ascites, we proposed that the ADRB1-PIK3CG-AVPR2 signal axis might be involved in the effects of the GS-GC combination on ascites. This proposal was further experimentally validated in a H22 hepatocellular carcinoma (HCC) ascites model. Collectively, this systems-level investigation integrated drug target prediction and network analysis to reveal the combination principles of the herbal pair of GS and GC. Experimental validation in an in vivo system provided convincing evidence that different combination designs of GS and GC might result in synergistic or antagonistic effect[...]



Methacryloxylethyl Cetyl Ammonium Chloride Induces DNA Damage and Apoptosis in Human Dental Pulp Cells via Generation of Oxidative Stress

2016

The polymerizable antibacterial monomer methacryloxylethyl cetyl ammonium chloride (DMAE-CB) has provided an effective strategy to combat dental caries. However, the application of such material raises the question about the biological safety and the question remains open. The mechanism of this toxic action, however, is not yet clearly understood. The present study aims at providing novel insight into the possible causal link between cellular oxidative stress and DNA damage, as well as apoptosis in human dental pulp cells exposed to DMAE-CB. The enhanced formation of reactive oxygen species and depletion of glutathione, as well as differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase in DMAE-CB-treated cells indicated oxidative stress. By using substances that can alter GSH synthesis, we found that GSH was the key component in the regulation of cell response towards oxidative stress induced by DMAE-CB. The increase in oxidative stress-sensitive 8-Oxo-2'-deoxyguanosine (8-OHdG) content, formation of γ-H2AX and cell cycle G1 phase arrest indicated that DNA damage occurred as a result of the interaction between DNA base and ROS beyond the capacities of antioxidant mechanisms in cells exposed to DMAE-CB. Such oxidative DNA damage thus triggers the activation of ataxia telangiectasia-mutated (ATM) signaling, the intrinsic apoptotic pathway, and destruction of mitochondrial morphology and function.




Dysregulation of the Low-Density Lipoprotein Receptor Pathway Is Involved in Lipid Disorder-Mediated Organ Injury

2016

The low-density lipoprotein receptor (LDLR) pathway is a negative feedback system that plays important roles in the regulation of plasma and intracellular cholesterol homeostasis. To maintain a cholesterol homeostasis, LDLR expression is tightly regulated by sterol regulatory element-binding protein-2 (SREBP-2) and SREBP cleavage-activating protein (SCAP) in transcriptional level and by proprotein convertase subtilisin/kexin type 9 (PCSK9) in posttranscriptional level. The dysregulation of LDLR expression results in abnormal lipid accumulation in cells and tissues, such as vascular smooth muscle cells, hepatic cells, renal mesangial cells, renal tubular cells and podocytes. It has been demonstrated that inflammation, renin-angiotensin system (RAS) activation, and hyperglycemia induce the disruption of LDLR pathway, which might contribute to lipid disorder-mediated organ injury (atherosclerosis, non-alcoholic fatty liver disease, kidney fibrosis, etc). The mammalian target of rapamycin (mTOR) pathway is a critical mediator in the disruption of LDLR pathway caused by pathogenic factors. The mTOR complex1 activation upregulates LDLR expression at the transcriptional and posttranscriptional levels, consequently resulting in lipid deposition. This paper mainly reviews the mechanisms for the dysregulation of LDLR pathway and its roles in lipid disorder-mediated organ injury under various pathogenic conditions. Understanding these mechanisms leading to the abnormality of LDLR expression contributes to find potential new drug targets in lipid disorder-mediated diseases.




Direct Reprogramming of Human Amniotic Fluid Stem Cells by OCT4 and Application in Repairing of Cerebral Ischemia Damage

2016

Amniotic fluid stem cells (AFSCs) are a type of fetal stem cell whose stemness encompasses both embryonic and adult stem cells, suggesting that they may be easily and efficiently reprogrammed into induced pluripotent stem cells (iPSCs). To further simplify the reprogramming process, the creation of AFSC-derived iPSCs using a single factor is desirable. Here we report the generation of one-factor human AFSC-iPSCs (AiPSCs) from human AFSCs by ectopic expression of the transcription factor OCT4. Just like human embryonic stem cells, AiPSCs exhibited similar epigenetic status, global gene expression profiles, teratoma formation and in vitro & in vivo pluripotency. Our results indicate that the OCT4 is necessary and sufficient to directly reprogram human AFSCs into pluripotent AiPSCs. Moreover, reflecting the similar memory characteristics of AFSCs and neural stem cells, we show that AiPSC membrane-derived vesicles (MVs) repair cerebral ischemia damage. We anticipate that the successful generation of one-factor AiPSCs will facilitate the creation of patient-specific pluripotent stem cells without the need for transgenic expression of oncogenes. Moreover, MVs from tissue-specific AiPSCs have potential in tissue repair, representing a novel application of iPSCs.




Upregulation of the Non-Coding RNA OTUB1-isoform 2 Contributes to Gastric Cancer Cell Proliferation and Invasion and Predicts Poor Gastric Cancer Prognosis

2016

Background: The deubiquitinase OTUB1 plays critical oncogenic roles and facilitates tumor progression in cancer. However, less is known regarding the aberrant expression, clinical significance and biological functions of the non-coding RNA OTUB1-isoform 2. We aimed to evaluate the OTUB1-isoform 2 levels in gastric cancer and their possible correlation with clinicopathologic features and patient survival to reveal its biological effects in gastric cancer progression.Methods: Total RNA extraction was performed on 156 gastric cancer case samples, and RT-qPCR was conducted. Chi-square test analysis was used to calculate the correlation between pathological parameters and the OTUB1-isoform 2 mRNA levels. Kaplan-Meier and Cox proportional hazards analyses were used to analyze the overall survival (OS) and disease-free survival (DFS) rates. Nuclear and cytoplasmic RNAs were isolated to detect the subcellular localization of OTUB1-isoform 2. We also assessed whether overexpression of OTUB1-isoform 2 influenced in vitro cell proliferation, cell cycle progression, tumor cell invasion and migration, as well as in vivo nude mouse xenograft and metastasis models.Results: The OTUB1-isoform 2 expression levels were higher in the gastric cancer samples than in the paratumorous gland samples. OTUB1-isoform 2 expression levels tightly correlated with tumor size, lymph node metastasis and TNM staging. Higher OTUB1-isoform 2 expression levels led to significantly poorer OS and DFS rates, and a multivariate analysis revealed that OTUB1-isoform 2 was an independent risk factor [...]



Mitochondrial Stat3, the Need for Design Thinking

2016

Stat3 has been studied extensively as a transcription factor, however the finding that Stat3 also localizes to mitochondria has opened a new area to discover non-classical functions. Here we review the current knowledge of mitochondrial Stat3 as a regulator of the electron transport chain (ETC) and its impact on mitochondrial production of ATP and ROS. We also describe recent findings identifying Stat3 as a regulator of mitochondrial Ca2+ homeostasis through its effect on the ETC. It is becoming evident that these non-classical functions of Stat3 can have a major impact on cancer progression, cardiovascular diseases, and inflammatory diseases. Therefore, mitochondrial Stat3 functions challenge the current design of therapies that solely target Stat3 as a transcription factor and suggest the need for “design thinking,” which leads to the development of novel strategies, to intervene the Stat3 pathway.




Glycogen Synthase Kinase 3β Influences Injury Following Cerebral Ischemia/Reperfusion in Rats

2016

Abnormal activation of GSK-3β is associated with psychiatric and neurodegenerative disorders. However, no study has examined the effect of GSK-3β on cerebral ischemia/reperfusion injury. We used oxygen-glucose deprivation/reoxygenation (OGD/R) and middle cerebral artery occlusion (MCAO) as models of ischemia/reperfusion in rats in vitro and in vivo. Our study showed that knockdown of GSK-3β with a GSK-3β siRNA virus improved injury and increased viability of neurons subjected to OGD/R. Levels of total Nrf2, nuclear Nrf2, and Nrf2 downstream proteins sulfiredoxin (Srx1) and thioredoxin (Trx1) increased after transfection with the GSK-3β siRNA virus. GSK-3β siRNA increased SOD activity and decreased MDA levels. Overexpression of GSK-3β with a pcDNA-GSK-3β virus showed opposite results. We also demonstrated that intracerebroventricular injection of GSK-3β siRNA in rats ameliorated neurological deficits, reduced brain infarct volume and water content, and reduced damage to cerebral cortical neurons after MCAO. Changes in total Nrf2, nuclear Nrf2, Srx1, Trx1, SOD, and MDA were similar to those observed in vitro. Our results show for the first time that GSK-3β can influence cerebral ischemia/reperfusion injury. The effects may be due to regulating the Nrf2/ARE pathway and decreasing oxidative stress. These results suggest a potential new drug target for clinical treatment of stroke.




Transcriptional Reactivation of OTX2, RX1 and SIX3 during Reprogramming Contributes to the Generation of RPE Cells from Human iPSCs

2016

Directed differentiation of human induced pluripotent stem cells (iPSCs) into retinal pigmented epithelium (RPE) holds great promise in cell replacement therapy for patients suffering from degenerative eye diseases, including age-related macular degeneration (AMD). In this study, we generated iPSCs from human dermal fibroblasts (HDFs) by electroporation with episomal plasmid vectors encoding OCT4, SOX2, KLF4, L-MYC together with p53 suppression. Intriguingly, cell reprogramming resulted in a metastable transcriptional activation and selective demethylation of neural and retinal specification-associated genes, such as OTX2, RX1 and SIX3. In contrast, RPE progenitor genes were transcriptionally silent in HDFs and descendant iPSCs. Overexpression of OCT4 and SOX2 directly stimulated the expression of OTX2, RX1 and SIX3 in HDFs and iPSCs. Luciferase and chromatin immunoprecipitation (ChIP) assays further identified an OCT4- and two SOX2-binding sites located in the proximal promoter of OTX2. Histone acetylation and methylation on the local promoter also participated in the reactivation of OTX2. The transcriptional conversion of RX1 and SIX3 genes partially attributed to DNA demethylation. Subsequently, iPSCs were induced into the RPE cells displaying the characteristics of polygonal shapes and pigments, and expressing typical RPE cell markers. Taken together, our results establish readily efficient and safe protocols to produce iPSCs and iPSC-derived RPE cells, and underline that[...]



Phylogenetic inference of calyptrates, with the first mitogenomes for Gasterophilinae (Diptera: Oestridae) and Paramacronychiinae (Diptera: Sarcophagidae)

2016

The complete mitogenome of the horse stomach bot fly Gasterophilus pecorum (Fabricius) and a near-complete mitogenome of Wohlfahrt's wound myiasis fly Wohlfahrtia magnifica (Schiner) were sequenced. The mitogenomes contain the typical 37 mitogenes found in metazoans, organized in the same order and orientation as in other cyclorrhaphan Diptera. Phylogenetic analyses of mitogenomes from 38 calyptrate taxa with and without two non-calyptrate outgroups were performed using Bayesian Inference and Maximum Likelihood. Three sub-analyses were performed on the concatenated data: (1) not partitioned; (2) partitioned by gene; (3) 3rd codon positions of protein-coding genes omitted. We estimated the contribution of each of the mitochondrial genes for phylogenetic analysis, as well as the effect of some popular methodologies on calyptrate phylogeny reconstruction. In the favoured trees, the Oestroidea are nested within the muscoid grade. Relationships at the family level within Oestroidea are (remaining Calliphoridae (Sarcophagidae (Oestridae, Pollenia + Tachinidae))). Our mito-phylogenetic reconstruction of the Calyptratae presents the most extensive taxon coverage so far, and the risk of long-branch attraction is reduced by an appropriate selection of outgroups. We find that in the Calyptratae the ND2, ND5, ND1, COIII, and COI genes are more phylogenetically informative compared with other mitochondrial protein-coding genes. Our study provides evidence that d[...]



Ebselen Is a Potential Anti-Osteoporosis Agent by Suppressing Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclast Differentiation In vitro and Lipopolysaccharide-Induced Inflammatory Bone Destruction In vivo

2016

Ebselen is a non-toxic seleno-organic drug with anti-inflammatory and antioxidant properties that is currently being examined in clinical trials to prevent and treat various diseases, including atherosclerosis, stroke, and cancer. However, no reports are available for verifying the pharmacological effects of ebselen on major metabolic bone diseases such as osteoporosis. In this study, we observed that ebselen suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in an osteoblast/osteoclast co-culture by regulating the ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin secreted by osteoblasts. In addition, ebselen treatment in the early stage of osteoclast differentiation inhibited RANKL-dependent osteoclastogenesis by decreasing the phosphorylation of IκB, PI3K, and Akt in early signaling pathways and by subsequently inducing c-Fos and nuclear factor of activated T-cells c1. Further, ebselen induced apoptosis of osteoclasts in the late stage of osteoclast differentiation. In addition, ebselen treatment suppressed filamentous actin ring formation and bone resorption activity of mature osteoclasts. Reflecting these in vitro effects, administration of ebselen recovered bone loss and its µ-CT parameters in lipopolysaccharide-mediated mouse model. Histological analysis confirmed that ebselen prevented trabecular bone matrix degradation an[...]



Hyperglycemia Differentially Affects Maternal and Fetal DNA Integrity and DNA Damage Response

2016

Objective: Investigate the DNA damage and its cellular response in blood samples from both mother and the umbilical cord of pregnancies complicated by hyperglycemia. Methods: A total of 144 subjects were divided into 4 groups: normoglycemia (ND; 46 cases), mild gestational hyperglycemia (MGH; 30 cases), gestational diabetes mellitus (GDM; 45 cases) and type-2 diabetes mellitus (DM2; 23 cases). Peripheral blood mononuclear cell (PBMC) isolation and/or leukocytes from whole maternal and umbilical cord blood were obtained from all groups at delivery. Nuclear and mitochondrial DNA damage were measured by gene-specific quantitative PCR, and the expression of mRNA and proteins involved in the base excision repair (BER) pathway were assessed by real-time qPCR and Western blot, respectively. Apoptosis was measured in vitro experiments by caspase 3/7 activity and ATP levels. Results: GDM and DM2 groups were characterized by an increase in oxidative stress biomarkers, an increase in nuclear and mitochondrial DNA damage, and decreased expression of mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1) involved in BER. The levels of hyperglycemia were associated with the in vitro apoptosis pathway. Blood levels of DNA damage in umbilical cord were similar among the groups. Newborns of diabetic mothers had increased expression of BER mRNA (APE1, POLβ and FEN1) and proteins (hOGG1, APE1, POLβ a[...]



Alternative Roles of STAT3 and MAPK Signaling Pathways in the MMPs Activation and Progression of Lung Injury Induced by Cigarette Smoke Exposure in ACE2 Knockout Mice

2016

Inflammation-mediated abnormalities in the renin-angiotensin system (RAS) and expression of matrix metalloproteinases (MMPs) are implicated in the pathogenesis of lung injury. Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue that displays antagonist effects on ACE/angiotensin II (Ang II) axis, could also play a protective role against lung diseases. However, the relationship between ACE2 and MMPs activation in lung injury is still largely unclear. The purpose of this study is to investigate whether MMPs activity could be affected by ACE2 and which ACE2 derived signaling pathways could be also involved via using a mouse model with lung injury induced by cigarette smoke (CS) exposure for 1 to 3 weeks. Wild-type (WT; C57BL/6) and ACE2 KO mice (ACE2-/-) were utilized to study CS-induced lung injury. Increases in the resting respiratory rate (RRR), pulmonary immunokines, leukocyte infiltration and bronchial hyperplasia were observed in the CS-exposed mice. Compared to WT mice, more serious physiopathological changes were found in ACE2-/- mice in the first week of CS exposure. CS exposure increased pulmonary ACE and ACE2 activities in WT mice, and significantly increased ACE in ACE2-/- mice. Furthermore, the activity of pulmonary MMPs was decreased in CS-exposed WT mice, whereas this activity was increased in ACE2-/- mice. CS exposure[...]