ALBERT

Description: Secreted phosphoprotein 24 kDa (Spp24) is an apatite- and BMP/TGF-β cytokine-binding phosphoprotein found in serum and many tissues, including bone. N-terminally intact degradation products ranging in size from 14 kDa to 23 kDa have been found in bone. The cleavage sites in Spp24 that produce these short forms have not been definitively identified, and the biological activities and mechanisms of action of Spp24 and its degradation products have not been fully elucidated. We found that the C-terminus of Spp24 is labile to proteolysis by furin, kallikrein, lactoferrin, and trypsin, indicating that both extracellular and intracellular proteolytic events could account for the generation of biologically-active Spp18, Spp16, and Spp14. We determined the effects of these truncation products on kinase-mediated signal transduction, gene expression, and osteoblastic differentiation in W-20-17 bone marrow stromal cells cultured in basal or pro-osteogenic media. After culturing for five days, all forms inhibited BMP-2-stimulated osteoblastic differentiation, assessed as induction of alkaline phosphatase activity, in basal, but not pro-osteogenic media. After 10 days, they also inhibited BMP-2-stimulated mineral deposition in pro-osteogenic media. Spp24 had no effect on Erk1/2 phosphorylation, but Spp18 stimulated short-term Erk1/2, MEK 1/2, and p38 phosphorylation. Pertussis toxin and a MEK1/2 inhibitor ablated Spp18-stimulated Erk 1/2 phosphorylation, indicating a role for G i proteins and MEK1/2 in the Spp18-stimulated Erk1/2 phosphorylation cascade. Truncation products, but not full-length Spp24, stimulated RUNX2, ATF4, and CSF1 transcription. This suggests that Spp24 truncation products have effects on osteoblastic differentiation mediated by kinase pathways that are independent of exogenous BMP/TGF-β cytokines. This article is protected by copyright. All rights reserved

Description: Similar to phosphorylation, transient conjugation of ubiquitin to target proteins (ubiquitination) mediated by the concerted action of ubiquitin ligases and de-ubiquitinating enzymes (DUBs) can affect substrate function. As obligate intracellular parasites, viruses rely on different cellular pathways for their own replication and the well conserved ubiquitin conjugating/de-conjugating system is not an exception. Viruses not only usurp the host proteins involved in the ubiquitination/de-ubiquitination process, but they also encode their own ubiquitin ligases and DUBs. Here we report that an N-terminal variant of the herpes simplex virus (HSV) type-1 large tegument protein VP1/2 (VP1/2 1–767 ), encompassing an active DUB domain (herpesvirus tegument ubiquitin specific protease, htUSP), and TSG101, a component of the endosomal sorting complex required for transport (ESCRT)-I, functionally interact. In particular, VP1/2 1–767 modulates TSG101 ubiquitination and influences its intracellular distribution. Given the role played by the ESCRT machinery in crucial steps of both cellular pathways and viral life cycle, the identification of TSG101 as a cellular target for the HSV-1 specific de-ubiquitinating enzyme contributes to the clarification of the still under debate function of viral encoded DUBs highly conserved throughout the Herpesviridae family. This article is protected by copyright. All rights reserved

Description: Sarcopenia and osteoporosis have recently been noted for their relationship with locomotive syndrome and increased number of older people. Sarcopenia is defined by decreased muscle mass and impaired muscle function, which may be associated with frailty. Several clinical data have indicated that increased muscle mass is related to increased bone mass and reduced fracture risk. Genetic, endocrine and mechanical factors as well as inflammatory and nutritional states concurrently affect muscle tissues and bone metabolism. Several genes, including myostatin and α-actinin 3, have been shown in a genome-wide association study (GWAS) to be associated with both sarcopenia and osteoporosis. Vitamin D, growth hormone and testosterone as well as pathological disorders, such as an excess in glucocorticoid and diabetes, affect both muscle and bone. Basic and clinical research of bone metabolism and muscle biology suggests that bone interacts with skeletal muscle via signaling from local and humoral factors in addition to their musculoskeletal function. However, the physiological and pathological mechanisms related to muscle and bone interactions remain unclear. We found that Tmem119 may play a critical role in the commitment of myoprogenitor cells to the osteoblast lineage. We also reported that osteoglycin and FAM5C might be muscle-derived humoral osteogenic factors. Other factors, including myostatin, osteonectin, insulin-like growth factor I, irisin and osteocalcin, may be associated with the interactions between muscle tissues and bone metabolism. This article is protected by copyright. All rights reserved

Description: The transition to a pathological phenotype such as Barrett's esophagus occurs via induction of resistance upon repeated contact with gastric refluxate in esophagus. This study examined the molecular changes within normal esophageal epithelial cells (EECs) under short-term acid loading and the role of these changes in defensive resistance against acidic cytotoxicity. After primary cultured EECs were exposed to pH 4-acidified medium (AM4), cell viability was determined by the MTT assay. Reactive oxygen species (ROS) and NAD(P)H oxidase (NOX) activity were measured. Activation of the mitogen-activated protein kinases (MAPKs) MEK/ERK1/2, p38 and JNK; phosphoinositol-3-kinase (PI3K)/Akt, and nuclear factor-kappa B (NF-κB) were detected by Western blot analysis or immunofluorescence staining. AM4 incubation induced intracellular ROS generation accompanied by increase in NOX activity, which was further increased by Na + /H + exchange-1 (NHE1)-dependent inhibition but was prevented by inhibition of NOX or mitochondria complex I. AM4 also induced phosphorylation of MEK/ERK1/2, p38 MAPK, PI3K/Akt, and nuclear translocation of NF-κB, and all these effects, except for p38 MAPK phosphorylation, were abolished by inhibition of ROS. ROS-dependent PI3K/Akt activation, which mediates NF-κB nuclear translocation, was inhibited by protein tyrosine kinase (PTK) inhibitors and NHE1-specific inhibitor. All inhibitors of NHE, ROS, PTK, PI3K or NF-κB further decreased AM4-induced cell viability. Acid loading in the presence of NHE1-dependent protection induced ROS generation by activating NOX and mitochondria complex I, which stimulated PTK/PI3K/Akt/NF-κB-dependent survival in EEC. Our data indicate that normal EEC initially respond to acid loading through intrinsic survival activation. This article is protected by copyright. All rights reserved

Description: The CRIPSR-Cas9 system consists of a site-specific, targetable DNA nuclease that holds great potential in gene editing and genome-wide screening applications. In order to apply the CRISPR-Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterised the rate of Cas9 off-target activity in typical Cas9 experiments in two human and one mouse cell lines. We analysed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ∼90 predicted off-target sites per gRNA. In a Cas9-based knock-out experiment, gRNAs induced detectable Cas9 cutting activity in all on-target sites and in only a few off-target sites genome-wide in human 293FT, human iPS cells and mouse ES cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on-target and off-target sites. In clonal Cas9 cutting analysis in mouse ES cells, bi-allelic Cas9 cutting was observed with low off-target activity. Our results show that off-target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off-target site. Off-target Cas9 activity can be minimized by selecting gRNAs with few off-target sites of near-complementarity. genesis 00:00–00, 2014. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The recent advances on ascidian pigment sensory organ development and function represent a fascinating platform to get insight on the basic programs of chordate eye formation. This review aims to summarize current knowledge, at the structural and molecular levels, on the two main building blocks of ascidian light sensory organs, i.e . pigment cells and photoreceptor cells. The unique features of these structures (e.g. simplicity and well characterized cell lineage) are indeed making it possible to dissect the developmental programs at single cell resolution and will soon provide a panel of molecular tools to be exploited for a deep developmental and comparative-evolutionary analysis. © 2014 Wiley Periodicals, Inc.

Description: Dynamic interactions with DNA allow replication protein A to direct single-stranded DNA-intermediates into different pathways for synthesis or repair. On pages 1156–1161 , Chen and Wold review recent discoveries that show that replication protein A (RPA), the major eukaryotic single-stranded DNA-binding protein, binds DNA dynamically and that this is important for correct processing of DNA intermediates. The cover shows a model of human RPA interacting with ssDNA based on the known structures of the domains of human RPA and the structure of Ustalago RPA bound to DNA. The three subunits of RPA are shown in blue (RPA1), red (RPA2), and green (RPA3) with ssDNA shown in black.

Description: The aim of this study was to investigate the involvement of exchange proteins directly activated by cyclic adenosine (ADO) monophosphate (EPAC) in 4-mer hyaluronan (HA) oligosaccharide-induced inflammatory response in mouse normal synovial fibroblasts (NSF). Treatment of NSF with 4-mer HA increased Toll-like receptor-4, TNF-alpha and IL-1beta mRNA expression and of the related proteins, as well as nuclear factor kappaB (NF-kB) activation. Addition to NSF, previously stimulated with 4-mer HA oligosaccharides, of ADO significantly reduced NF-kB activation, TNF-alpha and IL-1beta expression. The pre-treatment of NSF with cyclic ADO monophosphate and/or PKA and/or EPAC-specific inhibitors significantly inhibited the anti-inflammatory effect exerted by ADO. In particular, the EPAC inhibitor reduced the ADO effect to a major extent than the PKA inhibitor. These results mean that both PKA and EPAC pathways are involved in ADO-induced NF-kB inhibition although EPAC seems to be more involved than PKA. Copyright © 2014 John Wiley & Sons, Ltd.

Description: Temporally controlled induction of gene expression is a useful technique for analyzing gene function. To make such a technique possible in Ciona intestinalis embryos, we employed the cis -regulatory region of the heat-shock protein 70 (HSP70) gene Ci-HSPA1/6/7-like for heat-inducible gene expression in C. intestinalis embryos. We showed that Ci-HSPA1/6/7-like becomes heat shock-inducible by the 32-cell stage during embryogenesis. The 5'-upstream region of Ci-HSPA1/6/7-like , which contains heat-shock elements indispensable for heat-inducible gene expression, induces the heat shock-dependent expression of a reporter gene in the whole embryo from the 32-cell to the middle gastrula stages and in progressively restricted areas of embryos in subsequent stages. We assessed the effects of heat-shock treatments in different conditions on the normality of embryos and induction of transgene expression. We evaluated the usefulness of this technique through overexpression experiments on the well-characterized, developmentally relevant gene, Ci-Bra , and showed that this technique is applicable for inferring the gene function in C. intestinalis . © 2014 Wiley Periodicals, Inc.

Description: Osteolytic bone diseases are characterized by excessive osteoclast formation and activation. Protein kinase C (PKC)-dependent pathways regulate cell growth, differentiation and apoptosis in many cellular systems, and have been implicated in cancer development and osteoclast formation. A number of PKC inhibitors with anti-cancer properties have been developed, but whether they might also influence osteolysis (a common complication of bone invading cancers) is unclear. We studied the effects of the PKC inhibitor compound, GF109203X on osteoclast formation and activity, processes driven by receptor activator of NFκB ligand (RANKL). We found that GF109203X strongly and dose dependently suppresses osteoclastogenesis and osteoclast activity in RANKL-treated primary mouse bone marrow cells. Consistent with this GF109203X reduced expression of key osteoclastic genes, including cathepsin K, calcitonin receptor, tartrate resistant acid phosphatase (TRAP) and the proton pump subunit V-ATPase-d2 in RANKL-treated primary mouse bone marrow cells. Expression of these proteins is dependent upon RANKL-induced NF-κB and NFAT transcription factor actions; both were reduced in osteoclast progenitor populations by GF109203X treatment, notably NFATc1 levels. Furthermore, we showed that GF109203X inhibits RANKL-induced calcium oscillation. Together, this study shows GF109203X may block osteoclast functions, suggesting that pharmacological blockade of PKC-dependent pathways has therapeutic potential in osteolytic diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in Δψm, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Tumor cells display different bioenergetic profiles when compared to normal cells. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin A in breast cancer cells representing different stages of aggressiveness and metabolic profile. When testing the effect of NaB and TSA on viability of cells, it was shown that non-tumorigenic MCF-10A cells were less affected by increasing doses of the drugs than the tumorigenic, hormone dependent, tightly cohesive MCF-7, T-47D and the highly metastatic triple-negative MDA-MB 231 cells. T-47D cells were the most sensitive to treatment with both, NaB and TSA. Experiments measuring anchorage- independent growth of tumor cells showed that MCF-7, T-47D, and MDA-MB-231 cells were equally sensitive to the treatment with NaB. The NaB induced an attenuation of glycolysis, reflected by a decrease in lactate release in MCF-7 and T47D lines. Pyruvate kinase activity was significantly enhanced by NaB in MDA-MB-231 cells only. In contrast, the inhibitor enhanced lactate dehydrogenase activity specifically in T-47 D cells. Glucose-6-phosphate dehydrogenase activity was shown to be differentially modulated by NaB in the cell lines investigated: the enzyme was inhibited in MCF-7 cells, whereas in T-47D and MDA-MB-231 cells, G6PDH was activated. NaB and TSA were able to significantly increase the oxygen consumption by MDA-MB-231 and T-47D cells. Collectively the results show that epigenetic changes associated to acetylation of proteins in general affect the energy metabolism in all cancer cell lines and that mitochondria may occupy a central role in metastasis. This article is protected by copyright. All rights reserved

Description: Genomic and trascriptomic profiling has recently contributed details to the characterization of luminal B breast cancer. We explored the contribution of anthropometric, metabolic and molecular determinants to the multifaceted heterogeneity of this breast cancer subtype, with a specific focus on the association between body mass index (BMI), pre-treatment fasting glucose, hormone receptors and expression of human epidermal growth factor receptor 2 (HER2). Extensively annotated specimens were obtained from 154 women with luminal B breast cancer diagnosed at two Italian comprehensive cancer centres. Participants' characteristics were descriptively analyzed overall and by HER2 status (positive vs negative). BMI (〈25 vs ≥25), pre-treatment fasting glucose (〈median value of 94 mg/dl vs ≥94) and percentage of hormone receptors were tested for association with HER2 expression in regression models. In univariate models, BMI, fasting glucose and, at a lesser extent, percentage of estrogen receptors (ER) were significantly and inversely associated with HER2 expression (OR: 0.32, 95%CI: 0.16-0.66; 0.43, 0.23-0.0.82; 0.96, 0.94-0.97, respectively). The multivariate models confirmed the protective role of BMI and ER on HER2 expression, with luminal B HER2 positive patients being significantly less frequent among women within the highest category of BMI and percentage expression of ER compared with their counterparts (OR: 0.22, 95%CI: 0.09-0.53; 0.95, 0.93-0.97). In conclusions, BMI and percentage of ER representation are inversely associated with HER2 expression in luminal B breast cancers. Upon confirmatory findings, this might help identify patient subgroups who may best benefit from the use of interventions targeting insulin resistance in well depicted breast cancer scenarios. This article is protected by copyright. All rights reserved

Description: Histone deacetylases (HDACs) play a crucial role in the regulation of gene expression through remodeling of chromatin structures. However, the molecular mechanisms involved in this event remain unknown. In this study, we sought to examine whether HDAC inhibition-mediated protective effects involved HDAC4 sumoylation, degradation, and the proteasome pathway. Isolated neonatal mouse ventricular myocytes (NMVM) and H9c2 cardiomyoblasts were subjected to 48 hours of hypoxia (H) (1% O 2 ) and 2 hours of reoxygenation (R). Treatment of cardiomyocytes with trichostatin A (TSA) attenuated H/R-elicited injury, as indicated by a reduction of lactate dehydrogenase (LDH) leakage, an increase in cell viability, and decrease in apoptotic positive cardiomyocytes. MG132, a potent proteasome pathway inhibitor, abrogated TSA-induced protective effects, which was associated with the accumulation of ubiquitinated HDAC4. NMVM transduced with adenoviral HDAC4 led to an exaggeration of H/R-induced injury. TSA treatment resulted in a decrease in HDAC4 in cardiomyocytes infected with adenoviral HDAC4, and HDAC4-induced injury was attenuated by TSA. HDAC inhibition resulted in a significant reduction in reactive oxygen species (ROS) in cardiomyoblasts exposed to H/R, which was attenuated by blockade of the proteasome pathway. Cardiomyoblasts carrying wild type and sumoylation mutation (K559R) were established to examine effects of HDAC4 sumoylation and ubiquitination on H/R injury. Disruption of HDAC4 sumoylation brought about HDAC4 accumulation and impairment of HDAC4 ubiquitination in association with enhanced susceptibility of cardiomyoblasts to H/R. Taken together, these results demonstrated that HDAC inhibition stimulates proteasome dependent degradation of HDAC4, which is associated with HDAC4 sumoylation to induce these protective effects. This article is protected by copyright. All rights reserved

Description: Endothelial cells play a major role in the initiation and perpetuation of the inflammatory process in health and disease, including their pivotal role in leukocyte recruitment. The role of pro-inflammatory transcription factors in this process has been well-described, including NF-κB. However, much less is known regarding transcription factors that play an anti-inflammatory role in endothelial cells. Myocyte enhancer factor 2 C (MEF2C) is a transcription factor known to regulate angiogenesis in endothelial cells. Here, we report that MEF2C plays a critical function as an inhibitor of endothelial cell inflammation. Tumor necrosis factor (TNF)-α inhibited MEF2C expression in endothelial cells. Knockdown of MEF2C in endothelial cells resulted in the upregulation of pro-inflammatory molecules and stimulated leukocyte adhesion to endothelial cells. MEF2C knockdown also resulted in NF-κB activation in endothelial cells. Conversely, MEF2C overexpression by adenovirus significantly repressed TNF-α induction of pro-inflammatory molecules, activation of NF-κB, and leukocyte adhesion to endothelial cells. This inhibition of leukocyte adhesion by MEF2C was partially mediated by induction of KLF2. In mice, lipopolysaccharide (LPS)-induced leukocyte adhesion to the retinal vasculature was significantly increased by endothelial cell-specific ablation of MEF2C. Taken together, these results demonstrate that MEF2C is a novel negative regulator of inflammation in endothelial cells and may represent a therapeutic target for vascular inflammation. This article is protected by copyright. All rights reserved

Description: Ramathal et al. have employed an elegant xenotransplantation technique to study the fate of human induced pluripotent stem cells (hiPSCs) from fertile males and from males carrying Y chromosome deletions of the azoospermia factor ( AZF ) region. When placed in a mouse testis niche, hiPSCs from fertile males differentiate into germ cell-like cells (GCLCs). Highlighting the crucial role of cell autonomous factors in male sterility, hiPSCs derived from azoospermic males prove to be less successful under similar circumstances. Their studies argue that the agametic “Sertoli cell only” phenotype of two of the AZF deletions likely arises from a defect in the maintenance of germline stem cells (GSCs) rather than from a defect in their specification. These observations underscore the importance of the dialogue between the somatic niche and its inhabitant stem cells, and open up interesting questions concerning the functioning of the somatic niche and how it communicates to the GSCs. In Ramathal et al., human induced pluripotent stem cells (hiPSCs) from wild type and azoospermic men adopt germ cell fate in vitro. Upon xenotransplantation into mouse seminiferous tubules, these hiPSCs acquired germ cell-like (GCL) fate. Remarkably, samples from azoospermic males were less efficient in both contexts.

Description: Human adult stem cells, which are capable of self-renewal and differentiation into other cell types, can be isolated from various tissues. There are no ethical and rejection problems as in the case of embryonic stem cells, so they are a promising source for cell therapy. The human body contains a great amount of adipose tissue that contains high numbers of mesenchymal stem cells. Human adipose-derived stem cells (hADSCs) could be easily induced to form neuron-like cells, and because of its availability and abundance, we can use it for clinical cell therapy. On the other hand, T 3 hormone as a known neurotropic factor has important impressions on the nervous system. The aim of this study was to explore the effects of T 3 treatment on neural differentiation of hADSCs. ADSCs were harvested from human adipose tissue, after neurosphere formation, and during final differentiation, treatment with T 3 was performed. Immunocytochemistry, real-time RT-PCR, Western blotting techniques were used for detection of nestin, MAP2, and GFAP markers in order to confirm the effects of T 3 on neural differentiation of hADSCs. Our results showed an increase in the number of glial cells but reduction in neuronal cells number fallowing T 3 treatment. Copyright © 2014 John Wiley & Sons, Ltd.

Description: Palmitate induces insulin resistance and apoptosis in insulin target tissues. Rosiglitazone (RSG), a peroxisome proliferator-activated receptor γ (PPAR γ ) agonist, can activate both pro-apoptotic and anti-apoptotic pathways in different cells; however, its effect on palmitate-induced apoptosis in skeletal muscle cells remains to be elucidated. After differentiation of C2C12 cells, myotubes were treated with palmitate, RSG and GW9662 (PPAR γ antagonist). MTT and terminal deoxynucleotide transferase dUTP nick end labelling (TUNEL) assays and caspase-3 activity were used to investigate the apoptosis. To study the underlying mechanism, glucose uptake, gene expression and protein levels were evaluated. A total of 0.75 mM palmitate reduced cell viability by 43% and increased TUNEL-positive cells and caspase-3 activity by 15-fold and 6.6-fold, respectively. RSG (10 μM) could markedly decrease the level of TUNEL-positive cells and caspase-3 activity in palmitate-treated cells. The protective effect of RSG on apoptosis was abrogated by GW9662. To investigate the molecular mechanism of this effect, gene expression and protein level of protein tyrosine phosphatase 1B (PTP1B) were evaluated. Palmitate and RSG individually increased the expression and protein level of PTP1B, whereas combined treatment (palmitate and RSG) were able to further increase the expression of PTP1B in C2C12 cells. We also evaluated the effect of RSG on palmitate-induced insulin resistance in muscle cells. RSG could significantly improve glucose uptake by 0.4-fold in myotubes treated with palmitate. Moreover, RSG could restore the phosphorylation of Akt in palmitate-treated cells. These data suggest that RSG protects skeletal muscle cells against palmitate-induced apoptosis and this effect appears to be mediated via the PPAR γ -dependent and PTP1B-independent mechanisms. Copyright © 2014 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDY Saturated free fatty acids (FFAs), such as palmitate, have been shown to induce cellular apoptosis. Strategies for preventing the cytotoxic effect of palmitate are useful in reduction of diabetes complications. In this study, we introduced RSG as an agent that protects skeletal muscle cells against palmitate-induced apoptosis and insulin resistance. It appears that RSG protects skeletal muscle cells against palmitate-induced apoptosis via the PPARγ-dependent and PTP1B-independent mechanisms. Given the role of FFAs in skeletal muscle apoptosis, these findings support the idea that RSG can ameliorate diabetes complications such as skeletal muscle loss.

Description: The expression of Hes1 is increased following myocardial infarct and other ischemic cardiomyopathies, but the role of Hes1 in cardioprotection provided by ischemic postconditioning (IPost) remains unclear. In this study, we used gain and loss of function approaches to investigate the role of Hes1 in cardioprotection during IPost. Primary cardiac myocytes exposed to ischemia reperfusion injury (IRI) and IPost were used as the experimental model. The results showed that Hes1 expression was increased during myocardial IPost, and Hes1 promoted the viability while inhibited the apoptosis of cardiomyocytes. Moreover, Hes1 inhibited the opening of mitochondrial permeability transition pore (mPTP) and the generation of reactive oxygen species in primary cardiac myocytes exposed to IRI. Mechanistically, we found that Hes1-mediated cardioprotection was related to the downregulation of phosphatase and tensin homolog and the activation of phosphatidylinositol 3-kinase/Akt and signal transducer and activator of transcription 3 signalling. These data demonstrate that Hes1 is upregulated and mediates cardioprotection provided by IPost and suggest that Hes1 is a potential new target for the treatment of ischemic cardiomyopathy. Copyright © 2014 John Wiley & Sons, Ltd.

Description: The aim of this study was to study RAS-siRNA blocking RAS pathway and suppressing cell growth in human oesophageal squamous cell carcinoma in nude mice. The methods in this study was to construct RAS-siRNA expression vector, establish 40 oesophageal squamous cell carcinoma xenograft animal models and divided them into five groups: control group, siRNA control group, RAS-siRNA group, paclitaxel group and RAS-siRNA and paclitaxel group. We observed tumour growth in nude mice, studied histology by HE staining, tumour growth inhibition by TUNEL assay and detected the RAS, MAPK and cyclin D1 protein expression by immunohistochemistry and western blot. We have obtained the following results: (i) successfully established animal models; (ii) nude mice in each group after treatment inhibited tumour volume was significantly reduced compared with the control group ( p  〈 0.05); (iii) compared with the control group, the number of apoptotic cells were significantly increased in the siRNA control group and the RAS-siRNA group, and the number of apoptosis cells in the paclitaxel and RAS-siRNA group is significantly most than the paclitaxel group and RAS-siRNA group ( p  〈 0.05); and (iv) after treatment, RAS, MAPK and cyclin D1 expression in five groups was decreasing gradually. After adding paclitaxel, the protein expression in the paclitaxel and RAS-siRNA group was significantly lower than that of paclitaxel group, negative control and paclitaxel group ( p  〈 0.05). We therefore conclude that RAS-siRNA can block the RAS signal transduction pathway, reduce the activity of tumour cells, arrest tumour cell cycle, promote apoptosis, inhibit cell proliferation and increase tumour cell sensitivity to chemotherapeutic drugs. Copyright © 2014 John Wiley & Sons, Ltd.

Description: There is increasing evidence suggesting that oxidative stress plays an important role in the development of many chronic and degenerative conditions such as diabetic encephalopathy and depression. Considering that diabetic rats and mice present higher depressive-like behaviour when submitted to the forced swimming test and that treatment with insulin and/or clonazepam is able to reverse the behavioural changes of the diabetic rats, the present work investigated the antioxidant status, specifically total antioxidant reactivity and antioxidant potential of insulin and clonazepam, as well as the effect of this drugs upon protein oxidative damage and reactive species formation in cortex, hippocampus and striatum from diabetic rats submitted to forced swimming test. It was verified that longer immobility time in diabetic rats and insulin plus clonazepam treatment reversed this depressive-like behaviour. Moreover, data obtained in this study allowed to demonstrate through different parameters such as protein carbonyl content, 2′7′-dichlorofluorescein oxidation, catalase, superoxide dismutase, glutathione peroxidase assay, total radical-trapping antioxidant potential and total antioxidant reactivity that there is oxidative stress in cortex, hippocampus and striatum from diabetic rats under depressive-like behaviour and highlight the insulin and/or clonazepam effect in these different brain areas, restoring antioxidant status and protein damage. Copyright © 2014 John Wiley & Sons, Ltd.

Description: Propofol is widely used in paediatric anaesthesia and intensive care unit because of its essentially short-acting anaesthetic effect. Recent data have shown that propofol induced neurotoxicity in developing brain. However, the mechanisms are not extremely clear. To gain a better insight into the toxic effects of propofol on hippocampal neurons, we treated cells at the days in vitro 7 (DIV 7), which were prepared from Sprague–Dawley embryos at the 18th day of gestation, with propofol (0.1–1000 μM) for 3 h. A significant decrease in neuronal proliferation and a remarkable increase in neuroapoptosis were observed in DIV 7 hippocampal neurons as measured by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay and apoptosis assay respectively. Moreover, propofol treatment decreased the nuclear factor kappaB (NF-κB) p65 expression, which was accompanied by a reduction in B-cell lymphoma 2 (Bcl-2) mRNA and protein levels, increased caspase-3 mRNA and activation of caspase-3 protein. These results indicated that downregulation of NF-κB p65 and Bcl-2 were involved in the potential mechanisms of propofol-induced neurotoxicity. This likely led to the caspase-3 activation, triggered apoptosis and inhibited the neuronal growth and proliferation that we have observed in our in vitro systems. Copyright © 2014 John Wiley & Sons, Ltd.

Description: Gene transcription is strictly controlled by the interplay of regulatory events at gene promoters and gene-distal regulatory elements called enhancers. Despite extensive studies of enhancers, we still have a very limited understanding of their mechanisms of action and their restricted spatio-temporal activities. A better understanding would ultimately lead to fundamental insights into the control of gene transcription and the action of regulatory genetic variants involved in disease. Here, I review and discuss pros and cons of state-of-the-art genomics methods to localize and infer the activity of enhancers. Among the different approaches, profiling of enhancer RNAs yields the highest specificity and may be superior in detecting in vivo activity. I discuss their apparent similarities to promoters, which challenge the established view of enhancers and promoters as distinct entities, and present a unifying model of regulatory elements in transcriptional regulation, in which activity, transcriptional output and regulatory function is context specific. Profiling of enhancer RNAs (eRNAs) likely represents a paradigm shift in enhancer genomics, yielding high specificity in the localization of active enhancers. The similarities between enhancers and promoters are discussed and their established distinctions are deconstructed. Based on this, a unified model of regulatory elements in transcriptional regulation is presented.

Description: Analyses of the international human genome sequencing results in 2004 converged to a consensual number of ~20 000 protein-coding genes, spanning over 〈2% of the total genomic sequence. Therefore, the developmental and physiological complexity of human beings remains unaccounted if viewed only in terms of the number of protein-coding genes; the epigenetic influences involving chromatin remodelling and RNA interference and alternative precursor messenger RNA splicing of functional protein-coding transcripts as well as post-translational modifications of proteins increase the diversity and the functionality of the proteome and likely explain the increased complexity. In addition, there has been an explosion of research addressing possible functional roles for the other 98% of the human genome that does not encode proteins. In fact, 〉90% of the human genome is likely to be transcribed yielding a complex network of overlapping transcripts that include tens of thousands of long RNAs with little or no protein forming capacity; they are collectively called non-coding RNA. This review highlights the fundamental concepts of biological roles of non-coding RNA and their importance in regulation of cellular physiology under disease conditions like cancer. Copyright © 2014 John Wiley & Sons, Ltd.

Description: Since the discovery of brain asymmetry in a wide range of vertebrate species, it has become possible to study development and expression of lateralized behavior accurately in well-controlled experiments. Several species have emerged as useful models for investigating aspects of lateralization. Discussed here are: (1) the influence of exposure to light during embryonic development on lateralization, (2) effects of steroid hormones on lateralization, (3) developmental changes in which hemisphere is controlling behavior, and (4) asymmetry in memory formation and recall. The findings have bearing on understanding the development of hemispheric specialization in humans and are likely to provide insight into dysfunctional behavior associated with weak or absent lateralization and impaired interhemispheric communication (e.g., autism, schizophrenia, dyslexia). This review features research on chicks, pigeons and zebrafish, with the addition of some recent evidence of lateralization in bees. Discoveries made using these species have highlighted the interaction between experience, hormones and genetic factors during development, and have provided some of the first clear evidence of the advantage of having a lateralized brain. © 2014 Wiley Periodicals, Inc.

Description: Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome, which spontaneously resolves within several weeks or months after birth, may represent a very special form of leukemia arising in the fetal liver (FL). To explore the role of the fetal hematopoietic microenvironment in the pathogenesis of TAM, we examined the in vitro influences of stromal cells of human FL and fetal bone marrow (FBM) on the growth of TAM blasts. Both FL and FBM stromal cells expressed mesenchymal cell antigens (vimentin, α-smooth muscle actin, CD146 and nestin), being consistent with perivascular cells/mesenchymal stem cells that support hematopoietic stem cells. In addition, a small fraction of the FL stromal cells expressed an epithelial marker, cytokeratin 8, indicating that they could be cells in epithelial-mesenchymal transition (EMT). In the coculture system, stromal cells of the FL, but not FBM, potently supported the growth of TAM blast progenitors, mainly through humoral factors. High concentrations of hematopoietic growth factors were detected in culture supernatants of the FL stromal cells and a neutralizing antibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) almost completely inhibited the growth-supportive activity of the culture supernatants. These results indicate that FL stromal cells with unique characteristics of EMT cells provide a pivotal hematopoietic microenvironment for TAM blasts and that GM-CSF produced by FL stromal cells may play an important role in the pathogenesis of TAM.

Description: Because of the biological relevance of thiols and sulfides such as cysteine, homocysteine, glutathione and hydrogen sulfide, their detection has attracted a great deal of research interest. Fluorescent probes are emerging as a new strategy for thiol and hydrogen sulfide analysis due to their high sensitivity, low cost, and ability to detect and image thiols in biological samples. In this short review we have summarized recent advances in the development of thiol and hydrogen sulfide reactive fluorescent probes. These probes are compared and contrasted with regard to their designing strategies, mechanisms, photophysical properties, and/or reaction kinetics. Biological applications of these probes are also discussed. J. Cell. Biochem. 9999: XX–XX, 2014. © 2013 Wiley Periodicals, Inc.

Description: Liraglutide, a modified form of glucagon-like peptide-1 (GLP-1), has been found to improve beta cell function in type 2 diabetes (T2DM). However, the effect of liraglutide on beta cell function under lipotoxic stress and the underlying molecular mechanisms remain unclear. In the present study, we investigated the role of PI3K/Akt/FoxO1 signaling in liraglutide-involved beta cell protection in high free fatty acids (FFAs) condition. The apoptosis, proliferation, and insulin secretion capability of MIN6 cells and islets from C57BL/6J mice were evaluated when exposed to FFAs with/without liraglutide. The expression of effectors involved in PI3K/Akt/FoxO1signalling pathway was detected by real-time PCR and western blotting in MIN6 cells and islets from C57BL/6J mice. Liraglutide substantially inhibited the lipoapoptosis and improved the proliferation and insulin secretion of beta cells in high FFAs condition. Western blot revealed that the phosphorylation of Akt and FoxO1 was markedly decreased under lipid stress but was elevated when treated with liraglutide. Moreover, FFAs could up-regulate the expression levels of p27, Bax, Cidea but down-regulate the expression levels of Pdx-1, MafA, and NeuroD in beta cells, which was canceled by the addition of liraglutide. Moreover, LY294002, a PI3K inhibitor, could significantly abrogate all the protective actions of liraglutide against lipotoxicity. We concluded that liraglutide markedly improved beta cell function under lipid stress and that the protective action of liraglutide was mediated by activation of PI3K/Akt, which resulted in inactivation of FoxO1 along with the down-regulation of p27, Bax, Cidea and up-regulation of Pdx-1, MafA, and NeuroD expressions.

Description: The protective potential of chelators, i.e. N-acetyl cysteine (0.6 mg /kg, intraperitoneally) and dithiothreitol (15.4 mg kg −1 , intraperitoneally) with selenium (0.5 mg kg −1 , pre-oral) were evaluated individually and in combination against methylmercury-induced biochemical alterations and oxidative stress consequences. Forty-two male Sprague–Dawley rats were exposed with methylmercury (1.5 mg kg −1 , pre-oral) daily for 21 days followed by different treatments for five consecutive days. Administration of methylmercury caused significant enhancement in the release of transaminases, alkaline phosphatases and lactate dehydrogenases in serum. A significant increased was observed in lipid peroxidation level with a concomitant decreased in glutathione content after methylmercury exposure in liver, kidney and brain. Hepatic microsomal drug metabolizing enzymes (aniline hydroxylase and amidopyrine N-demethylase) of cytochrome p4502E 1 showed sharp depletion after methylmercury exposure. Alterations in histological changes in liver, kidney and brain were also noted in methylmercury administered group. All treated groups showed recovery pattern, but the combined treatments with N-acetyl cysteine and dithiothreitol in combination with selenium were more effective than that with either alone treatments in recovering blood biochemical changes after methylmercury toxicity. In conclusion, the results demonstrated that combination therapy may recover all blood biochemical alterations and offer maximum protection against methylmercury-induced toxicity. Copyright © 2014 John Wiley & Sons, Ltd.

Description: Comparing structural and evolutionary dynamics of proteins . On pages 209–218 Marsh and Teichmann discuss parallels between protein dynamics and evolution, and how we can gain insights into function from these studies. Buried residues, for example, are usually more evolutionarily conserved and evolve less quickly than exposed residues. These residues generally form more intramolecular contacts, and mutating them would therefore more likely lead to a destabilization of the protein. Having more intramolecular contacts on the other hand also means that these residues are less flexible to move around. In contrast, dynamic parts of a protein in real time are also dynamic over evolutionary time and evolve more quickly. The cover shows the buried (blue) and surface (red) residues for the receiver domain of sensor histidine kinase CKI1 from Arabidopsis .

Description: Trehalose-6-phosphate phosphatase (TPP) catalyzes the final step in the biosynthesis of the anti-stress sugar trehalose. An 82 kDa TPP enzyme was isolated from Candida utilis with 61% yield and 43-fold purification. The protein sequence, determined by N-terminal sequencing and MALDI-TOF analysis, showed significant homology with known TPP sequences from related organisms. The full length gene sequence of TPP of Candida utilis was identified using rapid amplification of cDNA ends - PCR reaction (RACE-PCR). The gene was cloned and expressed in E. coli BL21. Recombinant TPP enzyme was isolated using affinity chromatography. CD spectroscopy and steady state fluorescence revealed that the structural and conformational aspects were identical in both native and recombinant forms. The biochemical properties of the two forms were also similar. K m was determined to be ∼0.8 mM. Optimum temperature and pH was found to be 30 °C and 8.5 respectively. Activity was dependent on the presence of divalent cations and inhibited by metal chelators. Methylation mediated regulation of TPP enzyme and its effect on the overall survival of the organism under stress were investigated. The results indicated that enhancement of TPP activity by methylation at the Cysteine residues increased resistance of Candida cells against thermal stress. This work involves extensive investigations towards understanding the physico-chemical properties of the first TPP enzyme from any yeast strain. The mechanism by which methylation regulates its activity has also been studied. A correlation between regulation of trehalose synthesis and survivability of the organism under thermal stress was established. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: The microvascular network of the microcirculation works in tight communication with surrounding tissues to control blood supply and exchange of solutes. In cerebral circulation, microvascular endothelial cells constitute a selective permeability barrier that controls the environment of parenchymal brain tissue, which is known as the blood-brain barrier (BBB). Connexin- and pannexin-formed channels (gap junctions and hemichannels) play a central role in the coordination of endothelial and smooth muscle cell function and connexin-mediated signaling in endothelial cells is essential in the regulation of BBB permeability. Likewise, gap junction communication between astrocyte-end feet also contributes to maintain the BBB integrity, but the participation of hemichannels in this process cannot be discarded. Sympathetic and sensory perivascular nerves are also involved in the control and coordination of vascular function through the release of vasoconstrictor or vasodilator signals and by the regulation of gap junction communication in the vessel wall. Conversely, ATP release through pannexin-1-formed channels mediates the α1-adrenergic signaling. Furthermore, here we show that capsaicin-induced CGRP release from mesenteric perivascular sensory nerves induces pannexin-1-formed channel opening, which in turn leads to reduction of pannexin-1 and endothelial nitric oxide synthase (eNOS) expression along the time. Interestingly, blockade of CGRP receptors with CGRP 8–37 increased eNOS expression by ∼5 fold, suggesting that capsaicin-sensitive sensory nerves are involved in the control of key signaling proteins for vascular function. In this review, we discuss the importance of connexin-based channels in the control of BBB integrity and the functional interaction of vascular connexins and pannexins with the peripheral nervous system.

Description: We previously established a role for cancer-associated fibroblasts (CAFs) in enhancing the self-renewal and differentiation potentials of putative prostate cancer stem cells (CSCs). Our published work focused on androgen-dependent prostate cancer (ADPC) using the conditional Pten deletion mouse model. Employing the same model, we now describe the interaction of CAF and CSC in castration-resistant prostate cancer (CRPC). CAF isolated from ADPC (ADPCAF) and from CRPC (CRPCAF) were compared in terms of their ability to support organoid formation and tumor initiation by CSC from CRPC (CRPCSC) in vitro and in vivo. CRPCSC formed spheroids in vitro and well-differentiated glandular structures under the renal capsules of recipient mice in vivo more effectively in the presence of CRPCAF compared to ADPCAF. Furthermore, whereas CSC with CAF from ADPC formed mostly well-differentiated tumors in our previous study, we now show that CRPCSC, when combined with CRPCAF (but not ADPCAF) can form aggressive, poorly differentiated tumors. The potential of CRPCAF to support organoid/tumor formation by CRPCSC remained greater even when compared to 10-fold more ADPCAF, suggesting that paracrine factors produced specifically by CRPCAF preferentially potentiate the stemness and tumorigenic properties of the corresponding CSC. This apparently unique property of CRPCAF was notable when the CAF and CSC were grafted in either intact or castrated recipient mice. In both environments, CRPCAF induced in the epithelial compartment higher proliferative activity compared to ADPCAF, indicated by a higher Ki67 index. Factors released by CRPCAF to regulate CRPCSC may be targeted to develop novel therapeutic approaches to manage prostate cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Tat transactivating activity regulated by NAD + -dependent histone deacetylase sirtuin1 (SIRT1) connects HIV transcription with the metabolic state of the cell. Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the mammalian NAD + biosynthesis. Nampt, SIRT1 and AMPK were involved in inhibiting HIV-1 transactivation through redox-regulated pathway. Tanshinone IIA is a main lipid-soluble monomer derivative from the root of Salvia miltiorrhiza (Danshen) and tanshinone ⅡA possess a variety of biological activities through redox signaling pathway. Here we investigated the effect of tanshinone ⅡA on Tat-induced HIV-1 transactivation and the redox signaling pathway involved in it. As the results were shown, tanshinone ⅡA reversed Tat-induced reactive oxygen species (ROS) production and down-regulation of glutathione (GSH) levels in TZM-bl cells through up-regulation of Nrf2 expression. Tanshinone ⅡA reversed Tat-induced inhibition of SIRT1 activity but not SIRT1 protein expression. Tanshinone ⅡA reversed Tat-induced inhibition of Nampt protein expression and depletion of NAD + levels in TZM-bl cells in a dose-dependent manner. Tanshinone ⅡA-evoked Nampt expression was mediated by AMPK signaling pathway. Tanshinone ⅡA inhibited Tat-induced HIV-1 LTR transactivation dependent on AMPK-Nampt pathway. Collectively, our data provide new insights into understanding of the molecular mechanisms of tanshinone ⅡA inhibited Tat-regulated transcription, suggesting that targeting AMPK/Nampt/SIRT1 pathway could serve as new anti-HIV-1 agents.

Description: Osteoblast differentiation is tightly regulated by several factors including microRNAs (miRNAs). In this paper we report that pre-mir-15b is highly expressed in differentiated osteoblasts. The functional role of miR-15b in osteoblast differentiation was determined using miR-15b mimic/inhibitor and the expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP), type I collagen genes was decreased by miR-15b inhibitor. Runx2, a bone specific transcription factor is generally required for expression of osteoblast differentiation marker genes and in response to miR-15b inhibitor treatment, Runx2 mRNA expression was not changed; whereas its protein expression was decreased. Even though Smurf1 (SMAD specific E3 ubiquitin protein ligase 1), HDAC4 (histone deacetylase 4), Smad7, and Crim1 were found to be few of miR-15b's putative target genes, there was increased expression of only Smurf1 gene at mRNA and protein levels by miR-15b inhibitor. miR-15b mimic treatment significantly increased and decreased expressions of Runx2 and Smurf1 proteins, respectively. We further identified that the Smurf1 3’UTR is directly targeted by miR-15b using the luciferase reporter gene system. This is well documented that Smurf1 interacts with Runx2 and degrades it by proteasomal pathway. Hence, based on our results we suggest that miR-15b promotes osteoblast differentiation by indirectly protecting Runx2 protein from Smurf1 mediated degradation. Thus, this study identified that miR-15b can act as a positive regulator for osteoblast differentiation.

Description: The aim of this study is to determine the efficacy of tumor-targeting Salmonella typhimurium A1-R (A1-R) on a pancreatic cancer patient-derived orthotopic xenografts (PDOX). The PDOX model was originally established from a pancreatic cancer patient in SCID-NOD mice. The pancreatic cancer PDOX was subsequently transplanted by surgical orthotopic implantation (SOI) in transgenic nude red fluorescent protein (RFP) mice in order that the PDOX stably acquired red fluorescent protein (RFP)-expressing stroma for the purposes of imaging the tumor after passage to non-transgenic nude mice in order to visualize tumor growth and drug efficacy. The nude mice with human pancreatic PDOX were treated with A1-R or standard chemotherapy, including gemcitabine (GEM), which is first-line therapy for pancreatic cancer, for comparison of efficacy. A1-R treatment significantly reduced tumor weight, as well as tumor fluorescence area, compared to untreated control ( P  = 0.011), with comparable efficacy of GEM, CDDP, and 5-FU. Histopathological response to treatment was defined according to Evans’s criteria and A1-R had increased efficacy compared to standard chemotherapy. The present report is the first to show that A1-R is effective against a very low-passage patient tumor, in this case, pancreatic cancer. The data of the present report suggest A1-1 will have clinical activity in pancreatic cancer, a highly lethal and treatment-resistant disease and may be most effectively used in combination with other agents.

Description: Summary : The radiate sunflower inflorescence is composed by zygomorphic ray flowers and actinomorphic disk flowers. Studies performed on mutants identify HaCYC2c , a CYCLOIDEA ( CYC ) -like gene, as one of the key players controlling flower symmetry in sunflower. turf and tub mutants are characterized by a shift from zygomorphic to actinomorphic ray flowers, caused by insertion of transposable elements (TEs) in HaCYC2c gene. In dbl or Chry mutants, an insertion upstream the coding region of HaCYC2c causes the ectopic expression of the gene and the shift from actinomorphic to zygomorphic disk flowers. We focused on Chry2 mutant: a 1034 bp insertion placed 558 bp before the start codon of HaCYC2c was identified. The insertion is a truncated version of a CACTA TE. Unexpectedly, phenotypic and genetic co-segregation analysis in F 2 and F 3 progenies derived from the crosses Chry2 × turf and turf × Chry2 demonstrated that CACTA insertion is not always sufficient to alter the expression of HaCYC2c gene and generate Chry2 phenotype. F 3 plants homozygous for the CACTA insertion displayed either HaCYC2c transcription pattern identical to wild type plants or a normal heterogamous inflorescence. Stated these results, we conclude that a much more complex regulatory system stays behind the Chry2 phenotype. © 2014 Wiley Periodicals, Inc.

Description: Gestational factors play a role in the development of several neuropsychiatric disorders including schizophrenia and autism. In utero conditions influence future mental health through epigenetic mechanisms, which alter gene expression without affecting DNA coding sequence. Environmental factors account for at least 60% of the risk for developing major depression, and earlier onset of depressive illness has been observed over the past decades. I speculate that gestational factors may play a greater role in programing depression than previously recognized. Here, I examine recent evidence for a role for gestational factors in programing mood disorders, and how epigenetic mechanisms mediate this effect. How does the gestational environment transduce vulnerability to depression to the fetus? The in utero environment alters gene expression through epigenetic mechanisms, which mediate long-term effects on physiology and behavior without changing DNA sequence. I examine recent work suggesting that gestational environment programs depression in adult offspring via epigenetics.

Description: Recent findings have provided evidence for the existence of non-vertebrate acquired immunity. We survey these findings and propose that all living organisms must express both innate and acquired immunity. This is opposed to the paradigm that only vertebrates manifest the two forms of immune mechanism; other species are thought to use innate immunity alone. We suggest new definitions of innate and acquired immunity, based on whether immune recognition molecules are encoded in the inherited genome or are generated through somatic processes. We reason that both forms of immunity are similarly ancient, and have co-evolved in response to lifestyle, cost-benefit tradeoffs and symbiosis versus parasitism. However, different species have evolved different immune solutions that are not necessarily genetically related, but serve a similar general function – allowing individuals to learn from their own immune experience; survival of species is contingent on the acquired immune experience of its individuals. We propose that all organisms express both innate and adaptive/acquired immunity. Acquired immune manifestations vary between species, but all use recognition molecules not directly encoded in the inherited genome, and all serve a similar function – allowing individuals to learn from their own immune experience, thus promoting species survival.

Description: The standard representation of the Central Dogma (CD) of Molecular Biology conspicuously ignores metabolism. However, both the metabolites and the biochemical fluxes behind any biological phenomenon are encrypted in the DNA sequence. Metabolism constrains and even changes the information flow when the DNA-encoded instructions conflict with the homeostasis of the biochemical network. Inspection of adaptive virulence programs and emergence of xenobiotic-biodegradation pathways in environmental bacteria suggest that their main evolutionary drive is the expansion of their metabolic networks towards new chemical landscapes rather than perpetuation and spreading of their DNA sequences. Faulty enzymatic reactions on suboptimal substrates often produce reactive oxygen species (ROS), a process that fosters DNA diversification and ultimately couples catabolism of the new chemicals to growth. All this calls for a revision of the CD in which metabolism (rather than DNA) has the leading role. The central dogma of Molecular Biology has shortcomings that need revision: (i) metabolism is the next step of the information flow, (ii) metabolism has the predominant role (thereby the hierarchy of actors should be flipped upside down) and (iii) metabolism feedbacks on DNA by means of reactive oxygen species.

Description: If validated, diet-derived foreign microRNA absorption and function in consuming vertebrates would drastically alter our understanding of nutrition and ecology. RNA interference (RNAi) mechanisms of Caenorhabditis elegans are enhanced by uptake of environmental RNA and amplification and systemic distribution of RNAi effectors. Therapeutic exploitation of RNAi in treating human disease is difficult because these accessory processes are absent or diminished in most animals. A recent report challenged multiple paradigms, suggesting that ingested microRNAs (miRNAs) are transferred to blood, accumulate in tissues, and exert canonical regulation of endogenous transcripts. Independent replication of these findings has been elusive, and multiple disconfirmatory findings have been published. In the face of mounting negative results, any additional positive reports must provide the proverbial “extraordinary proof” to support such claims. In this article, we review the evidence for and against a significant role for dietary miRNAs in influencing gene expression, and make recommendations for future studies. A report that foreign microRNAs from the diet rival endogenous vertebrate miRNAs in abundance and function has been refuted by multiple independent studies, although low-level uptake may occur. We present the current evidence for and against the paradigm-challenging but uncorroborated dietary xenomiR hypothesis, and discuss remaining questions in the field.

Description: Literature on maternal exposures and the risk of epigenetic changes or diseases in the offspring is growing. Paternal contributions are often not considered. However, some animal and epidemiologic studies on various contaminants, nutrition, and lifestyle-related conditions suggest a paternal influence on the offspring's future health. The phenotypic outcomes may have been attributed to DNA damage or mutations, but increasing evidence shows that the inheritance of environmentally induced functional changes of the genome, and related disorders, are (also) driven by epigenetic components. In this essay we suggest the existence of epigenetic windows of susceptibility to environmental insults during sperm development. Changes in DNA methylation, histone modification, and non-coding RNAs are viable mechanistic candidates for a non-genetic transfer of paternal environmental information, from maturing germ cell to zygote. Inclusion of paternal factors in future research will ultimately improve the understanding of transgenerational epigenetic plasticity and health-related effects in future generations. Animal and epidemiologic studies on various environmental exposures suggest that transgenerational epigenetic changes can be induced through the paternal germ line, ultimately affecting health status of the offspring. This essay suggests the existence of epigenetic windows of susceptibility to environmental insults during spermatogenesis or other early developmental processes.

Description: Expression of smooth muscle alpha-actin (SMαA) is essential for myofibroblast-mediated wound contraction following tissue injury. The Pur α/β and YB-1 transcriptional repressors govern the DNA-binding activity of serum response factor (SRF) and phosphorylated Smad3 (pSmad3) transcriptional activators during induction of SMαA gene expression in human pulmonary myofibroblasts. In quiescent fibroblasts, Pur α exhibited a novel function in enhancing stability of pre-existing SRF complexes with SMαA core promoter DNA whereas Pur β was more effective in disrupting SRF-DNA interaction. Pur proteins were less efficient competitors of pre-existing, core-promoter complexes containing both SRF and pSmad3 in nuclear extracts from TGFβ1-activated myofibroblasts. TGFβ1 signaling dissociated a SRF/Pur protein complex with concurrent formation of a transient pSmad3/MRTF-A/Pur β complex during early phase myofibroblast differentiation. Pur β was replaced by Pur α in the pSmad3/MRTF-A complex in mature myofibroblasts. Combining all three repressors potently inhibited SRF and pSmad3 binding to promoter DNA in quiescent fibroblasts and TGFβ1-activated myofibroblasts, respectively. The results point to dynamic interplay between transcriptional activators and repressors in regulating SMαA gene output during myofibroblast differentiation. Therapeutic targeting of nucleoprotein complexes regulating the SMαA promoter may prevent excessive myofibroblast accumulation associated with chronic cardiopulmonary fibrosis and dysfunctional tissue remodeling.

Description: Several general anesthetics produce their sedative effect by activating endogenous sleep pathways. We propose that general anesthesia is a two-step process targeting sleep circuits at low doses, and synaptic release mechanisms across the entire brain at the higher doses required for surgery. Our hypothesis synthesizes data from a variety of model systems, some which require sleep (e.g. rodents and adult flies) and others that probably do not sleep (e.g. adult nematodes and cultured cell lines). Non-sleeping systems can be made insensitive (or hypersensitive) to some anesthetics by modifying a single pre-synaptic protein, syntaxin1A. This suggests that the synaptic release machinery, centered on the highly conserved SNARE complex, is an important target of general anesthetics in all animals. A careful consideration of SNARE architecture uncovers a potential mechanism for general anesthesia, which may be the primary target in animals that do not sleep, but a secondary target in animals that sleep. Volatile general anesthetics such as isoflurane produce loss of behavioral responsiveness in all animals. We propose that this can be explained as a two-step process: activation of endogenous sleep pathways at low doses followed by attenuation of synaptic release at the drug concentrations required for surgery.

Description: This study evaluated the effects of HgCl 2 on renal parameters in nonlactating and lactating rats and their pups, as well as the preventive role of ZnCl 2 . Rats received 27 mg kg −1 ZnCl 2 for five consecutive days and 5 mg kg −1 HgCl 2 for five subsequent days (s.c.). A decrease in δ -aminolevulinic acid dehydratase ( δ -ALA-D) activity in the blood and an increase in urine protein content in renal weight as well as in blood and urine Hg levels were observed in lactating and nonlactating rats from Sal―Hg and Zn―Hg groups. ZnCl 2 prevented partially the δ -ALA-D inhibition and the proteinuria in nonlactating rats. Renal Hg levels were increased in all HgCl 2 groups, and the ZnCl 2 exposure potentiated this effect in lactating rats. Nonlactating rats exposed to HgCl 2 exhibited an increase in plasma urea and creatinine levels, δ -ALA-D activity inhibition and histopathological alterations (necrosis, atrophic tubules and collagen deposition) in the kidneys. ZnCl 2 exposure prevented the biochemical alterations. Hg-exposed pups showed lower body and renal weight and an increase in the renal Hg levels. In conclusion, mercury-induced nephrotoxicity differs considerably between lactating and nonlactating rats. Moreover, prior exposure with ZnCl 2 may provide protection to individuals who get exposed to mercury occupationally or accidentally. Copyright © 2014 John Wiley & Sons, Ltd.

Description: ABSTRACT Targeted mutagenesis of genes-of-interest, or gene- knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases ( TALENs ) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis. © 2014 Wiley Periodicals, Inc.

Description: Parthenolide is the main bioactive component in feverfew, a common used herbal medicine, and has been extensively studied in relation to its anti-cancer properties. However there have been very few in-depth studies of the activities of this compound at the molecular level. Here, we showed that parthenolide increased reactive oxygen species (ROS), induced cell death, activated AMPK and autophagy, and led to M phase cell cycle arrest in breast cancer cells. Removal of ROS inhibited all parthenolide-associated events, such as cell death, AMPK activation, autophagy induction and cell cycle arrest. Blockade of autophagy relieved cell cycle arrest, whereas inhibition of AMPK activity significantly repressed the induction of both autophagy and cell cycle arrest. These observations clearly showed that parthenolide-driven ROS activated AMPK-autophagy pathway. Furthermore, inhibition of either AMPK or autophagy significantly potentiated parthenolide-induced apoptosis. Therefore, our results show that parthenolide activates both apoptosis pathway and AMPK-autophagy survival pathway through the generation of ROS, and that suppression of AMPK or autophagy can potentially enhance the anti-cancer effect of parthenolide on breast cancer cells. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.

Description: The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time-point, miR-sequencing analysis revealed that 20 miRs were upregulated (〉twofold) while 20 miRs were downregulated (〉threefold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR-4448, -4708, and -4773, which were significantly downregulated (〉fivefold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs that directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP-2) osteogenic signal, were upregulated by Ti with nanotopography. Overexpression of miR-4448, -4708, and 4773 in MC3T3-E1 pre-osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP-2 displayed a higher osteogenic effect on MC3T3-E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP-2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR-SMAD-BMP-2 circuit governs the osteoblast differentiation induced by Ti with nanotopography. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Polycomb repressive complex 2 (PRC2) is a critical epigenetic regulator in many biological processes, including maintenance of cell identity, stem cell self-renewal, differentiation, and deregulation of PRC2 is often observed in human cancers and diseases. Here we report that KDM5B (PLU-1/JARID1B), a histone lysine demethylase of Jumonji family, associates with PRC2 and colocalizes with PRC2 in nuclear bodies, and their physical association is dependent on direct interaction between KDM5B and the SUZ12 component of PRC2. Interestingly, co-occupancy of KDM5B and PRC2 was evidenced at the conserved cis -regulatory DNA element on retinoic acid (RA) responsive genes. Transcription readout and in vitro pull-down experiments suggest that KDM5B is an essential co-activator, but not a co-repressor, for the RA signaling, and the interface between KDM5B's JMJC domain and retinoic acid receptor α (RARα) is crucial for RA-mediated gene expression. Detailed chromatin immunoprecipitation assays addressed the seemingly paradox by revealing a biphasic effect of KDM5B on RA-induced gene activation through decoupled H3K4me3 demethylation and PRC2-antagonizing activities. These results demonstrate that KDM5B and PRC2 regulate RA signaling cascade in a cooperative and orchestrated fashion. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.

Description: The discovery of traces of a blood meal in the abdomen of a 50-million-year-old mosquito reminds us of the insights that the chemistry of fossils can provide. Ancient DNA is the best known fossil molecule. It is less well known that new fossil targets and a growing database of ancient gene sequences are paralleled by discoveries on other classes of organic molecules. New analytical tools, such as the synchrotron, reveal traces of the original composition of arthropod cuticles that are more than 400 my old. Pigments such as melanin are readily fossilized, surviving virtually unaltered for ∼200 my. Other biomarkers provide evidence of microbial processes in ancient sediments, and have been used to reveal the presence of demosponges, for example, more than 635 mya, long before their spicules appear in the fossil record. Ancient biomolecules are a powerful complement to fossil remains in revealing the history of life. Ancient biomolecules range from hundreds of thousand-year-old DNA to lipids and structural macromolecules that survive for billions of years. Extracted from fossils and sedimentary rocks, they reveal organism relationships, past environments, and the origin of fossil fuels. In the oldest rocks they may be the only evidence of particular life forms.

Description: The mechanisms that lead to the onset of organoselenium intoxication are still poorly understood. Therefore, in the present study, we investigated the effect of acute administration of 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one on some parameters of oxidative stress and on the activity of creatine kinase (CK) in different brain areas and on the behaviour in the open field test of 90-day-old male rats. Animals ( n  = 10/group) were treated intraperitoneally with a single dose of the organoselenium (125, 250 or 500 µg kg −1 ), and after 1 h of the drug administration, they were exposed to the open field test, and behaviour parameters were recorded. Immediately after they were euthanized, cerebral cortex, hippocampus and cerebellum were dissected for measurement of thiobarbituric acid reactive substances (TBARS), carbonyl, sulfhydryl, catalase (CAT), superoxide dismutase (SOD) and CK activity. Our results showed that the dose of 500 µg kg −1 of the organoselenium increased the locomotion and rearing behaviours in the open field test. Moreover, the organochalcogen enhanced TBARS in the cerebral cortex and cerebellum and increased the oxidation of proteins (carbonyl) only in the cerebral cortex. Sulfhydryl content was reduced in all brain areas, CAT activity enhanced in the hippocampus and reduced in the cerebellum and SOD activity increased in all brain structures. The organoselenium also inhibited CK activity in the cerebral cortex. Therefore, changes in motor behaviour, redox state and energy homeostasis in rats treated acutely with organoselenium support the hypotheses that the brain is a potential target for the organochalcogen action. Copyright © 2014 John Wiley & Sons, Ltd.

Description: Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta-cells, an event linked to apoptotic loss of β -cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage-inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin-producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA-activated protein kinase-like ER-localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2 α (eIF2 α ) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate-induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2 α phosphorylation sensitizes insulin-producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels. Copyright © 2014 John Wiley & Sons, Ltd.

Description: As neural stem cells differentiate into neurons during neurogenesis, the proteome of the cells is restructured by de novo expression and selective removal of regulatory proteins. The control of neurogenesis at the level of gene regulation is well documented and the regulation of protein abundance through protein degradation via the Ubiquitin/26S proteasome pathway is a rapidly developing field. This review describes our current understanding of role of the proteasome pathway in neurogenesis. Collectively, the studies show that targeted protein degradation is an important regulatory mechanism in the generation of new neurons. © 2014 Wiley Periodicals, Inc.

Description: The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole-exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10-nucleotide deletion in the BCL2-associated athanogene 3 ( BAG3 ) gene ((Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C-terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end-stage HF undergoing cardiac transplantation when compared with non-failing controls. Diminished levels of BAG3 protein may be associated with both familial and non-familial forms of dilated cardiomyopathy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Cardiosphere-derived cells (CDCs) were cultured from human, murine and rat hearts. Diluted supernatant (conditioned-medium) of the cultures improved the contractile behavior of isolated rat cardiomyocytes (CMCs). This effect is mediated by the paracrine release of cytokines. The present study tested the hypothesis, that the cardiovascular state of the donor‘s heart influences this effect on CMCs and tries to identify the responsible factors. CDCs were cultured from human tissue samples of cardiac surgery and from murine and rat hearts. The supernatants of cultured CDCs from hypertensive humans and rats showed a higher improvement of the contractile behavior of CMCs compared to CDCs of normotensive origin. Subsequently, the cytokine profile of the supernatants was analyzed. Among the cytokines elevated in supernatants originating from hypertensive humans or rats was Interleukin-6. CDCs were also generated from Interleukin-6 -/- -mice and their wildtype littermates. The supernatant of the cultured Interleukin-6 -/- -CDCs had no effect on the contractile behavior, whereas the supernatant of the Interleukin-6 +/+ -CDCs showed a positive effect. To confirm the hypothesis that Interleukin-6 contributes to the paracrine effects, CMCs were incubated with Interleukin-6. It improved the contractile function in a concentration dependent way. Finally, the effect of the supernatant of cultured CDCs derived from a hypertensive human sample could be abolished by simultaneous incubation with a specific Interleukin-6 antibody. CDCs release cytokines that improve the contractile behavior of CMCs. This effect is more intense in CDCs from hypertensive donors. Interleukin-6 is involved in this phenomenon. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The encounter of amateur science with synthetic biology has led to the formation of several amateur/do-it-yourself biology (DIYBio) groups worldwide. Although media outlets covered DIYBio events, most seemed only to highlight the hope, hype, and horror of what DIYBio would do in the future. Here, we analyze the European amateur biology movement to find out who they are, what they aim for and how they differ from US groups. We found that all groups are driven by a core leadership of (semi-)professional people who struggle with finding lab space and equipment. Regulations on genetic modification limit what groups can do. Differences between Europe and the US are found in the distinct regulatory environments and the European emphasis on bio-art. We conclude that DIYBio Europe has so far been a responsible and transparent citizen science movement with a solid user base that will continue to grow irrespective of media attention. Here, we analyzed the European do-it-yourself biology (DIYBio) community consisting of enthusiastic biotechnologists, artists, and designers. We conclude that DIYBio Europe has so far been a responsible and transparent citizen science movement with a solid user base that will continue to grow irrespective of media attention.

Description: HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity , Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design. Cellular proteins including cyclophilin A (CypA) interact with the capsid (CA) of HIV-1. Recent work shows that wild-type CA prevents the induction of an interferon (IFN) response. Increased CypA binding to mutated CA results in abortive infection and activation of the innate immune sensor cGAS that detects exposed cDNA.

Description: Cleavage of the cell–cell adhesion molecule, PTPµ, occurs in human glioblastoma multiforme brain tumor tissue and glioma cell lines. PTPµ cleavage is linked to increased cell motility and growth factor independent survival of glioma cells in vitro. Previously, PTPµ was shown to be cleaved by furin in the endoplasmic reticulum to generate membrane associated E- (extracellular) and P- (phosphatase) subunits, and by ADAMs and the gamma secretase complex at the plasma membrane. We also identified the presence of additional extracellular and intracellular PTPµ fragments in brain tumors. We set out to biochemically analyze PTPµ cleavage in cancer cells. We determined that, in addition to the furin-processed form of PTPµ, a pool of 200 kDa full-length PTPµ exists at the plasma membrane that is cleaved directly by ADAM to generate a larger shed form of the PTPµ extracellular segment. Notably, in glioma cells, full-length PTPµ is also subject to calpain cleavage, which generates novel PTPµ fragments not found in other immortalized cells. We also observed glycosylation and phosphorylation differences in the cancer cells. Our data suggest that an additional serine protease also contributes to PTPµ shedding in glioma cells. We hypothesize that a “protease storm” occurs in cancer cells whereby multiple proteases converge to reduce the presence of cell–cell adhesion molecules at the plasma membrane and to generate protein fragments with unique biological functions. As a consequence, the “protease storm” could promote the migration and invasion of tumor cells. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.

Description: Mesenchymal stem cells (MSCs) have shown a great potential for cell-based therapy and many different therapeutic purposes. Despite the recent advances in the knowledge of MSCs biology, their biochemical and molecular properties are still poorly defined. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5′-nucleotidase (eNT/CD73) are widely expressed enzymes that hydrolyze extracellular nucleotides, generating an important cellular signaling cascade. Currently, studies have evidenced the relationship between the purinergic system and the development, maintenance, and differentiation of stem cells. The objective of this study is to identify the NTPDases and eNT/CD73 and compare the levels of nucleotide hydrolysis on MSCs isolated from different murine tissues (bone marrow, lung, vena cava, kidney, pancreas, spleen, skin, and adipose tissue). MSCs from all tissues investigated expressed the ectoenzymes at different levels. In MSCs from pancreas and adipose tissue, the hydrolysis of triphosphonucleosides was significantly higher when compared to the other cells. The diphosphonucleosides were hydrolyzed at a higher rate by MSC from pancreas when compared to MSC from other tissues. The differential nucleotide hydrolysis activity and enzyme expression in these cells suggests that MSCs play different roles in regulating the purinergic system in these tissues. Overall MSCs are an attractive adult-derived cell population for therapies, however, the fact that ecto-nucleotide metabolism can affect the microenvironment, modulating important events, such as immune response, makes the assessment of this metabolism an important part of the characterization of MSCs to be applied therapeutically. J. Cell. Biochem. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: Recent technological advances allow for high throughput profiling of biological systems in a cost-efficient manner. The low cost of data generation is leading us to the “big data” era. The availability of big data provides unprecedented opportunities, but it also brings out challenges in data mining and analysis. In this review, we introduce key concepts in the analysis of big data, including both “machine learning” algorithms as well as “unsupervised” and “supervised” examples of each. We note packages for the R programming language that are available perform machine learning analyses. In addition to programming based solutions, we review webservers that allow users with limited or no programming background to perform these analyses on large data compendia. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Endometriosis is estimated to affect 10% of women during the reproductive years. The lack of a non-invasive diagnostic test significantly contributes to the long delay between onset of the symptoms and definitive diagnosis of endometriosis. This case–control study was conducted to identify specific endometriosis antigens using 2D gel analysis in women with endometriosis (n = 5) and without endometriosis (n = 5). Differentially expresses spots were analyzed using matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (nanoLC-ESI-MS/MS) with MASCOT analysis, in order to identify the corresponding proteins. ELISAs were performed on a different cohort of endometriosis (n = 120) and healthy patients (n = 20) in order to confirm the differential expression of the identified proteins. ROC analysis of ELISA results confirmed the statistical significance of the differential expression for one of these proteins: Zn-alpha2-glycoprotein ( P  = 0.019). We propose the analysis of the expression level of this protein in the serum as a new non-invasive diagnostic test for endometriosis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: We recently described a novel form of stress-associated bidirectional plasticity at GABA synapses onto hypothalamic parvocellular neuroendocrine cells (PNCs), the apex of the hypothalamus-pituitary-adrenal axis. This plasticity may contribute to neuroendocrine adaptation. However, this GABA synapse plasticity likely does not translate into a simple more and less of inhibition because the ionic driving force for Cl − , the primary charge carrier for GABA A receptors, is dynamic. Specifically, stress impairs a Cl − extrusion mechanism in PNCs. This not only renders the steady-state GABA response less hyperpolarizing but also makes PNCs susceptible to the activity-dependent accumulation of Cl − . Accordingly, GABA synapse plasticity impacts both the robustness of GABA voltage response and dynamic Cl − loading, imposing nonlinear influences on PNC excitability during circuit activities. This theoretical consideration predicts roles for GABA transmission far more versatile than canonical inhibition. We propose potential impacts of GABA synapse plasticity on the experience-dependent fine-tuning of neuroendocrine stress responses. Hypothalamic parvovellular neuroendocrine cells (PNCs) form the apex of the hypothalamus-pituitary-adrenal axis, controlling the corticosteroid (CORT) response to stress. GABAergic inputs onto PNCs undergo multiple forms of plasticity following stress. We propose a hypothesis that GABA synapse plasticity impacts Cl − homeostasis, generates excitatory GABA responses, and tunes neuroendocrine response.

Description: Replication of the main chromosome in the halophilic archaeon Haloferax volcanii was recently reported to continue despite deletion of all active replication origins. Equally surprising, the deletion strain grew faster than the parent strain. It was proposed that origin-less H. volcanii duplicate their chromosomes via recombination-dependent replication. Here, we recall our present knowledge of this mode of chromosome replication in different organisms. We consider the likelihood that it accounts for the viability of H. volcanii deleted for its main specific replication origins, as well as possible alternative interpretations of the results. The selective advantages of having defined chromosome replication origins are discussed from a functional and evolutionary perspective. Hawkins et al. (Nature 503:544–7, 2013) reported the surprising finding that deleting all three replication origins from the archaea Haloferax volcanii main chromosome does not impair viability and rather increases growth rate. We discuss the authors' proposal that replication is then initiated from recombination intermediates, and present alternative hypotheses.

Description: The purinergic signalling system, which utilises ATP, related nucleotides and adenosine as transmitter molecules, appeared very early in evolution: release mechanisms and ATP-degrading enzymes are operative in bacteria, and the first specific receptors are present in single cell eukaryotic protozoa and algae. Further evolution of the purinergic signalling system resulted in the development of multiple classes of purinoceptors, several pathways for release of nucleotides and adenosine, and a system of ectonucleotidases controlling extracellular levels of purinergic transmitters. The purinergic signalling system is expressed in virtually all types of tissues and cells, where it mediates numerous physiological reactions and contributes to pathological responses in a variety of diseases. Purinergic chemical signalling appears early in evolution in species without a nervous system followed by purinergic neurotransmission in animals that developed a nervous system. Widespread purinoceptor expression on most mammalian cell types suggests ATP and adenosine have been retained as successful, compared to more restricted distributions of other chemical messengers.

Description: Although combination chemotherapy and radiotherapy have become the standard of care in numerous tumors, the mechanisms of interaction are often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigate their mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation-induced cytotoxicity before and after cisplatin treatment. Expression levels of a panel of genes were determined by real-time RT-PCR. Cytotoxic effect was evaluated by flow cytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the two treatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells. Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from 16 to 〉100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet-shaped nucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin only induced weak cytotoxicity. Our findings highlight the efficacy of the sequence radiation → cisplatin in reducing cell proliferation and in inducing apoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: This review summarizes and integrates our current understanding of how sea stars make gametes. Although little is known of the mechanism of germ line formation in these animals, recent results point to specific cells and to cohorts of molecules in the embryos and larvae that may lay the ground work for future research efforts. A coelomic outpocketing forms in the posterior of the gut in larvae, referred to as the posterior enterocoel (PE), that when removed, significantly reduces the number of germ cell later in larval growth. This same PE structure also selectively accumulates several germ-line associated factors – vasa, nanos, piwi – and excludes factors involved in somatic cell fate. Since its formation is relatively late in development, these germ cells may form by inductive mechanisms. When integrated into the morphological observations of germ cells and gonad development in larvae, juveniles, and adults, the field of germ line determination appears to have a good model system to study inductive germ line determination to complement the recent work on the molecular mechanisms in mice. We hope this review will also guide investigators interested in germ line determination and regulation of the germ line in how these animals can help in this research field. The review is not intended to be comprehensive – sea star reproduction has been studied over 100 years and many reviews are comprehensive in their coverage of, for example, seasonal growth of the gonads in response to light, nutrient, and temperature. Rather the intent of this review is to help the reader focus on new experimental results attached to the historical underpinnings of how the germ cell functions in sea stars with particular emphasis to clarify the important areas of priority for future research. © 2014 Wiley Periodicals, Inc.

Description: Lung development follows a stereotypic program orchestrated by key interactions among epithelial and mesenchymal tissues. Deviations from this developmental program can lead to pulmonary diseases including bronchopulmonary dysplasia and pulmonary hypertension. Significant efforts have been made to examine the cellular and molecular basis of the tissue interactions underlying these stereotypic developmental processes. Genetically engineered mouse models, lung organ culture, and advanced imaging techniques are a few of the tools that have expanded our understanding of the tissue interactions that drive lung development. Intimate crosstalk has been identified between the epithelium and mesenchyme, distinct mesenchymal tissues, and individual epithelial cells types. For interactions such as the epithelial-mesenchymal crosstalk regulating lung specification and branching morphogenesis, the key molecular players, FGF, BMP, WNT, and SHH, are well established. Additionally, VEGF regulation underlies the epithelial-endothelial crosstalk that coordinates airway branching with angiogenesis. Recent work also discovered a novel role for SHH in the epithelial-to-mesenchymal (EMT) transition of the mesothelium. In contrast, the molecular basis for the crosstalk between upper airway cartilage and smooth muscle is not yet known. In this review we examine current evidence of the tissue interactions and molecular crosstalk that underlie the stereotypic patterning of the developing lung and mediate injury repair. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.

Description: The epithelial lining of the respiratory system originates from a small group of progenitor cells in the ventral foregut endoderm of the early embryo. Research in the last decade has revealed a number of paracrine signaling pathways that are critical for the development of these respiratory progenitors. In the post genomic era the challenge now is to figure out at the genome wide level how these different signaling pathways and their downstream transcription factors interact in a complex “gene regulatory network” (GRN) to orchestrate early lung development. In this prospective we review our growing understanding of the GRN governing lung specification. We discuss key gaps in our knowledge and describe emerging opportunities that will soon provide an unprecedented understanding of lung development and accelerate our ability to apply this knowledge to regenerative medicine. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.

Description: Insufficiency of surfactants is a core factor in respiratory distress syndrome (RDS), which causes apnea and neonatal death, particularly in preterm infants. Surfactant proteins are secreted by alveolar type II cells in the lung epithelium, the differentiation of which is regulated by Fgf10 elaborated by the adjacent mesenchyme. However, the molecular regulation of mesenchymal Fgf10 during lung development has not been fully understood. Here we show that Pbx1, a homeodomain transcription factor, is required in the lung mesenchyme for the expression of Fgf10 . Mouse embryos lacking Pbx1 in the lung mesenchyme show compact terminal saccules and perinatal lethality with failure of postnatal alveolar expansion. Mutant embryos had severely reduced expression of Fgf10 and surfactant genes ( Spa , Spb , Spc , and Spd ) that are essential for alveolar expansion for gas exchange at birth. Molecularly, Pbx1 directly binds to the Fgf10 promoter and cooperates with Meis and Hox proteins to transcriptionally activate Fgf10 . Our results thus show how Pbx1 controls Fgf10 in the developing lung. © 2014 Wiley Periodicals, Inc.

Description: Drosophila is a classical model to study body patterning, however Left-Right (L/R) asymmetry had remained unexplored, until recently. The discovery of the conserved myosin ID gene as a major determinant of L/R asymmetry has revealed a novel L/R pathway involving the actin cytoskeleton and the adherens junction. In this process, the HOX gene Abdominal-B plays a major role through the control of myosin ID expression and therefore symmetry breaking. In this review, we present organs and markers showing L/R asymmetry in Drosophila and discuss our current understanding of the underlying molecular genetic mechanisms. Drosophila represents a valuable model system revealing novel strategies to establish L/R asymmetry in invertebrates and providing an evolutionary perspective to the problem of laterality in bilateria. © 2014 Wiley Periodicals, Inc.

Description: Differentiation of mesenchymal stem cells (MSCs) to hepatocyte-like cells is associated with morphological and biological changes. In this study, the effect of hepatogenic differentiation on fatty acid profile and the expression of proliferator-activated receptors- α (PPAR- α ) have been studied. For this purpose, MSCs isolated from human umbilical cord were differentiated into hepatocyte-like cells on selective culture media. The morphological and biochemical changes, PPAR- α expression and reactive oxygen species (ROS) levels were studied during the differentiation process. Besides, the cells were processed to determine changes in fatty acid profile using gas chromatography analysis. The results showed that hepatic differentiation of the MSCs is associated with a decrease in major polyunsaturated fatty acids in mature hepatocytes, whereas there was an increase in the saturated fatty acid (SFA) levels during hepatocyte maturation. The differentiation-dependent shift in the ratio of SFA/USFA was associated with changes in albumin and PPAR- α expression, whereas changes in fatty acid profile were independent of ROS production and lipid peroxidation in differentiating cells. In conclusion, these data may suggest that hepatocyte formation during the stem cell differentiation is associated with a shift in the fatty acid profile that is probably a normal phenomenon in hepatogenic differentiation of the MSCs. Copyright © 2014 John Wiley & Sons, Ltd.

Description: Magoh encodes a core component of the exon junction complex (EJC), which binds mRNA and regulates mRNA metabolism. Magoh is highly expressed in proliferative tissues during development. EJC components have been implicated in several developmental disorders including TAR syndrome, Richieri-Costa-Pereira Syndrome, and intellectual disability. Existing germline null Magoh mice are embryonic lethal as homozygotes and perinatal lethal as heterozygotes, precluding detailed analysis of embryonic and postnatal functions. Here we report the generation of a new genetic tool to dissect temporal and tissue specific roles for Magoh in development and adult homeostasis. This Magoh conditional allele has two LoxP sites flanking the second exon. Ubiquitous Cre-mediated deletion of the floxed allele in a heterozygous mouse ( Magoh del /+ ) causes 50% reduction of both Magoh mRNA and protein. Magoh del /+ mice exhibit both microcephaly and hypopigmentation, thus phenocopying germline haploinsufficient Magoh mice. Using Emx1 - Cre , we further show that conditional Magoh deletion in neural progenitors during embryonic development also causes microcephaly. We anticipate this novel conditional allele will be a valuable tool for assessing tissue specific roles for Magoh in mammalian development and postnatal processes. © 2014 Wiley Periodicals, Inc.

Description: Anatomical left-right (L/R) handedness asymmetry in C. elegans is established in the four-cell embryo as a result of anteroposterior skewing of transverse mitotic spindles with a defined handedness. This event creates a chiral embryo and ultimately an adult body plan with fixed L/R positioning of internal organs and components of the nervous system. While this “dextral” configuration is invariant in hermaphrodites, it can be reversed by physical manipulation of the early embryo or by mutations that interfere with mitotic spindle orientation, which leads to viable, mirror-reversed (sinistral) animals. During normal development of the C. elegans male, the gonad develops on the right of the midline, with the gut bilaterally apposed on the left. However, we found that in males of the laboratory N2 strain and Hawaiian (“Hw”) wild isolate, the gut/gonad asymmetry is frequently reversed in a temperature-dependent manner, independent of normal embryonic chirality. We also observed sporadic errors in gonad migration occurring naturally during early larval stages of these and other wild strains; however, the incidence of such errors does not correlate with the frequency of L/R gut/gonad reversals in these strains. Analysis of N2/Hw hybrids and recombinant inbred advanced intercross lines (RIAILs) indicate that the L/R organ reversals are likely to result from recessively acting variations in multiple genes. Thus, unlike the highly reproducible L/R asymmetries of most structures in hermaphrodites, the L/R asymmetry of the male C. elegans body plan is less rigidly determined and subject to natural variation that is influenced by a multiplicity of genes. © 2014 Wiley Periodicals, Inc.

Description: The thickness of epicardial adipose tissue (EAT), which is an inflammatory source for coronary artery disease (CAD), correlates with insulin resistance. One trigger factor is impaired adipogenesis. Here, our aim was to clarify the underlying mechanisms of insulin resistance on EAT-mesenchymal cells (MC). EAT and subcutaneous adipose tissue (SAT) were collected from 19 patients who were undergoing heart surgery. Their dedifferentiated adipocytes (DAs) and/or MCs were cultured. After the induction of adipogenesis or stimulation with insulin, the expression of adipokines was analyzed using real-time polymerase chain reaction (PCR). Colorimetric assays were performed to measure glucose levels and proliferation rate. Proteins modifications were detected via the proteomic approach and Western blot. Our results showed lower adipogenic ability in EAT-MCs than in SAT-MCs. Maximum adiponectin levels were reached within 28–35 days of exposure to adipogenic inducers. Moreover, the adipogenesis profile in EAT-MCs was dependent on the patients' clinical characteristics. The low adipogenic ability of EAT-MCs might be associated with an insulin-resistant state because chronic insulin treatment reduced the inflammatory cytokine expression levels, improved the glucose consumption, and increased the post-translational modifications (PTMs) of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1). We found lower adipogenic ability in EAT-MCs than in SAT-MCs. This lower ability level was dependent on gender and the presence of diabetes, obesity, and CAD. Low adipogenesis ability and insulin resistance in EAT-MCs might shed light on the association between EAT dysfunction and cardiovascular disease. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The primitive node is the ‘hub' of early left-right patterning in the chick embryo: (1) it undergoes asymmetrical morphogenesis immediately after its appearance at stage 4; (2) it is closely linked to the emerging asymmetrical expression of nodal and shh at stage 5; and (3) its asymmetry is spatio-temporally related to the emerging notochord, the midline barrier maintaining molecular left-right patterning from stage 6 onwards. Here, we study the correlation of node asymmetry to notochord marker expression using high-resolution histology, and we test pharmacological inhibition of shh signaling using cyclopamine at stages 4 and 5. Just as noggin expression mirrors an intriguing structural continuity between the right node shoulder and the notochord, shh expression in the left node shoulder confirms a similar continuity with the future floor plate. Shh inhibition at stage 4 or 5 suppressed nodal in both its paraxial or lateral plate mesoderm domains, respectively, and resulted in randomized heart looping. Thus, the ‘primordial' paraxial nodal asymmetry at stage 4/5 (1) appears to be dependent on, but not instructed by, shh signaling, and (2) may be fixed by asymmetrical roots of the notochord and the floor plate, thereby adding further twists to the node's pivotal role during left-right patterning. © 2014 Wiley Periodicals, Inc.

Description: Myocardial infarction (MI) is defined as cardiac cell death due to prolonged ischemia. Although necrotic cell death was considered to be solely responsible for myocyte death during MI, it was recently revealed that apoptosis also plays its part in this death process. Our laboratory has recently shown that endoplasmic reticulum (ER) stress-induced apoptosis is the predominant route for apoptosis during MI and the conventional mitochondrial pathway is bypassed by activation of a small heat shock protein α-crystallin B (CRYAB). Since CRYAB is a direct target of P38 mitogen-activated protein kinase (MAPK) cascade, we were prompted to check the role of P38 MAPK in 20-week-old male Wister rats immediately after infarct formation. Interestingly, parallel activation of mitochondrial apoptotic pathway with an increase in ER stress-induced apoptotic load was observed along with decreased activation of CRYAB and Nrf2 (a pro-survival protein activated in response to ER stress) in MI rats treated with SB203580, a specific inhibitor of P38α and P38β compared to the MI alone. As a cumulative effect, this inhibitor treatment also resulted in significant increase in the levels of caspase3 activity and TUNEL positivity, the end point apoptotic markers. Furthermore, SB203580-treated hypoxic adult cardiomyocytes showed formation of desmin aggregates which were previously associated with impaired cardiac function. Thus, this study shows for the first time the precise mechanism by which P38 MAPK plays a pro-survival role and confers protection of cardiomyocytes, during infarct formation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Colon cancer remains one of the lethal malignancies in the world. Aberrant activation of canonical Wnt/β-catenin signaling pathway has been observed in colon cancer. In contrast, the non-canonical Wnt signaling functions remain obscure. Wnt5a is a representative non-canonical Wnt ligand which has gained extensive attention nowadays. Wnt5a has been shown to play an important role in EMT in prostate cancer and melanoma, but its role in colon cancer is still ambiguous. Here we have evaluated Wnt5a expression in a large cohort of 217 colon cancers by immunohistochemistry and analyzed its correlation with clinicopathologic characteristics. We found that expression of Wnt5a was diminished significantly in majority of primary colon cancers and negatively related with EMT biomarkers. To further enlighten the mechanism which Wnt5a regulates EMT in vitro , we established ectopic Wnt5a expression models. Protein analysis demonstrated that Wnt5a inhibited EMT and antagonized canonical Wnt signaling in colon cancer cells. Overexpression of Wnt5a impaired cell motility and invasion and inhibited cell proliferation by manipulating Bax. Moreover, Wnt5a suppressed the tumor growth in nude mice and impaired tumorigenicity in vivo . Wnt5a also induced intracellular calcium and activated non-canonical Wnt/Ca 2+ signaling in colon cancer. In summary, although Wnt5a was down-regulated in majority of colon cancers, enhanced Wnt5a expression predict preferable outcome in colon cancer patients. Our findings indicate that Wnt5a might act as tumor suppressor by inhibiting cell proliferation and attenuating EMT in colon cancer cells. Wnt5a could be used as a novel prognostic marker and/or therapeutic target for colon cancer in the future. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Genomes are inherently unstable because of the need for DNA sequence variation as a substrate for evolution through natural selection. However, most multicellular organisms have postmitotic tissues, with limited opportunity for selective removal of cells harboring persistent damage and deleterious mutations, which can therefore contribute to functional decline, disease, and death. Key in this process is the role of genome maintenance, the network of protein products that repair DNA damage and signal DNA damage response pathways. Genome maintenance is beneficial early in life by swiftly eliminating DNA damage or damaged cells, facilitating rapid cell proliferation. However, at later ages accumulation of unrepaired damage and mutations, as well as ongoing cell depletion, promotes cancer, atrophy, and other deleterious effects associated with aging. As such, genome maintenance and its phenotypic sequelae provide yet another example of antagonistic pleiotropy in aging and longevity. DNA damage has since long been suspected to be a major cause of aging. Here, I will discuss the dual role of genome maintenance systems in providing DNA sequence variation in the germline as the substrate for evolution and mediating DNA damage-induced aging phenotypes in the soma.

Description: Disruption of Nodal in the lateral plate mesoderm (LPM) usually leads to left-right (LR) patterning defects in multiple organs. However, whether the LR patterning of organs is always regulated in a coupled way has largely not yet been elucidated. In addition, whether other crucial regulators exist in the LPM that coordinate with Nodal in regulating organ LR patterning is also undetermined. In this paper, after briefly summarizing the common process of LR patterning, the most puzzling question regarding the initiation of asymmetry is considered and the divergent mechanisms underlying the uncoupled LR patterning in different organs are discussed. On the basis of cases in which different organ LR patterning is determined in an uncoupled way via an independent mechanism or at a different time, we propose that there are other critical factors in the LPM that coordinate with Nodal to regulate heart LR asymmetry patterning during early LR patterning. Also watch the Video Abstract . Based on the fact that distinct mechanisms determine different organ left-right (LR) patterning in an uncoupled way, we hypothesize that other critical factors in the lateral plate mesoderm (LPM) may coordinate with Nodal to regulate heart LR asymmetry patterning during LR patterning, and this can be evaluated by our proposed model.

Description: Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that belongs to the interleukin-6 family and is expressed by multiple tissue types. This study analyzed the effect of LIF on osteoblast differentiation using primary murine bone marrow stromal cells (BMSCs). Colony-forming unit-osteoblast formation by BMSCs was significantly suppressed by LIF treatment. To clarify the mechanism underlying the LIF suppressive effect on osteoblast differentiation, we analyzed the downstream signaling pathway of LIF. LIF/signal transducer and activator of transcription 3 (STAT3) signaling induces the expression of suppressor of cytokine signaling 3 (SOCS3). SOCS3 knockdown experiments have previously demonstrated that short-hairpin SOCS3-BMSCs reversed the LIF suppressive effect. Our results demonstrated that LIF suppresses osteoblast differentiation through the LIF/STAT3/SOCS3 signaling pathway. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.

Description: The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin-mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier-disruptive effect of thrombin in HUVECs by a concentration-dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A 2A and A 2B are expressed at the highest level among the four receptor subtypes (A 2B  〉 A 2A  〉 A 1  〉 A 3 ) on HUVECs. The barrier-protective effect of adenosine in response to thrombin was recapitulated by the A 2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A 2A receptor and by the A 2A -specific antagonists, ZM-241385 and SCH-58261. The thrombin-induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP-dependent manner, providing a molecular mechanism through which adenosine exerts a barrier-protective function. Adenosine also inhibited thrombin-mediated activation of NF-κB and decreased adhesion of monocytic THP-1 cells to stimulated HUVECs via down-regulation of expression of cell surface adhesion molecules, VCAM-1, ICAM-1, and E-selectin. Moreover, adenosine inhibited thrombin-induced elevated expression of proinflammatory cytokines, IL-6 and HMGB-1; and chemokines, MCP-1, CXCL-1, and CXCL-3. Taken together, these results suggest that adenosine may inhibit thrombin-mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Dental pulp originating from the neural crest is considered a better source of postnatal stem cells for cell-based therapies in neurodegenerative diseases. Dental Pulp Stem Cells (DPSCs) have been shown to differentiate into cell-types of cranial neural crest ontology; however, their ability to differentiate to functional neurons of the central nervous system remains to be studied. We hypothesized that midbrain cues might commit DPSCs to differentiate to functional dopaminergic cell-type. As expected, DPSCs in their naïve state spontaneously expressed early and mature neuronal markers like nestin, musashi12, β tubulin III and Map2ab. On exposure to midbrain cues (sonic hedgehog, fibroblast growth factor 8 and basic fibroblast growth factor), DPSCs showed upregulation of dopaminergic neuron-specific transcription factors Nuclear Receptor related protein 1 (Nurr1), Engrailed 1 (En1) and paired-like homeodomain transcription factor 3 (Pitx3) as revealed by real-time RT-PCR. Immunofluorescence and flow cytometry analysis showed enhanced expression of mature neuronal marker Map2ab and dopaminergic-neuronal markers [tyrosine hydroxylase (TH), En1, Nurr1 and Pitx3], with nearly 77% of the induced DPSCs positive for TH. Functional studies indicated that the induced DPSCs could secrete dopamine constitutively and upon stimulation with potassium chloride (KCl) and adenosine triphosphate (ATP), as measured by dopamine ELISA. Additionally, the induced DPSCs showed intracellular Ca 2+ influx in the presence of KCl, unlike control DPSCs. ATP-stimulated Ca 2+ influx was observed in control and induced DPSCs, but only the induced cells secreted dopamine. Our data clearly demonstrates for the first time that DPSCs in the presence of embryonic midbrain cues show efficient propensity towards functional dopaminergic cell-type. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: One of the central concerns of Evolutionary Developmental biology is to understand how the specification of cell types can change during evolution. In the last decade, developmental biology has progressed towards a systems level understanding of cell specification processes. In particular, the focus has been on determining the regulatory interactions of the repertoire of genes that make up gene regulatory networks (GRNs). Echinoderms provide an extraordinary model system for determining how GRNs evolve. This review highlights the comparative GRN analyses arising from the echinoderm system. This work shows that certain types of GRN sub-circuits or motifs, i.e. those involving positive feedback, tend to be conserved and may provide a constraint on development. This conservation may be due to a required arrangement of transcription factor binding sites in cis regulatory modules. The review will also discuss ways in which novelty may arise, in particular through the co-option of regulatory genes and sub-circuits. The development of the sea urchin larval skeleton, a novel feature that arose in echinoderms, has provided a model for study of co-option mechanisms. Finally, the types of GRNs that can permit the great diversity in the patterns of ciliary bands and their associated neurons found among these taxa are discussed. The availability of genomic resources is rapidly expanding for echinoderms, including genome sequences not only for multiple species of sea urchins, but also a species of sea star, sea cucumber, and brittle star. This will enable echinoderms to become a particularly powerful system for understanding how developmental GRNs evolve. © 2014 Wiley Periodicals, Inc.

Description: Gene retrocopies are generated by reverse transcription and genomic integration of mRNA. As such, retrocopies present an important exception to the central dogma of molecular biology, and have substantially impacted the functional landscape of the metazoan genome. While an estimated 8,000–17,000 retrocopies exist in the human genome reference sequence, the extent of variation between individuals in terms of retrocopy content has remained largely unexplored. Three recent studies by Abyzov et al., Ewing et al. and Schrider et al. have exploited 1,000 Genomes Project Consortium data, as well as other sources of whole-genome sequencing data, to uncover novel gene retrocopies. Here, we compare the methods and results of these three studies, highlight the impact of retrocopies in human diversity and genome evolution, and speculate on the potential for somatic gene retrocopies to impact cancer etiology and genetic diversity among individual neurons in the mammalian brain. Three recent studies have employed whole genome sequencing data to identify novel gene retrocopies in humans, by exploiting distinguishing retrocopy hallmarks including exon-exon junctions and genomic locations distal to parent genes. Gene retrocopies contribute to human genetic diversity.

Description: Photodynamic therapy (PDT) utilizes light, a photosensitizer, and molecular oxygen as a treatment modality for a variety of cancers. We have recently combined ruthenium(II) polypyridyl groups with a zinc(II) centered porphyrin as a new photosensitizer for the treatment of melanoma. In-vitro studies have indicated that this photosensitizer is toxic to melanoma cells when irradiated with low energy light; however, it is non-toxic to normal cells under similar conditions. To determine the toxicity and cell viability of this compound in-vivo we present herein a study using Drosophila melanogaster. In the absence of light the new photosensitizer shows no discernible effects to fly larvae at various concentrations of compound and stages of larval development. When the larvae were fed the photosensitizer it was observed, by fluorescence microscopy, that the compound passes through the cell membrane and localizes in the cytosol at lower concentrations and the nucleus at slightly higher concentrations indicating that the compound is not immediately metabolized. © 2014 Wiley Periodicals, Inc.

Description: Mechanical stimuli are important in directing the fate of stem cells; the effects of mechanical stimuli reported in recent research are reviewed here. Stem cells normally undergo two fundamental processes: proliferation, in which their numbers multiply, and differentiation, in which they transform into the specialized cells needed by the adult organism. Mechanical stimuli are well known to affect both processes of proliferation and differentiation, although the complete pathways relating specific mechanical stimuli to stem cell fate remain to be elucidated. We identified two broad classes of research findings and organized them according to the type of mechanical stress (compressive, tensile or shear) of the stimulus. Firstly, mechanical stress of any type activates stretch-activated channels (SACs) on the cell membrane. Activation of SACs leads to cytoskeletal remodelling and to the expression of genes that regulate the basic growth, survival or apoptosis of the cells and thus regulates proliferation. Secondly, mechanical stress on cells that are physically attached to an extracellular matrix (ECM) initiates remodelling of cell membrane structures called integrins. This second process is highly dependent on the type of mechanical stress applied and result into various biological responses. A further process, the Wnt pathway, is also implicated: crosstalk between the integrin and Wnt pathways regulates the switch from proliferation to differentiation and finally regulates the type of differentiation. Therefore, the stem cell differentiation process involves different signalling molecules and their pathways and most likely depends upon the applied mechanical stimulation. Copyright © 2014 John Wiley & Sons, Ltd.

Description: The environment can have a long-lasting influence on an individual's physiology and behavior. While some environmental conditions can be beneficial and result in adaptive responses, others can lead to pathological behaviors. Many studies have demonstrated that changes induced by the environment are expressed not only by the individuals directly exposed, but also by the offspring sometimes across multiple generations. Epigenetic alterations have been proposed as underlying mechanisms for such transmissible effects. Here, we review the most relevant literature on these changes and the developmental stages they affect the most. We discuss current evidence for transgenerational effects of prenatal and postnatal factors on bodily functions and behavioral responses, and the potential epigenetic mechanisms involved. We also discuss the need for a careful evaluation of the evolutionary importance with respect to health and disease, and possible directions for future research in the field. Evidence for epigenetic inheritance of acquired traits in mammals is growing. Transgenerational transmission is based on mechanisms involving DNA-methylation, histone post-translational modifications, and non-coding small RNAs in the germline. These epigenetic modifications constitute potential vehicles for effects of early life stress and nutrition on brain and behavior across generations.