ALBERT

Beschreibung: Photodynamic therapy (PDT) is a non-thermal technique for inducing tumour damage following administration of a light-activated photosensitizing drug (PS). In a previous work we found that PDT induces cytoskeleton changes in HB4a-Ras cells (human mammary breast carcinoma HB4a cells transfected with the RAS oncogene). In the present work we have studied the migratory and invasive features and the expression of proteins related to these processes on HB4a-Ras cells after 3 successive cycles of PDT using different PSs: 5-aminolevulinic acid (ALA), Verteporfin (Verte), m -tetrahydroxyphenylchlorin ( m -THPC) and Merocyanine 540 (MC). A slight (1.25- to -2 fold) degree of resistance was acquired in cell populations subjected to the three successive PDT treatments. However, complete cell killing was achieved after a light dose increase. Regardless of the PS employed, all the PDT-treated populations had shorter stress fibres than the untreated control HB4a-Ras cells, and the number of dorsal stress fibres was decreased in the PDT-treated populations. E-Cadherin distribution, which was already aberrant in HB4a-Ras cells, became even more diffuse in the PDT-treated populations, though its expression was increased in some of them. The strong migratory and invasive ability of HB4a-Ras cells in vitro was impaired in all the PDT-treated populations, with a behaviour that was similar to the parental non-tumoral HB4a cells. MMP-2 and MMP-9 metalloproteinase activities were also impaired in the PDT-treated populations. The evidence presented herein suggests that the cells surviving PDT would be less metastatic than the initial population. These findings encourage the use of PDT in combination with other treatments such as intraoperative or post-surgery therapeutic procedures. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Mechanotransduction is a key process by which cells perceive extracellular mechanical cues / intercellular physical interactions and transform them into intracellular biochemical signals. This physiological process is crucial during bone development and bone remodeling throughout childhood and adult life, whereas several aberrations during this process have emerged as a distinct pathogenic molecular entity in bone maladies and tumor formation. The present review focuses on recent advances regarding the mechanobiology of osteosarcoma, the most common type of bone cancer. Special emphasis is given on the mechano-responsive signal transduction pathways underlying osteosarcoma pathology and on specific mechanosensitive molecules engaged in osteosarcoma development. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Protandim and 6-gingerol, two potent nutraceuticals, have been shown to decrease free radicals production through enhancing endogenous antioxidant enzymes. In this study, we evaluated the effects of these products on the expression of different factors involved in osteoarthritis (OA) process. Human OA chondrocytes were treated with 1 ng/ml IL-1β in the presence or absence of protandim (0-10 μg/ml) or 6-gingerol (0-10 μM). OA was induced surgically in mice by destabilization of the medial meniscus (DMM). The animals were treated weekly with an intraarticular injection of 10 μl of vehicle or protandim (10 μg/ml) for 8 weeks. Sham-operated mice served as controls. In vitro , we demonstrated that protandim and 6-gingerol preserve cell viability and mitochondrial metabolism and prevented 4-hydroxynonenal (HNE)-induced cell mortality. They activated Nrf2 transcription factor, abolished IL-1β-induced NO, PGE 2 , MMP-13, and HNE production as well as IL-β − induced GSTA4-4 down-regulation. Nrf2 overexpression reduced IL-1β-induced HNE and MMP-13 as well as IL-1β-induced GSTA4-4 down-regulation. Nrf2 knockdown following siRNA transfection abolished protandim protection against oxidative stress and catabolism. The activation of MAPK and NF-κB by IL-1β was not affected by 6-gingerol. In vivo , we observed that Nrf2 and GSTA4-4 expression was significantly lower in OA cartilage from humans and mice compared to normal controls. Interestingly, protandim administration reduced OA score in DMM mice. Altogether, our data indicate that protandim and 6-gingerol are essential in preserving cartilage and abolishing a number of factors known to be involved in OA pathogenesis. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Epithelial morphogenesis in the mammary gland proceeds as a consequence of complex cell behaviors including apoptotic cell death and epithelial-mesenchymal transition (EMT); the extracellular matrix (ECM) protein laminin is crucially involved. Syntaxins mediate intracellular vesicular fusion, yet certain plasmalemmal members have been shown to possess latent extracellular functions. In this study, the extracellular subpopulation of syntaxin-4, extruded in response to the induction of differentiation or apoptosis in mammary epithelial cells, was detected. Using a tetracycline-repressive transcriptional system and clonal mammary epithelial cells, SCp2, we found that the expression of cell surface syntaxin-4 elicits EMT-like cell behaviors. Intriguingly, these cells did not up-regulate key transcription factors associated with the canonical EMT such as snail, slug , or twist , and repressed translation of E-cadherin. Concurrently, the cells completely evaded the cellular aggregation/rounding triggered by a potent EMT blocker laminin-111. We found that the recombinant form of syntaxin-4 not only bound to laminin but also latched onto the glycosaminoglycan (GAG) side chains of syndecan-1, a laminin receptor that mediates epithelial morphogenesis. Thus, temporal extracellular extrusion of syntaxin-4 emerged as a novel regulatory element for laminin-induced mammary epithelial cell behaviors. This article is protected by copyright. All rights reserved

Beschreibung: Glial cell line-derived neurotrophic factor (GDNF) is known to mediate multiple biological activities such as promotion of cell motility and proliferation, and morphogenesis. However, little is known about its effects on periodontal ligament (PDL) cells. Recently, we reported that GDNF expression is increased in wounded rat PDL tissue and human PDL cells (HPDLCs) treated with proinflammatory cytokines. Here, we investigated the associated expression of GDNF and the proinflammatory cytokine interleukin-1 beta (IL-1β) in wounded PDL tissue, and whether HPDLCs secrete GDNF which affects neurocytic differentiation. Rat PDL cells near the wounded area showed intense immunoreactions against an anti-GDNF antibody, where immunoreactivity was also increased against an anti-IL-1β antibody. Compared with untreated cells, HPDLCs treated with IL-1β or tumor necrosis factor-alpha showed an increase in the secretion of GDNF protein. Conditioned medium of IL-1β-treated HPDLCs (IL-1β-CM) increased neurite outgrowth of PC12 rat adrenal pheochromocytoma cells. The expression levels of two neural regeneration-associated genes, growth-associated protein-43 (Gap-43) and small proline-rich repeat protein 1A (Sprr1A), were also upregulated in IL-1β-CM-treated PC12 cells. These stimulatory effects of IL-1β-CM were significantly inhibited by a neutralizing antibody against GDNF. In addition, U0126, a MEK inhibitor, inhibited GDNF-induced neurite outgrowth of PC12 cells. These findings suggest that an increase of GDNF in wounded PDL tissue might play an important role in neural regeneration probably via the MEK/ERK signaling pathway. This article is protected by copyright. All rights reserved

Beschreibung: Herpes simplex viruses can cause uncommon systemic complications as acute liver failure or urinary tract dysfunctions. Diphenyl diselenide, (PhSe) 2 , a classical studied organic selenium compound, has a novel antiviral action against HSV-2 infection and well-known antioxidant and anti-inflammatory properties. This study aimed to investigate if (PhSe) 2 reduces oxidative stress and systemic toxicity caused by HSV-2 infection in mice. Adult BALB/c mice were pre-treated with (PhSe) 2 (5 mg kg −1 /day, intragastric, i.g.) during 5 days; at day 6 mice were infected with HSV-2 (10 µl-10 5 PFU/ml −1 ) and post-treated with (PhSe) 2 for more 5 days. At day 11, they were killed and samples of liver and kidney were obtained to determine: reactive species (RS); malondialdehyde (MDA) and non-protein thiols (NPSH) levels; the activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). The activities of adenosine deaminase (ADA), Na + /K + -ATPase (liver and kidney); alanine aminotransferase (ALT), aspartate aminotransferase (AST) and the levels of urea (plasma) were determined as markers of hepatic and renal toxicity. The results revealed that (PhSe) 2 treatment was effective against the increase of renal and hepatic oxidative stress in infected mice and also normalized hepatic and renal ADA activity. It recovered the activity of Na + /K + - and was not effective against the increase in urea levels in infected mice. Different from (PhSe) 2 , acyclovir (positive control), caused an increase in ADA activity and a decrease in hepatic CAT activity. Considering the interest of alternative therapies to treat HSV-2 infections and secondary complications, (PhSe) 2 become a notable candidate. This article is protected by copyright. All rights reserved

Beschreibung: Diclofenac is the most commonly used phenylacetic acid derivative non-steroidal anti-inflammatory drug (NSAID) that demonstrates significant analgesic, antipyretic, and anti-inflammatory effects. Several epidemiological studies have demonstrated anti-proliferative activity of NSAIDs and examined their apoptotic induction effects in different cancer cell lines. However, the precise molecular mechanisms by which these pharmacological agents induce apoptosis and exert anti-carcinogenic properties are not well known. Here, we have observed that diclofenac treatment induces proteasome malfunction and promotes accumulation of different critical proteasome substrates, including few pro-apoptotic proteins in cells. Exposure of diclofenac consequently elevates aggregation of various ubiquitylated misfolded proteins. Finally, we have shown that diclofenac treatment promotes apoptosis in cells, which could be because of mitochondrial membrane depolarization and cytochrome c release into cytosol. This study suggests possible beneficial insights of NSAIDs-induced apoptosis that may improve our existing knowledge in anti-proliferative interspecific strategies development. This article is protected by copyright. All rights reserved

Beschreibung: Apolipoprotein A-I (ApoA-I) is a key component of High Density Lipoproteins which possess anti-atherosclerotic and anti-inflammatory properties. Insulin is a crucial mediator of the glucose and lipid metabolism that has been implicated in atherosclerotic and inflammatory processes. Important mediators of insulin signaling such as Liver X Receptors (LXRs) and Forkhead Box A2 (FOXA2) are known to regulate apoA-I expression in liver. Forkhead Box O1 (FOXO1) is a well-known target of insulin signaling and a key mediator of oxidative stress response. Low doses of insulin were shown to activate apoA-I expression in human hepatoma HepG2 cells. However, the detailed mechanisms for these processes are still unknown. We studied the possible involvement of FOXO1, FOXA2, LXRα and LXRβ transcription factors in the insulin-mediated regulation of apoA-I expression. Treatment of HepG2 cells with high doses of insulin (48 hours, 100 nM) suppresses apoA-I gene expression. siRNAs against FOXO1, FOXA2, LXRβ or LXRα abrogated this effect. FOXO1 forms a complex with LXRβ and insulin treatment impairs FOXO1/LXRβ complex binding to hepatic enhancer and triggers its nuclear export. Insulin as well as LXR ligand TO901317 enhance the interaction between FOXA2, LXRα and hepatic enhancer. These data suggest that high doses of insulin downregulate apoA-I gene expression in HepG2 cells through redistribution of FOXO1/LXRβ complex, FOXA2 and LXRα on hepatic enhancer of apoA-I gene. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Chronic inflammation and metabolic reprogramming have been proposed as hallmarks of cancer development. Currently, many of the functional clues between these two phenomena are studied under the integrative view of functional stroma-epithelia interaction. It has been proposed that stromal cells, due to their abundance and avidity for glucose, are able to modify the metabolic behavior of an entire solid tumor”. In the present study, using a mammary stromal cell line derived from healthy tissue subjected to long-term culture in low (5 mM) or high (25 mM) glucose, we found that the hyperglycemic condition favors the establishment of a pro-inflammatory and pro-oxidant environment characterized by the induction of the COX-2/PGE2 axis. In this condition, epithelial migration was stimulated. Moreover, we also found that stromal-derived PGE2, acting as a stimulator of IL-1 epithelial expression was one of the factors that promote the acquisition of motile properties by epithelial cells and the maintenance of a COX-2/PGE2-dependent inflammatory condition. Overall, our work provides experimental evidence that glucose stimulates a tumor inflammatory environment that, as a result of a functional cross-talk between stroma and epithelia, may be responsible for tumor progression. This article is protected by copyright. All rights reserved

Beschreibung: The neural crest is a population of cells in the vertebrate embryo that gives rise to a wide range of tissues and cell types, including components of the peripheral nervous system and the craniofacial skeleton as well as melanocytes and the adrenal medulla. Aberrations in neural crest development can lead to numerous diseases, including cancers such as melanoma and neuroblastoma. Cancer stem cells (CSCs) have been identified in these neural crest-derived tumors, and these CSCs demonstrate resistance to treatment and are likely key contributors to disease relapse. Patients with neural crest-derived tumors often have poor outcomes due to frequent relapses, likely due to the continued presence of residual treatment-resistant CSCs, and therapies directed against these CSCs are likely to improve patient outcomes. CSCs share many of the same genetic and biologic features of primordial neural crest cells, and therefore a better understanding of neural crest development will likely lead to the development of effective therapies directed against these CSCs. Signaling through STAT3 has been shown to be required for neural crest development, and granulocyte colony stimulating factor (GCSF)-mediated activation of STAT3 has been shown to play a role in the pathogenesis of neural crest-derived tumors. Expression of the cell surface marker CD114 (the receptor for GCSF) has been identified as a potential marker for CSCs in neural crest-derived tumors, suggesting that CD114 expression and function may contribute to disease relapse and poor patient outcomes. In this review we review the processes of neural crest development and tumorigenesis and we discuss the previously identified markers for CSC subpopulations identified in neural crest tumors and their role in neural crest tumor biology. We also discuss the potential for CD114 and downstream intracellular signaling pathways as potential targets for CSC-directed therapy. This article is protected by copyright. All rights reserved

Beschreibung: Human topoisomerase I is partitioned between the nucleolus and the nucleoplasm in the interphase cells. Under unstressed conditions it is concentrated in the first compartment but nucleolar concentration of the full length protein is lost after inactivation of relaxation activity. Due to the above, subnuclear localization of topoisomerase I is linked with DNA relaxation activity of topoisomerase I. Looking for other factors responsible for subnuclear distribution of topoisomerase I, we studied here localization of the fluorescently tagged fragments and point mutants of topoisomerase I in HeLa cells. We found that two regions of topoisomerase I, the N-terminal and the linker domains, were critical for subnuclear localization of the enzyme. The linker domain and the distal region of the N-terminal domain directed topoisomerase I to the nucleolus, whereas the remaining region of the N-terminal domain was responsible for the nucleoplasmic localization. The effects exhibited by the regions which contributed to nuclear distribution of topoisomerase I were independent of DNA relaxation activity. Localization mutations in both domains complemented one another giving the wild type phenotype for the double mutant. These results suggest a two-stage model of regulation of partitioning of topoisomerase I between the nucleolus and the nucleoplasm. The first stage is a net of interactions provided by the N-terminal and the linker domains. The other stage, accessible only if the first net is balanced, is driven by DNA relaxation activity. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT ZC3H12D belongs to a recently discovered family of proteins containing four members of which the most studied and best described is the RNase ZC3H12A (MCPIP1/Regnase-1). ZC3H12A is a crucial negative regulator of inflammation. It accelerates the turnover of transcripts of a spectrum of proinflammatory cytokines, as well as its own mRNA. The biological role of ZC3H12D is less clear, although it was shown that this member of ZC3H12 family is also involved in the regulation of inflammation. Here, we show that ZC3H12A and ZC3H12D recognize a set of common target mRNAs encoding proteins that play important roles in the course of the inflammation. Similarly to ZC3H12A, ZC3H12D participates in the 3'UTR-dependent regulation of the turnover of mRNAs encoding interleukin-6 (IL-6), tumor necrosis factor (TNF) and immediate early response 3 gene ( IER3 ). The ZC3H12A mRNA is also among the identified ZC3H12D targets. Using the combination of immunofluorescence with single molecule RNA fluorescence in situ hybridization (smRNA FISH) we have shown that ZC3H12D protein interacts with the ZC3H12A transcript. The direct binding of these two molecules in vivo was further confirmed by RNA immunoprecipitation. Simultaneously, overexpression of ZC3H12D increases the turnover rate of transcripts containing ZC3H12A 3'UTR. Using reporter gene assays we have confirmed that the Asp95 residue present in the NYN/PIN-like domain is crucial for ZC3H12D biological activity. We have also revealed that ZC3H12D recognizes the same structural elements present in the 3'UTRs of the investigated transcripts, as ZC3H12A. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has driven efforts to develop Hh pathway inhibitors as anti-cancer agents. The majority of these agents antagonize Smoothened (Smo), a plasma membrane-associated signal transducer molecule. However, several such Smo antagonists have failed in clinical trials to benefit patients with cancers that arise from aberrant Hh signaling (which often bypasses Smo). In this study, we report that a naturally occurring oxysterol, 20α, 22( R )-dihydroxycholesterol (Oxy16), a known metabolite in the biosynthesis of steroid hormones, strongly inhibits Hh signaling induced in C3H10T1/2 embryonic fibroblasts and NIH3T3-E1 fibroblasts through a mechanism that is independent of liver X receptor (LXR) activation. We demonstrate that Oxy16 inhibits Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells, indicating that its inhibitory effect on Hh signaling is epistatic to Sufu. We further demonstrate that Oxy16 inhibits Gli1 transcriptional activity in NIH3T3-E1 cells overexpressing Gli1 and a Gli-dependent reporter construct. Altogether, data presented here suggest that Oxy16 may be a suitable starting point for the development of new drugs that inhibit Hh signaling downstream of Smo. By targeting aberrant Hh signaling, such novel Hh pathway inhibitors could significantly broaden the range of clinical applications compared to existing Smo antagonists. Furthermore, the present study adds a new facet to the spectrum of Hh pathway modulation that naturally occurring oxysterol derivatives are capable of, ranging from allosteric activation of the pathway via Smo binding to inhibition of the pathway downstream of Smo. This article is protected by copyright. All rights reserved

Beschreibung: Reduced insulin receptor protein levels have been reported in the kidney cortex from diabetic humans and animals. We recently reported that targeted deletion of insulin receptor (IR) from proximal tubules (PT) resulted in hyperglycemia in non-obese mice. To elucidate the mechanism, we examined human proximal tubule cells (hPTC) and C57BL/6 mice fed with high-fat diet (HFD, 60% fat for 20 weeks). Immunoblotting revealed a significantly lower protein level of IR in HFD compare to normal chow diet (NCD). Furthermore, a blunted rise in p-AKT 308 levels in the kidney cortex of HFD mice was observed in response to acute insulin (0.75 IU/kg body weight, i.p) relative to NCD n = 8/group, p 〈 0.05). Moreover, we found significantly higher transcript levels of phosphoenolpyruvate carboxykinase (PEPCK, a key gluconeogenic enzyme) in the kidney cortex from HFD, relative to mice on NCD. The higher level of PEPCK in HFD was confirmed by immunoblotting. However, no significant differences were observed in cortical glucose-6-phosphatase (G6Pase) or fructose-1,6, bisphosphosphatase (FBPase) enzyme transcript levels. Furthermore, we demonstrated insulin inhibited glucose production in hPTC treated with cyclic AMP and dexamethasone (cAMP/DEXA) to stimulate gluconeogenesis. Transcript levels of the gluconeogenic enzyme PEPCK were significantly increased in cAMP/DEXA-stimulated hPTC cells (n = 3, p 〈 0.05), and insulin attenuated this upregulation Furthermore, the effect of insulin on cAMP/DEXA–induced gluconeogenesis and PEPCK induction was significantly attenuated in IR (siRNA) silenced hPTC (n = 3, p 〈 0.05). Overall the above data indicate a direct role for IR expression as a determinant of PT-gluconeogenesis. Thus reduced insulin signaling of the proximal tubule may contribute to hyperglycemia in the metabolic syndrome via elevated gluconeogenesis. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Osteoblasts are essential for maintaining skeletal architecture and modulating bone microenvironment homeostasis. From numerous associated investigations, the BMP-2 pathway has been well-defined as a vital positive modulator of bone homeostasis. Gremlin2 (Grem2) is a bone morphogenetic protein (BMP) antagonists. However, the effect of Grem2 on the BMP-2-induced osteogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs) remains ambiguous. This study aimed to analyze the procedure in vitro and in vivo. The differentiation of hBMSCs was assessed by determining the expression levels of several osteoblastic genes, as well as the enzymatic activity and calcification of alkaline phosphatase. We found that Grem2 expression was upregulated by BMP-2 within the range of 0–1 µg/mL, and significant increases were evident at 48, 72, and 96 h after BMP-2 treatment. Si-Grem2 increased the BMP-2-induced osteogenic differentiation of hBMSCs, whereas over-expression of Grem2 had the opposite trend. The result was confirmed using a defective femur model. We also discovered that the BMP-2/Smad/Runx2 pathway played an important role in the process. This study showed that si-Grem2 increased the BMP-2-induced osteogenic differentiation of hBMSCs via the BMP-2/Smad/Runx2 pathway. This article is protected by copyright. All rights reserved

Beschreibung: The expression of Caudal -related homeobox transcription factor 2 (CDX2) is impaired by tumor necrosis factor-α (TNF-α)-mediated activation of nuclear factor-κB (NF-κB) in ulcerative colitis (UC). Laminin subunit γ2 (LAMC2) is an epithelial basement membrane protein implicated in cell migration, proliferation, differentiation as well as tumor invasion and intestinal inflammation, and its expression is enhanced by TNF-α in a NF-κB-dependent regulation of the recently identified LAMC2 enhancer. The aim was to determine whether CDX2 is involved in the basal regulation of LAMC2 in epithelial cells and to assess the influence of inflammation. Transcriptional regulation of LAMC2 was examined by reporter gene assays, overexpression, and shRNA-mediated knock-down of CDX2 . CDX2-DNA interactions were assessed by chromatin immunoprecipitation on Caco-2 cells without or with TNF-α as well as in purified colonic human epithelial cells. Immunohistochemical staining and quantitative reverse-transcription polymerase chain reaction analyses were used to measure the expression of CDX2 and LAMC2 in colonic biopsies from healthy controls and patients with UC. These data indicate that CDX2 directly regulates LAMC2 gene expression through interaction with elements in the LAMC2 promoter region. We further revealed an inverse effect of inflammation on CDX2 and LAMC2. The data presented provide a novel insight into how CDX2 is implicated in the transcriptional regulation of LAMC2 in intestinal epithelial cells, a function that is impaired during mucosal inflammation where a high level of TNF-α is present. This article is protected by copyright. All rights reserved

Beschreibung: Human multipotent mesenchymal stromal cells (hMSCs) possess the ability to differentiate into osteoblasts, and they can be utilized as a source for bone regenerative therapy. Osteoinductive pretreatment, which induces the osteoblastic differentiation of hMSCs in vitro, has been widely used for bone tissue engineering prior to cell transplantation. However, the molecular basis of osteoblastic differentiation induced by osteoinductive medium (OIM) is still unknown. Therefore, we used a next-generation sequencer to investigate the changes in gene expression during the osteoblastic differentiation of hMSCs. The hMSCs used in this study possessed both multipotency and self-renewal ability. Whole-transcriptome analysis revealed that the expression of zinc finger and BTB domain containing 16 (ZBTB16) was significantly increased during the osteoblastogenesis of hMSCs. ZBTB16 mRNA and protein expression was enhanced by culturing the hMSCs with OIM. Small interfering RNA (siRNA)-mediated gene silencing of ZBTB16 decreased the activity of alkaline phosphatase (ALP); the expression of osteogenic genes, such as osteocalcin (OCN) and bone sialoprotein (BSP); and the mineralized nodule formation induced by OIM. siRNA-mediated gene silencing of Osterix (Osx), which is known as an essential regulator of osteoblastic differentiation, markedly downregulated the expression of ZBTB16. In addition, chromatin immunoprecipitation (ChIP) assays showed that Osx associated with the ZBTB16 promoter region containing the GC-rich canonical Sp1 sequence, which is the specific Osx binding site. These findings suggest that ZBTB16 acts as a downstream transcriptional regulator of Osx and can be useful as a late marker of osteoblastic differentiation. This article is protected by copyright. All rights reserved

Beschreibung: Phototherapy is the most common therapy used for severe jaundice. There is increasing evidence that phototherapy can directly affect the expression and function of cell surface receptors including adhesion molecules, cytokines, and growth factor receptors. In this study, the effect of two infantile phototherapy regimens, including single and intensive phototherapy was investigated on biological features of circulation endothelial progenitor cells (cEPCs) as well as on serum secretion of two important chemotactic cytokines, SDF-1 and VEGF. Sixty infants diagnosed with severe hyperbilirubinemia and exposed to phototherapy were enrolled in this study. cEPCs were isolated before and after phototherapy and then migratory, proliferative, tubulogenic, and functional properties of these cells were analyzed. Our results revealed that intensive phototherapy markedly increased the release of EPCs into the circulation, and augmented the serum concentrations of both SDF-1 and VEGF cytokines. Cell proliferation, tubulogenic, and migratory properties of cEPCs isolated and expanded from infants with intensive phototherapy were significantly improved. cEPCs from infants with intensive phototherapy also showed greater levels of acetylated low-density lipoprotein and lectin binding. Overall, our results showed that the intensive phototherapy regimen can mobilize functional EPCs into the circulation through up-regulation of serum levels of VEGF and SDF-1, indicating phototherapy as an effective modality for improvement of stem cell mobilization in the therapeutic regenerative medicine. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT The mammalian hairless (Hr) protein plays critical roles in skin and brain tissues, but how it interacts with DNA and partner protein is only now being defined. Our initial tests of four consensus response elements, revealed that rat Hr can specifically bind to a consensus p53 response element (p53RE), 5'-AGACATGCCTAGACATGCCT-3', but not to response elements for NF-κB, TCF4 or Sp1. We then employed ChIP assays which verified that human HR binds to a p53RE of the GADD45A gene in both HEK293 (embryonic kidney) and U87 (glioblastoma) cells. Further, HR was shown to interact directly with the p53 protein in a co-immunoprecipitation assay. Cotransfections with p53RE reporter gene constructs revealed that rat Hr can boost p53-mediated transactivation of a reporter gene linked to the GADD45A p53RE, but blunts p53-mediated transactivation when the reporter gene is linked to a p21 promoter fragment containing a p53RE, with implications for the regulation of these two cell cycle control genes. Finally, our investigations of HR phosphorylation revealed that rat Hr is a substrate for PKC, but not PKA, and that human HR is phosphorylated in intact U87 cells at Ser-416, located in a highly conserved region which partially fulfills the criteria of a PKC site. We propose that mammalian Hr is a phosphoprotein which can exert cross-talk with the p53 pathway with important implications for the regulation of cell proliferation and differentiation in tissues such as skin and brain where Hr is highly expressed. This article is protected by copyright. All rights reserved

Beschreibung: The majority of advanced breast cancers have genetic alterations that are potentially targetable with drugs. Through initiatives such as The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), data can be mined to provide context for next-generation sequencing (NGS) results in the landscape of advanced breast cancer. Therapies for targets other than estrogen receptor alpha (ER) and HER2, such as cyclin-dependent kinases CDK4 and CDK6, were recently approved based on efficacy in patient subpopulations, but no predictive biomarkers have been found, leaving clinicians to continue a trial-and-error approach with each patient. Next-generation sequencing identifies potentially actionable alterations in genes thought to be drivers in the cancerous process including phosphatidylinositol 3-kinase (PI3K), AKT, fibroblast growth factor receptors (FGFRs), and mutant HER2. Epigenetically-directed and immunologic therapies have also shown promise for the treatment of breast cancer via histone deacetylases (HDAC) 1 and 3, programmed T cell death 1 (PD-1), and programmed T cell death ligand 1 (PD-L1). Identifying biomarkers to predict primary resistance in breast cancer will ultimately affect clinical decisions regarding adjuvant therapy in the first-line setting. However, the bulk of medical decision-making is currently made in the secondary resistance setting. Herein, we review the clinical potential of PI3K, AKT, FGFRs, mutant HER2, HDAC1/3, PD-1, and PD-L1 as therapeutic targets in breast cancer, focusing on the rationale for therapeutic development and the status of clinical testing. This article is protected by copyright. All rights reserved

Beschreibung: Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer related deaths among women worldwide. The purpose of this study is to evaluate the cytotoxic effects and possible molecular mechanisms of the antiproliferative properties of the antiangiogenic 1-aryl-3-[3-(thieno[3,2- b ]pyridin-7-ylthio)phenyl]ureas 1a - e , prepared earlier by us, on two human breast cancer cell lines of distinct histological types: hormone-dependent MCF-7 (ER positive), and hormone independent MDA-MB-231 (ER/PR/HER2 negative), this latter being the most aggressive and difficult to treat. Our findings clearly demonstrated that compounds 1a - e suppress breast cancer cell survival, proliferation, migration and colony formation at very low concentrations, not showing cytotoxicity in normal human mammary cells (MCF-10A). TUNEL assay demonstrated that compounds 1a - e induced apoptosis in MDA-MB-231, but not in MCF-7 at the concentrations tested. PI3K/Akt and MAPK/Erk cell signaling pathways were investigated using Western blot analysis, revealing that these compounds decrease their activity in both breast cancer cell lines. Compounds 1b (R 2  = F) 1c (R 2  = Me) and 1e (R 1  = Cl, R 2  = CF 3 ) were the most effective particularly in MDA-MB-231 cells. Overall, 1c and 1e compounds are the most promising antitumor compounds. These findings, together with the antiangiogenic activity previously described by us, render these compounds a relevant breakthrough for cancer therapy. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Tau is a microtubule-associated protein implicated in the pathogenesis of Alzheimer's disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto-assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post-translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol-induced hyperosmotic stress promotes Tau proteolysis in SH-SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of µ-calpain, the proteasome system and caspase-3. Tau proteolysis was completely prevented by caspase-3 inhibition but unaffected by neither the proteasome system nor µ-calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH-SY5Y cells, which was efficiently avoided by the inhibition of caspase-3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase-3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH-SY5Y cells. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest-specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the number and duration of G 1 phase in PCa cells. Importantly, GAS6 further increased the number of G 1 arrested cells during docetaxel chemotherapy. GAS6 altered the signals of key cell cycle regulators: Cyclin B1 (G 2 /M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry) were all downregulated, while p27, p21, Cyclin D1, and CDK4 (G 0 /G 1 phase) were upregulated. Importantly, these signaling events were further accentuated during docetaxel treatment in the presence of GAS6. Moreover, the apoptotic response of PCa cells to GAS6 was examined during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis. However, this apoptotic response was abrogated in PCa cell cultures in the presence of GAS6 or GAS6 secreted from co-cultured osteoblasts. Similarly, the GAS6-expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. In addition, docetaxel induced significant levels of Caspase-3 and PARP cleavages in PCa cells, while GAS6 protected PCa cells from docetaxel-induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which may have important implications for targeting metastatic disease. This article is protected by copyright. All rights reserved

Beschreibung: A causal link between benign prostatic hyperplasia (BPH) and prostate cancer has long been suspected but not widely accepted. A new model is proposed that supports such a connection. In contrast to the prevailing wisdom, our model, that draws on dynamical systems theory, suggests that in response to stress, epithelial cells in the unstable gland can give rise to both types of diseases via a phenotypic switching mechanism. The central idea is that phenotypic switching is a stochastic process which exploits the plasticity of the epithelial cell. It is driven by ‘noise’ contributed by the conformational dynamics of proteins that are intrinsically disordered. In a system that is noisy when stressed, disorder promotes promiscuity, unmasks latent information, and rewires the network to cause phenotypic switching. Cells with newly acquired phenotypes can transcend the traditional zonal boundaries to give rise to BPH or prostate cancer depending on the microenvironment. Establishing causality between the two diseases may provide us with an opportunity to better understand their etiology and guide prevention and treatment strategies. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Curcumin has demonstrated valuable therapeutic potential against a variety of human cancers including osteosarcoma. However, the molecular mechanisms underlying its anti-tumor effect remain to be poorly understood. By RNA sequence profiling, we found that curcumin significantly down-regulates the expression of estrogen-related receptor alpha (ERRα) in osteosarcoma cells. Overexpression of ERRα diminished curcumin-activated apoptotic cell death and scavenged curcumin-induced reactive oxygen species (ROS), while ERRα silencing sensitized osteosarcoma cells to curcumin, resulting in increased inhibition of cell proliferation. In addition, we found that curcumin suppressed the ERRα gene expression through upregulation of miR-125a. Data from this study revealed a novel mechanism for curcumin-mediated apoptotic cell death, which involves tumor cell killing via activating miR-125a/ERRα pathway. Our studies also provide further support for osteosarcoma therapy by targeting ERRα alone or in combination with curcumin. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Muscarinic acetylcholine receptors (mAChRs) regulate diverse cellular functions, including cell growth and proliferation, via multiple signaling pathways. Previously, we showed that mAChRs stimulate the MEK1/2-ERK1/2-RSK pathway in SNU-407 colon cancer cells and subsequently promote cell proliferation. In this study, we provide evidence that the PI3K-Akt-mTORC1-S6K1 pathway is activated by mAChRs in SNU-407 cells and that this pathway is associated with protein biosynthesis and cell proliferation. When the cells were treated with the cholinergic agonist carbachol, Akt was activated in a dose- and time-dependent fashion. This carbachol effect was almost completely blocked by the PI3K inhibitor LY294002, implying that PI3K is responsible for the Akt activation. S6K1, a major downstream target of mTORC1, was also activated by carbachol in a temporal profile similar to that of the Akt activation. This carbachol-stimulated S6K1 activation was abrogated by LY294002 or the mTORC1 inhibitor rapamycin, supporting the notion that mAChRs mediate S6K1 activation via the PI3K-Akt-mTORC1 pathway. We observed that global protein biosynthesis, monitored by puromycin incorporation, was strongly increased by carbachol in an atropine-sensitive manner. Inhibition experiments indicated that the ERK1/2 and mTORC1 signaling pathways may be involved in carbachol-stimulated global protein biosynthesis. We also found that treating SNU-407 cells with LY294002 or rapamycin significantly suppressed carbachol-stimulated cell proliferation. In the presence of the MEK1/2 inhibitor U0126, cell proliferation was further reduced by rapamycin treatment. Our data thus suggest that both the MEK1/2-ERK1/2 and mTORC1 pathways play important roles in mAChR-mediated cell proliferation in SNU-407 colon cancer cells. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT MSC-like populations derived from induced pluripotent stem cells (iPSC-MSC) serve as an alternative stem cell source due to their high proliferative capacity. In this study, we assessed the immunomodulatory potential of iPSC-MSC generated from periodontal ligament (PDL) and gingival (GF) tissue. The iPSC-MSC lines exhibited a similar level of suppression of mitogen-stimulated peripheral blood mononuclear cells (PBMNC) proliferation compared to their respective parental fibroblast populations in vitro . Moreover, iPSC-MSC demonstrated the ability to suppress T-cells effector cells, Th1/Th2/Th17 populations, and increase levels of Treg cells. In order to investigate the mechanisms involved, expression of common MSC-derived soluble factors known to supress lymphocyte proliferation were assessed in iPSC-MSC cultured with PBMNC with direct cell-cell contact or separated in transwells. Real-time PCR analysis of factors known to be involved in MSC mediated immune regulation, found a general trend of elevated IDO1 and IL6 transcript levels in iPSC-MSC lines and their respective primary cells co-cultured with activated PBMNC, with a wide range of gene expression levels between the different mesenchymal cell types. The results suggest that different iPSC-MSC may be useful as a potential alternative source of cells for future clinical use in therapeutic applications because of their potent immunosuppressive properties. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Several studies have shown the degradation of the extracellular matrix at the site of neuroinflammation and increased release of degradation products of glycosaminoglycans. Among these, low molecular weight fragments of hyaluronan (HA) may play a key role in the events leading to neuroinflammation and/or neuronal degeneration. Small HA fragments are able to induce inflammation by stimulating both TLR-2 and TLR-4 as well as CD44 receptors. This stimulation culminates in the nuclear translocation of NF-kB that in turn induces the production of pro-inflammatory intermediates such as TNF-α and IL-1β. The potential of HA fragments, as mediators of inflammation, it has been poorly investigated in neuron-like SH-SY5Y cells so, the aim of this study was to investigate the neuroinflammatory effects of very small HA oligosaccharides, the involvement of TLR-2, TLR-4 and CD44 and the production of α-synuclein in such cells. The addition of HA fragments to cell cultures up-regulated TLR-2, TLR-4 and CD44 levels, induced NF-kB activity and increased both TNF-α and IL-β as well as α-synuclein production. On blocking the activity of TLR-2, TLR-4 and CD44 the levels of inflammatory parameters and of α-synuclein were significantly reduced. Since several data have shown as α-synuclein, produced from neurons, is able to initiates ex novo or to maintain an existing neuroinflammatory response, which has been suggested as one of the principal components involved in neurodegenerative pathologies, as PD, we suggest that HA pathways should be given careful consideration when devising future anti-neuroinflammatory strategies to defend against the onset of neurodegenerative disorders. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Cancer cell lines have become a reliable tool in genetic and biochemical studies of breast cancer. Here, we described the behavior and novel molecular characterization of two cell lines derived from DMBA-induced rat mammary tumor, LA7 and RBA. LA7 cells have been identified as myoepithelial cells with stem cell properties, whereas the RBA cell line are epithelial cells that present mutational activated H-Ras, but are much less known. We evaluated the proliferation rate and molecular markers, several signaling pathways status related to proliferation, survival, inflammation and apoptosis, as well as migration capacity, global DNA methylation levels, and stem cells populations. In fact, we found the A/T transversion in the c-Ha-Ras codon 61 as the activator mutation origin described in RBA cells. LA7 and RBA cells showed a high proliferation rate associated with overexpression of Cyclin D1, and resistance to apoptotic signals due to lack of expression of Bad. Moreover, neither of these two cell lines expressed steroid receptors, but they showed high migration capacity, all in accordance with an aggressive phenotype. We found global DNA methylation levels in LA7 and RBA cells lower than reference tissues analyzed, in addition to the presence of different stem cells populations in RBA cell line that differed in the expression of CD44 and CD24. These results revealed a malignant behavior associated with cancer stem cell phenotype. Since this profile is similar to a human triple-negative basal-like tumor, their extensive characterization presented herein increases their value as a good in vitro model. This article is protected by copyright. All rights reserved

Beschreibung: Cancer therapeutics is a hot subject and PI3K class 1A isoforms (p110α and p110β) are pursued as major targets. Genetic analysis, biochemical approaches and structural studies have demonstrated crucial roles for these isoforms in several physiological processes. p110α is critical for insulin signaling whereas p110β is essential for the growth and differs from p110α in many ways. Here, we have generated and expressed GFP-fusion proteins of p110α and p110β in HEK293 cells to examine their subcellular localization and their impact on downstream signalling. In HEK293 cells, p110β GFP-fusion protein is translocated into the nucleus whereas p110α-GFP stays exclusivelly in the cytoplasm. This study demonstrates that p110α and p110β oncogenecity, kinase activity and interaction with p85 regulatory subunit does not have any impact on their subcellular localization. PI3K pathway specific inhibitor, LY294002, abrogated PI3K signaling by reducing pAkt levels, however, the subcellular localization of p110α and p110β remained unchanged. Moreover, we analyzed the expression of recombinant p110α and p110β in a panel of human cancer cells and observed remarkable differences in their expression levels. The differential expression of recombinant p110α and p110β was observed to be mainly regulated by the endogenous levels of pAkt. Unlike in HEK293, p110α showed nuclear localization in cancer cells in a similar fashion to p110β. Furthermore, we observed the PI3K signaling activities in low pAkt expressing cells is mediated by PDK1 and S6K proteins. Finally, p110α and p110β were seen to play an essential role in promoting the cell cycle progression in MCF-7 and HCT-116 cells. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Recent studies describing the mutational landscape of head and neck squamous cell carcinoma (HNSCC) on a genomic scale by our group and others, including The Cancer Genome Atlas, have provided unprecedented perspective for understanding the molecular pathogenesis of HNSCC progression and response to treatment. These studies confirmed that mutations of the TP53 tumor suppressor gene were the most frequent of all somatic genomic alterations in HNSCC, alluding to the importance of the TP53 gene in suppressing the development and progression of this disease. Clinically, TP53 mutations are significantly associated with short survival time and tumor resistance to radiotherapy and chemotherapy in HNSCC patients, which makes the TP53 mutation status a potentially useful molecular factor for risk stratification and predictor of clinical response in these patients. In addition to loss of wild-type p53 function and the dominant-negative effect on the remaining wild-type p53, some p53 mutants often gain oncogenic functions to promote tumorigenesis and progression. Different p53 mutants may possess different gain-of-function properties. Herein we review the most up-to-date information about TP53 mutations available via The Cancer Genome Atlas-based analysis of HNSCC and discuss our current understanding of the potential tumor-suppressive role of p53, focusing on gain-of-function activities of p53 mutations. We also summarize our knowledge regarding use of the TP53 mutation status as a potential evaluation or stratification biomarker for prognosis and a predictor of clinical response to radiotherapy and chemotherapy in HNSCC patients. Finally, we discuss possible strategies for targeting HNSCCs bearing TP53 mutations. This article is protected by copyright. All rights reserved

Beschreibung: SIRT3, the major deacetylase in mitochondria, plays a crucial role modulating ROS production and scavenging by regulating key proteins implicated in mitochondrial turnover and in antioxidant defenses. Therefore, SIRT3 could confer resistance to chemotherapy-induced oxidative stress, leading to a lower ROS production and a higher cell survival. Our aim was to analyze whether SIRT3 silencing in breast cancer cells through a specific siRNA could increase oxidative stress and thus compromise the antioxidant response, resulting in a sensitization of the cells to cisplatin (CDDP) or tamoxifen (TAM). For this purpose, we studied cell viability, ROS production, apoptosis and autophagy in MCF-7 and T47D cell lines treated with these cytotoxic compounds, these either alone, or in combination with SIRT3 silencing. Moreover, protein levels regulated by SIRT3 were also examined and survival curves were analyzed to study the importance of SIRT3 expression for the overall survival of breast cancer patients. When SIRT3 was silenced and combined with cytotoxic treatments, cell viability was highly decreased, and was accompanied by a significant increase in ROS production. While TAM treatment increased autophagic cell death, CDDP significantly triggered apoptosis, whereas SIRT3 silencing produced an enhancement of these two action mechanisms. SIRT3 knockdown also affected PGC-1α and TFAM (mitochondrial biogenesis), and MnSOD and IDH 2 (antioxidant defenses) protein levels. Finally, survival curves showed that higher SIRT3 expression is correlated to a poorer prognosis for patients with grade 3 breast cancer. In conclusion, SIRT3 could be a therapeutic target for breast cancer, improving the effectiveness of CDDP and TAM treatments. This article is protected by copyright. All rights reserved

Beschreibung: Stimulation of glucose transport is markedly impaired in cardiomyocytes exposed to free fatty acids (FFA), despite relative preservation of canonical insulin- or metabolic stress signaling. We determined whether Focal Adhesion Kinase (FAK) activity is required for stimulation of glucose transport in cardiomyocytes, and whether FAK downregulation participates in FFA-induced impairment of glucose transport stimulation. Glucose transport, measured in isolated cultured cardiomyocytes, was acutely stimulated either by insulin treatment, or by metabolic inhibition with oligomycin resulting in AMP-activated kinase (AMPK) activation. FAK activity was inhibited pharmacologically by preincubation with PF-573,228 (PF). FAK activity was assessed from its autophosphorylation on residue Y397, and from the phosphorylation of its target paxillin on Y118. Y397 FAK phosphorylation was reduced in cultured cardiomyocytes chronically exposed to FFA. Preincubation with PF prior to determination of glucose transport resulted in a significant reduction of oligomycin-stimulated glucose transport, with a lesser reduction in insulin-stimulated glucose transport. Insulin and AMPK signaling was unaffected by PF preincubation. siRNA-mediated FAK knockdown also resulted in reduced oligomycin-stimulated glucose transport. Chronic treatment of FFA-exposed cardiomyocytes with phenylephrine or a phorbol ester restored FAK activity and improved glucose transport. In conclusion, stimulation of glucose transport in cardiomyocytes requires FAK activity prior to stimulation. The chronic reduction of FAK activity in cardiomyocytes exposed to FFA contributes to the loss of glucose transport responsiveness to insulin or metabolic inhibition. This article is protected by copyright. All rights reserved

Beschreibung: Diabetes mellitus is a metabolic disorder characterized by hyperglycemia. We investigated the effect of a prior thirty days voluntary exercise protocol on STZ-diabetic CF1 mice. Glycemia, and the liver and skeletal muscle glycogen, mitochondrial function and redox status were analyzed up to five days after STZ injection. Animals were engaged in the following groups: Sedentary vehicle (Sed Veh), Sedentary STZ (Sed STZ), Exercise Vehicle (Ex Veh) and Exercise STZ (Ex STZ). Exercise prevented fasting hyperglycemia in the Ex STZ group. In the liver: There was decreased on glycogen level in Sed STZ group but not in EX STZ group. STZ groups showed decreased mitochondrial oxygen consumption compared to vehicle groups, whereas mitochondrial H2O2 production was not different between groups. Addition of ADP to the medium did not decrease H2O2 production in Sed STZ mice. Exercise increased GSH level. Sed STZ group increased nitrite levels compared to other groups. In quadriceps muscle: glycogen level was similar between groups. The Sed STZ group displayed decreased O2 consumption, and exercise prevented this reduction. The H2O2 production was higher in Ex STZ when compared to other groups. Also, GSH level decreased whereas nitrite levels increased in the Sed STZ compared to other groups. The PGC1 α levels increased in Sed STZ, Ex Veh and Ex STZ groups. In summary, prior exercise training prevents hyperglycemia in STZ-induced diabetes associated with increased liver glycogen storage, and oxygen consumption by the mitochondria of skeletal muscle implying in increased oxidative/biogenesis capacity, and improved redox status of both tissues. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT High mobility group box protein-1 (HMGB1) is released from cells under various pathological conditions and it plays a pivotal role as an alarmin signaling tissue damage. Little is known about the impact of HMGB1 in bone repair and remodeling. To this aim, we focused on HMGB1-induced effects on the in vitro osteoblast model SaOS-2. Cell proliferation was stimulated with a maximum at concentration of 2.5 nM, and such a dose also stimulated cell migration and scratch wound healing. We then characterized the modulatory effect of HMGB1 on bone biology, by using osteogenesis/mineralization assays, a PCR array, and the analysis of a series of osteogenic markers. We performed also a proteomic screening using SWATH-MS on SaOS-2 cell exposed to HMGB1 and we provide evidence for proteins modulated in HMGB1 exposed cells. Taken together, our data demonstrate that SaOS-2 cell proliferation, migration, and osteogenic differentiation were increased by HMGB1. We therefore propose that HMGB1 could be a potent bone-remodeling signal but the physiological meaning of this property remains to be more ascertained. This article is protected by copyright. All rights reserved

Beschreibung: Phospho-tyrosine signaling networks control numerous biological processes including cellular differentiation, cell growth and survival, motility and invasion. Aberrant regulation of the tyrosine kinome is a hallmark of malignancy and influences all stages of breast cancer progression; from initiation to the development of metastatic disease. The success of specific tyrosine kinase inhibitors strongly validates the clinical relevance of tyrosine phosphorylation networks in breast cancer pathology. However, a significant degree of redundancy exists within the tyrosine kinome. Numerous receptor and cytoplasmic tyrosine kinases converge on a core set of signaling regulators, including adaptor proteins and tyrosine phosphatases, to amplify pro-tumorigenic signal transduction pathways. Mutational activation, amplification or overexpression of one or more components of the tyrosine kinome represents key contributing events responsible for the tumor heterogeneity that is observed in breast cancers. It is this molecular heterogeneity that has become the most significant barrier to durable clinical responses due to the development of therapeutic resistance. This review focuses on recent literature that supports a prominent role for specific components of the tyrosine kinome in the emergence of unique breast cancer subtypes and in shaping breast cancer plasticity, sensitivity to targeted therapies and the eventual emergence of acquired resistance. This article is protected by copyright. All rights reserved

Beschreibung: It is a common complication to develop a secondary lymphedema after surgery or radiation, for example after axillary lymph node dissection due to breast cancer and current therapies are mainly symptomatic. Since these surgical procedures result in both, loss of adipose tissue and loss of lymphatic nodes and vessels, tissue engineering could be a new promising approach, to create an adipose tissue substitute comprised with a lymphatic network. We have conducted co-culture experiments to investigate the effects of human adipose-derived stem cells (ASCs) on human lymphatic endothelial cells (LECs) in terms of gene expression profile, proliferation, migration, and tube formation in vitro. In this respect, both cell types were co-cultured either indirectly or directly with or without the recombinant growth factor VEGF-C. Indirect co-cultures were performed with the aid of a transwell chamber. In case of direct co-culture, immunomagnetic separation by CD31 magnetic beads allowed examination of the LEC population. Direct and indirect co-culture of ASCs induced mRNA expression of lymphatic marker genes, proliferation and migration by LECs without affecting tube formation. Thus, we have shown that co-culture of ASCs with LECs might be a feasible approach that could be used in cell-based tissue engineering therapies to heal or improve a secondary lymphedema. This article is protected by copyright. All rights reserved

Beschreibung: Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated to RNA related molecules. This article is protected by copyright. All rights reserved

Beschreibung: The in vitro osteogenic differentiation has been intensively studied. However, it is not yet clear precisely how osteogenesis can be optimized. Changes in extracellular Ca 2+ concentration ([Ca 2+ ] e ), as well as modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the effects of a combined treatment of ATPγ-S and high [Ca 2+ ] e (5.35 mM) on osteogenic differentiation and function of primary cell cultures from rat calvaria. Our results indicate that ATPγ-S stimulates cell transition from the G 0 to S phase of cell cycle, involving the PI3K signaling pathway. Treatment with 10 or 100 µM ATPγ-S and [Ca 2+ ] e (ATP-[Ca 2+ ] e ) for 48 h increases cell number significantly above the control. ATPγ-S treatment in osteogenic medium containing [Ca 2+ ] e stimulates the gene expression of BMP-4, BMP-5 and OPN at 16, 48 and 72 h respectively above control. In same conditions, treatment for 6 days with 10 µM UTP or 100 µM UDP significantly increased the ALP activity respect to control. Cells grown in osteogenic medium showed a statistically significant increase in calcium deposits at 15 and 18 days, for 10 µM ATPγ-S treatment, and at 18 and 22 days, for [Ca 2+ ] e treatment, respect to control but ATP-[Ca 2+ ] e treatment shown a significant greater mineralization at 15 days respect to ATPγ-S, and at 18 days respect to both agonists. In conclusion, we demonstrated that an osteogenic medium containing 10 µM ATPγ-S and 5.35 mM [Ca 2+ ] e enhance osteogenesis and mineralization by rat primary calvarial cells cultures. This article is protected by copyright. All rights reserved

Beschreibung: The flap region in aspartic proteases, is a unique structural feature to this class of enzymes, and found to have a profound impact on protein overall structure, function and dynamics. Understanding the structure and dynamic behaviour of the flap regions is crucial in the design of selective inhibitors against aspartic proteases. Cathepsin-D, an aspartic protease enzyme, has been implicated in a long list of degenerative diseases as well as breast cancer progression. Presented herein, for the first time, is a comprehensive description of the conformational flap dynamics of cathepsin-D using a comparative 50ns “multiple” molecular dynamics simulations. Diverse collective metrics were proposed to accurately define flap dynamics. These are: distance d 1 between the flap tips residues (Gly79 and Met301); dihedral angle φ; in addition to TriCα angles Gly79-Asp33-Asp223, θ 1 , and Gly79-Asp223-Met301, θ 2 . The maximum distance attained throughout the simulation was 17.42Å and 11.47Å for apo and bound cathepsin-D respectively, while the minimum distance observed was 8.75Å and 6.32Å for apo and bound cathepsin-D, respectively. The movement of the flap as well as the twist of the active pocket can properly be explained by measuring the angle, θ 1, between Gly79-Asp33-Met301 and correlating it with the distance Cα of the flap tip residues. The asymmetrical opening of the binding cavity was best described by the large shift of -6.26 o to +20.94 o in the dihedral angle, φ, corresponding to the full opening of the flap at a range of 31-33ns. A wide-range of post-dynamic analyses was also applied in this report to supplement our findings. We believe that this report would augment current efforts in designing potent structure-based inhibitors against cathepsin-D in the treatment of breast cancer and other degenerative diseases. This article is protected by copyright. All rights reserved

Beschreibung: ACTH treatment has been proven to promote paxillin dephosphorylation and increase soluble protein tyrosine phosphatase (PTP) activity in rat adrenal zona fasciculata (ZF). Also, in-gel PTP assays have shown the activation of a 115-kDa PTP (PTP115) by ACTH. In this context, the current work presents evidence that PTP115 is PTP-PEST, a PTP that recognizes paxillin as substrate. PTP115 was partially purified from rat adrenal ZF and PTP-PEST was detected through Western blot in bioactive samples taken in each purification step. Immunohistochemical and RT-PCR studies revealed PTP-PEST expression in rat ZF and Y1 adrenocortical cells. Moreover, a PTP-PEST siRNA decreased the expression of this phosphatase. PKA phosphorylation of purified PTP115 isolated from non-ACTH-treated rats increased K M and V M . Finally, in-gel PTP assays of immunoprecipitated paxillin from control and ACTH-treated rats suggested a hormone-mediated increase in paxillin-PTP115 interaction, while PTP-PEST and paxillin co-localize in Y1 cells. Taken together, these data demonstrate PTP-PEST expression in adrenal ZF and its regulation by ACTH/PKA and also suggest an ACTH-induced PTP-PEST-paxillin interaction. This article is protected by copyright. All rights reserved

Beschreibung: Insight into the processes controlling adipogenesis is important in the battle against the obesity epidemic and its related disorders. The transcriptional regulatory cascade involved in adipocyte differentiation has been extensively studied, however the mechanisms driving the transcription activation are still poorly understood. In this study we explored the involvement of DNA methylation in transcriptional regulation during adipocyte differentiation of primary human mesenchymal stem cells (hMSCs). Genome-wide changes in DNA methylation were measured using the Illumina 450K BeadChip. In addition, expression of 84 adipogenic genes was determined, of which 43 genes showed significant expression changes during the differentiation process. Among these 43 differentially expressed genes, differentially methylated regions (DMRs) were detected in only 3 genes. By comparing genome-wide DNA methylation profiles in undifferentiated and differentiated adipocytes 793 significant DMRs were detected. Pathway analysis revealed the adipogenesis pathway as the most statistically significant, although only a small number of genes were differentially methylated. Genome-wide DNA methylation changes for single probes were most often located in intergenic regions, and underrepresented close to the transcription start site. In conclusion, DNA methylation remained relatively stable during adipocyte differentiation, implying that changes in DNA methylation are not the underlying mechanism regulating gene expression during adipocyte differentiation. This article is protected by copyright. All rights reserved

Beschreibung: Post mortem studies on familial and sporadic Parkinson's disease patient striatal tissue have shown that nearly 90% of α-synuclein deposited in Lewy-bodies is phosphorylated at serine-129 (pSyn-129) as opposed to only 4% in normal human brain. We aimed to find the influence of endogenous neurotoxin 6-hydroxydopamine (6-OHDA) on α-synuclein phosphorylation, resting vesicles and vesicular dopamine release. The relative distribution of pSyn-129+ cells in apoptotic and non-apoptotic populations at different 6-OHDA concentrations was assessed along with changes in oxidant-antioxidant system, mitochondrial membrane-potential and intracellular-Ca 2+ . Exposing SH-SY5Y cells to different concentrations of 6-OHDA for 48 hours showed cell-death and apoptosis. Immunocytochemical analysis indicated an increase in pSyn-129 with increasing 6-OHDA concentration, and ELISA-estimation showed a significant increase in the pSyn-129 to α-synuclein ratio. FACS analysis also showed a significant increase in pSyn-129; and at sub-lethal 6-OHDA concentrations, pSyn-129+ cells were primarily distributed in the non-apoptotic population, suggesting that phosphorylation of α-synuclein precedes apoptosis. At higher 6-OHDA concentrations, the pSyn-129+ cell count significantly increased in the apoptotic population and decreased in the non-apoptotic population. Cytosolic co-localization of α-synuclein and ubiquitin was noticed at higher doses of 6-OHDA. FACS analysis showed decrease in vesicular monoamine transporter-2 (VMAT2) expression in 6-OHDA-treated cells, confirmed by reduction in functional dopamine-release on KCl and ATP stimulation. Significant decrease in VMAT2 expression and vesicular dopamine-release were observed with the lower 6-OHDA concentration, together with mild occurrence of apoptosis and significant increase in phosphorylated α-synuclein. This suggests that at sub-lethal 6-OHDA concentrations, the decrease in resting vesicles (VMAT2) and vesicular dopamine release are not attributable to apoptotic cell death and occur concomitantly with the phosphorylation of α-synuclein. This article is protected by copyright. All rights reserved

Beschreibung: Fisetin (3,3',4',7-tetrahydroxyflavone), a flavonoid found in a number of fruits and vegetables, has diverse biological activities, including cytotoxic effects on cancer cells. In this study, we investigated the effect of fisetin on triple-negative breast cancer (TNBC) cells. TNBC has a poorer prognosis than other types of breast cancer and treatment options for this disease are limited. Fisetin inhibited the growth of MDA-MB-468 and MDA-MB-231 TNBC cells, as well as their ability to form colonies, without substantially affecting the growth of non-malignant cells. In addition, fisetin inhibited the growth of estrogen receptor-bearing MCF-7 breast cancer cells and human epidermal growth factor receptor 2-overexpressing SK-BR-3 breast cancer cells. Fisetin inhibited TNBC cell division and induced apoptosis, which was associated with mitochondrial membrane permeabilization and the activation of caspase-9 and caspase-8, as well as the cleavage of poly(ADP-ribose) polymerase-1. Induction of caspase-dependent apoptosis by fisetin was confirmed by reduced killing of TNBC cells in the presence of the pan-caspase inhibitors Z-VAD-FMK and BOC-D-FMK. Decreased phosphorylation of histone H3 at serine 10 in fisetin-treated TNBC cells at G2/M phase of the cell cycle suggested that fisetin-induced apoptosis was the result of Aurora B kinase inhibition. Interestingly, the cytotoxic effect of cisplatin, 5-fluorouracil, and 4-hydroxycyclophosphamide metabolite of cyclophosphamide on TNBC cells was increased in the presence of fisetin. These findings suggest that further investigation of fisetin is warranted for possible use in the management of TNBC. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Solid tumours contend with, and adapt to, a hostile micro-environment that includes limited availability of nutrient fuels and oxygen. The presence of hypoxia (O 2  〈 5%) stabilises the transcription factor Hif1 and results in numerous cellular adaptations including increased flux of glucose through glycolysis. Increasingly more sophisticated analysis of tumour oxygenation has revealed large gradients of oxygen tension and significant regions under severe hypoxia (O 2 ∼ 0.1%). The present investigation has demonstrated a significant increase in the glycolytic flux rate when tumour spheroids were exposed to 0.1% O 2 . The severe hypoxia was associated with uniform pimonidazole adduct formation and elevated levels of Hif1α and c-Myc. This resulted in elevated expression of GLUT and MCT transporters, in addition to increased activity of PFK1 in comparison to that observed in normoxia. However, the protein expression and enzymatic capacity of HK2, G6PDH, PK and LDH were all reduced by severe hypoxia. Clearly the effects of exposure to severe hypoxia lead to a significantly abridged Hif1 response, yet one still able to elevate glycolytic flux and prevent loss of intermediates to anabolism. This article is protected by copyright. All rights reserved

Beschreibung: Both CD4+ T lymphocytes and macrophages are the major targets of human immunodeficiency virus type 1 (HIV-1), however, they respond differently to HIV-1 infection. We hypothesized that HIV-1 infection alters gene expression in CD4+ T cells and monocyte-derived macrophages (MDMs) in a cell specific manner and microRNAs (miRNAs) in part play a role in cell specific gene expression. Results indicate that 183 and 31 genes were differentially regulated in HIV-1 infected CD4+ T cells and MDMs respectively, compared to their mock-infected counterparts. Among the differentially expressed genes, cell cycle regulatory gene, p21 (CDKN1A) was upregulated in virus infected CD4+ T cells both at the mRNA and protein level in CD4+ T cells whereas no consistent change was observed in MDMs. Productively infected CD4+ T cells express higher amount of p21 compared to bystander cells. In determining the mechanism(s) of cell type specific regulation of p21, we found that the miRNAs miR-106b and miR-20a that target p21 were specifically downregulated in HIV-1 infected CD4+ T cells. Overexpression of these two miRNAs reduced p21 expression significantly in HIV-1 infected CD4+ T cells. These findings provide a potential mechanism, by which, HIV-1 could exploit host cellular machineries to regulate selective gene expression in target cells. This article is protected by copyright. All rights reserved

Beschreibung: Vascularization is important for bone development, fracture healing and engineering of artificial bone tissue. In the context of bone tissue engineering, it was shown that coimplantation of human primary umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) results in the formation of functional blood vessels and enhanced bone regeneration. Implanted endothelial cells do not only contribute to blood vessel formation, but also support proliferation, cell survival and osteogenic differentiation of coimplanted hOBs. These effects are partially mediated by direct heterotypic cell contacts. In a previous report we could show that cocultivated hOBs strongly increase the expression of genes involved in extracellular matrix (ECM) formation in HUVECs, suggesting that ECM may be involved in the intercellular communication between hOBs and HUVECs. The present study aimed at investigating whether comparable changes occur in hOBs. We therefore performed a microarray analysis of hOBs cultivated in direct contact with HUVECs, revealing 1004 differentially expressed genes. The differentially expressed genes could be assigned to the functional clusters ECM, proliferation, apoptosis and osteogenic differentiation. The microarray data could be confirmed by performing quantitative real time RT-PCR on selected genes. Furthermore, we could show that the ECM produced by HUVECs increased the expression of the osteogenic differentiation marker alkaline phosphatase (ALP) in hOBs. In summary, our data demonstrate that HUVECs provoke complex changes in gene expression patterns in cocultivated hOBs and that ECM plays and important role in this interaction. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Constitutive activation of mutant K ras ( Kirsten rat sarcoma viral oncogene homologue ) and disassembly of E-cadherin–catenin complex (E-cadherin, α-catenin, β-catenin and γ-catenin) play an important role in apoptosis, differentiation and cell proliferation. In this study the expression pattern of K ras and E-cadherin–catenin complex has been evaluated in normal and mutant colorectal cancer cell lines with an object to determine its impact on disassociation of cells from one another. We addressed the expression analysis of K ras with reference to its association with adherence molecules in two colorectal cancer cell lines i.e . Caco-2 (wild type K ras served as a control) and DLD1 (heterozygous mutation at codon 13) at message level by qRT-PCR and translational level by western blotting. Compared to the control Caco-2 cell lines, the K ras in DLD1 cell lines showed slightly higher values while α-catenin showed a slight lower (1.3 folds), β-catenin and E-cadherin showed significantly lower expression (4.2 fold decrease).. It can be inferred that a possible cross talk exists between K ras and adherent junction mediated signalling. Mutation at codon 13 (G to D) leads to the overexpression of K ras and reduced expression of adherent junction complex resulting in metastasis. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Uterine fibroids (leiomyomas) are very common benign tumors grown on the smooth muscle layer of the uterus, present in up to 75% of reproductive-age women and causing significant morbidity in a subset of this population. Although the etiology and biology of uterine fibroids are unclear, strong evidence supports that proliferation, angiogenesis and fibrosis are involved in their formation and growth. Currently the only cure for uterine fibroids is hysterectomy; the available alternative therapies have limitations. Thus, there is an urgent need for developing a novel strategy for treating this condition. The green tea polyphenol epigallocatechin gallate (EGCG) inhibits the growth of uterine leiomyoma cells in vitro and in vivo , and the use of a green tea extract (containing 45% EGCG) has demonstrated clinical activity without side effects in women with symptomatic uterine fibroids. However, EGCG has a number of shortcomings, including low stability, poor bioavailability and high metabolic transformations under physiological conditions, presenting challenges for its development as a therapeutic agent. We developed a prodrug of EGCG (Pro-EGCG or 1 ) which shows increased stability, bioavailability and biological activity in vivo as compared to EGCG. We also synthesized prodrugs of EGCG analogs, compounds 2a and 4a , in order to potentially reduce their susceptibility to methylation/inhibition by catechol- O -methyltransferase. Here, we determined the effect of EGCG, Pro-EGCG, and 2a and 4a on cultured human uterine leiomyoma cells, and found that 2a and 4a have potent antiproliferative, antiangiogenic and antifibrotic activities. This article is protected by copyright. All rights reserved

Beschreibung: Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation and signaling pathways with key roles in stemness. Culturing mESCs with 2 μM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-β/β-catenin, was observed, in addition to activation of PI3K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation. This article is protected by copyright. All rights reserved

Beschreibung: An ideal tracheal scaffold must withstand luminal collapse yet be flexible, have a sufficient degree of porosity to permit vascular and cellular ingrowth, but also be airtight and must facilitate growth of functional airway epithelium to avoid infection and aid in mucocilliary clearance. Finally, the scaffold must also be biocompatible to avoid implant rejection. Over the last 40 years, efforts to design and manufacture the airway have been undertaken worldwide but success has been limited and far apart. As a result, tracheal resection with primary repair remains the Gold Standard of care for patients presenting with airway disorders and malignancies. However, the maximum resectable length of the trachea is restricted to 30% of the total length in children or 50% in adults. Attempts to provide autologous grafts for human application have also been disappointing for a host of different reasons, including lack of implant integration, insufficient donor organs and poor mechanical strength resulting in an unmet clinical need. The two main approaches researchers have taken to address this issue have been the development of synthetic scaffolds and the use of decellularised organs. To date, a number of different decellularisation techniques and a variety of materials, including polyglycolic acid (PGA) and nanocomposite polymers have been explored. The findings thus far have shown great promise, however there remain a significant number of caveats accompanying each approach. That being said, the possibilities presented by these two approaches could be combined to produce a highly successful, clinically viable hybrid scaffold. This article aims to highlight advances in airway tissue engineering and provide an overview of areas to explore and utilise in accomplishing the aim of a developing an ideal tracheal prosthesis. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT There are two major types of mouse xenograft models of cancer: subcutaneous implantation and orthotopic implantation. Subcutaneous transplant models are widely used, with both cancer cell lines and human-tumor specimens. Recently, subcutaneous models of patient tumors, termed patient-derived xenographs (PDX) have become highly popular and have acquired such names as “Avatar” and “Xenopatients”. However, such s.c. models rarely metastasize and are therefore not patient-like. In contrast, orthotopic models have the capability to metastasize. If intact fragments of tumor tissue are implanted by surgical orthotopic implantation (SOI), the metastatic potential can match that of the donor patient. The present study images in real time, using green fluorescent protein (GFP) expression, the very different tumor behavior at the orthotopic and subcutaneous sites of human prostate cancer PC-3 in athymic nude mice. By day-2 after tumor implantation, the orthotopic tumor is already highly vascularized and the cancer cells have begun to migrate out of the tumor. In contrast, the subcutaneous tumor only begins to be vascularized by day-3 and cells do not migrate from the tumor. Angiogenesis is much more extensive in the orthotopic tumor throughout the two-week observation period. The orthotopic PC-3-GFP tumor progresses very rapidly and distinct metastasis have appeared in lymph nodes by day 3 which rapidly appear in many areas of the abdominal cavity including portal lymph nodes by day-7. At day-14, no invasion or metastasis was observed with the s.c. tumor even when the animal was extensively explored. This article is protected by copyright. All rights reserved

Beschreibung: The role of vitamin D as a treatment option for neoplastic diseases, once considered to have a bright future, remains controversial. The preclinical studies discussed herein show compelling evidence that Vitamin D Derivatives (VDDs) can convert some cancer and leukemia cells to a benign phenotype, by differentiation/maturation, cell cycle arrest or induction of apoptosis. Further, there is considerable, though still evolving, knowledge of the molecular mechisms underlying these changes. However, the attempts to clearly document that the treatment outcomes of human neoplastic diseases can be positively influenced by VDDs have been, so far, disappointing. The clinical trials to date of VDDs, alone or combined with other agents, have not shown consistent results. It is our contention, shared by others, that there were limitations in the design or execution of these trials which have not yet been fully addressed. Based on the connection between upregulation of JNK by VDDs and DNA repair, we propose a new avenue of attack on cancer cells by increasing the toxicity of the current, only partially effective, cancer chemotherapeutic drugs by combining them with VDDs. This can impair DNA repair and thus kill the malignant cells, warranting a comprehensive study of this novel concept. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT We studied epigenetics, distribution pattern, kinetics and diffusion of proteins recruited to spontaneous and γ-radiation-induced DNA lesions. We showed that PML deficiency leads to an increased number of DNA lesions, which was accompanied by changes in histone signature. In PML wt cells, we observed two mobile fractions of 53BP1 protein with distinct diffusion in spontaneous lesions. These protein fractions were not detected in PML-deficient cells, characterized by slow-diffusion of 53BP1. Single particle tracking analysis revealed limited local motion of 53BP1 foci in PML double null cells and local motion 53BP1 foci was even more reduced after γ-irradiation. However, radiation did not change co-localization between 53BP1 nuclear bodies and interchromatin granule -associated zones (IGAZs), nuclear speckles or chromocenters. This newly observed interaction pattern imply that 53BP1 protein could be a part of not only DNA repair, but also process mediated via components accumulated in IGAZs, nuclear speckles or paraspeckles. Together, PML deficiency affected local motion of 53BP1 nuclear bodies and changed composition and a number of irradiation-induced foci. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT The facile nature of mesenchymal stem cell (MSC) acquisition in relatively large numbers has made Wharton's jelly (WJ) tissue an alternative source of MSCs for regenerative medicine. However, freezing of such tissue using dimethyl sulfoxide (DMSO) for future use impedes its clinical utility. In this study, we compared the effect of two different cryoprotectants (DMSO & cocktail solution) on post-thaw cell behavior upon freezing of WJ tissue following two different freezing protocols [Conventional (-1°C/min) & programmed]. The programmed method showed higher cell survival rate compared to conventional method of freezing. Further, cocktail solution showed better cryoprotection than DMSO. Post-thaw growth characteristics and stem cell behavior of Wharton's jelly mesenchymal stem cells (WJMSCs) from WJ tissue cryopreserved with a cocktail solution in conjunction with programmed method (Prog-Cock) were comparable with WJMSCs from fresh WJ tissue. They preserved their expression of surface markers, pluripotent factors, and successfully differentiated in vitro into osteocytes, adipocytes, chondrocytes, and hepatocytes. They also produced lesser annexin-V-positive cells compared to cells from WJ tissue stored using cocktail solution in conjunction with the conventional method (Conv-Cock). Real-time PCR and western blot analysis of post-thaw WJMSCs from Conv-Cock group showed significantly increased expression of pro-apoptotic factors (BAX, p53, and p21) and reduced expression of anti-apoptotic factor (BCL2) compared to WJMSCs from the fresh and Prog-Cock group. Therefore, we conclude that freezing of fresh WJ tissue using cocktail solution in conjunction with programmed freezing method allows for an efficient WJ tissue banking for future MSC-based regenerative therapies. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT The aim of this study was to investigate preventive effects of the lipophilic vitamin C derivative, 6- O -palmitoylascorbate (PlmtVC) against X-ray radiation-induced harmful events. Free radical scavenging activity tests showed that both fresh and old (being kept at 37°C for 72 hr) solutions of PlmtVC showed significantly higher abilities for scavenging both DPPH and peroxyl radical (ROO·) radicals than L-ascorbic acid (L-AA) under the same conditions, suggesting that PlmtVC is an antioxidant more efficient and stable than L-AA. Irradiation with X-ray (15 Gy) increased intracellular ROS production, lipid peroxidation and protein carbonylation, in human keratinocytes HaCaT, all of which were repressed, especially for intracellular ROS more markedly, by PlmtVC than by L-AA. After X-ray (15 Gy)-irradiation, caspase 3/7 activation and TUNEL-detected DNA-strand-breakages characteristic of apoptosis obviously increased in HaCaT cells or 3D-skin tissue equivalents, respectively, both of which were prevented more appreciably by PlmtVC than by L-AA. PlmtVC also noticeably prevented cumene hydroperoxide-induced generation of cellular ROS in epidermis parts of 3D-skin equivalents. Thus, PlmtVC prevents X-ray-induced diverse harmful effects, through its antioxidant activity and the palmitoyl moiety-based lipophilicity, more efficiently than L-AA. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Our laboratory pioneered patient-derived orthotopic xenograft (PDOX) mouse models using surgical orthotopic implantation (SOI). PDOX models are patient-like, in contrast to the ectopic subcutaneous-transplant cancer models. In the present study, we demonstrate that an undifferentiated pleomorphic soft-tissue sarcoma (UPS-STS) PDOX model acquired bright RFP-expressing stroma through one passage in red fluorescent protein (RFP) transgenic mice, which upon passage to non-colored nude mice was non-invasively imageable. A PDOX nude mouse model of UPS-STS was established in the biceps femoris of nude mice. After the tumors grew to a diameter of 10 mm, the tumors were subsequently-passaged to RFP transgenic mice, and after tumor growth were then passaged to non-transgenic nude mice. Tumors were divided into small fragments and transplanted in the biceps femoris at each passage. The OV100 Small Animal Fluorescence Imaging System and FV1000 laser scanning confocal microscope were used to image RFP fluorescence in the UPS-STS PDOX models. UPS-STS PDOX tumors, previously-grown in RFP transgenic nude mice for only one passage, had very bright fluorescence and after passage to non-transgenic nude mice were non-invasively imageable. FV1000 confocal imaging revealed diffusely-distributed bright RFP stromal cells in the PDOX tumor, both in RFP transgenic mice and after passage to non-transgenic mice. These results demonstrate a powerful method to make the PDOX UPS-STS model brightly-fluorescent for non-invasive imaging, as well as for confocal microscopy of individual stromal cells associated with the tumor. The RFP-labeled UPS PDOX has the potential to rapidly screen for novel effective agents for individual patients, including stroma-targeting drugs, whereby the stromal cells are a visual target. This article is protected by copyright. All rights reserved

Beschreibung: Several environmental pollutants (EPs) have been associated with biological and molecular processes leading to adverse human health effects, including different types of cancer. Nevertheless, the effects exerted on tumor glucose metabolism are unclear. To evaluate the effects on cellular and molecular mechanisms, namely glucose metabolism, MCF-7 cells were exposed to EPs during short- and long-term exposures. The effect of both, organochlorine pesticides and plasticizing agents, on glucose uptake by MCF-7 cells was not dose-dependent and was affected by time of exposure. The ΣHCH and BPA increased glucose uptake after 20 min. Long-term exposure to 250 nM of organochlorine pesticides ( p,p '-DDE and ΣHCH) and BPA increased cell proliferation. However, only the organochlorine pesticides were able to increase lactate production, without a concomitant higher glucose uptake or glycolytic enzymes transcription. Given their distinct persistent profiles, the biological significance of their exposure should be considered accordingly. This article is protected by copyright. All rights reserved

Beschreibung: Epidemiological and clinical data suggest adverse cardiovascular outcomes with respect to vitamin D deficiency. Here, we explored the effects of vitamin D in atherosclerotic plaque calcification in vivo by utilizing vitamin D receptor ( Vdr )-deficient mice in an Apoe -/- background. Animals were fed a high-fat diet (HFD) for either 12 or 18 weeks and then examined for atherosclerotic plaque development. In order to prevent calcium deficiency, Vdr -/- and Apoe -/- ;Vdr -/- animals were fed a high-calcium rescue diet prior to initiation of the HFD feeding and supplemented with high-calcium water during HFD feeding. Although calcium supplementation improved bone mass in Vdr -/- and Apoe -/- ;Vdr -/- mice, neither strain was fully rescued. Systemic inflammatory responses observed in the absence of VDR were exaggerated in Apoe -/- mice. Whereas, hyperlipidemic profiles seen in Apoe -/- mice were ameliorated in absence of VDR. Micro-computed tomography (µCT) analysis revealed that 6 out of 8 Apoe -/- animals developed atherosclerotic plaque calcification following 12 weeks of HFD feeding and 100% of the mice developed plaque calcification after 18 weeks. In contrast, although atherosclerotic lesions were evident in Apoe -/- ;Vdr -/- mice at 12 and 18 weeks of HFD challenge, none of these animals developed plaque calcification at either time point. The active vitamin D hormone, 1,25(OH) 2 D 3 likely increased calcification in aortic smooth muscle cells perhaps by directly modulating expression of Alpl , Rankl and Opg . Our data suggest that the absence of VDR inhibits atherosclerotic plaque calcification in hypercholesterolemic Apoe -/- mice, providing additional insight into the role of vitamin D in atherosclerotic plaque calcification. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Our incomplete understanding of the critical changes that accompany the earliest stages of tumor initiation provides a substantial hurdle for the development of novel intervention strategies for cancer prevention. Premalignant lesions are inherently difficult to characterize given their diminutive size, creating technical obstacles for accurate genetic profiling. Here, we describe an approach combining laser-capture microdissection (LCM) with targeted RNA-sequencing to study the transcriptional state of epithelial and stromal cells during the earliest detectable stage of human colorectal neoplasia, the aberrant crypt foci (ACF). We provide a robust and reproducible workflow for RNA isolation, library preparation, and expression profiling of laser-captured cells from frozen OCT-embedded tissue specimens. It is anticipated that the methodological approach outlined in this report will provide a framework for a broad range of microgenomics analyses that can be routinely applied to many other premalignant tissues. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Osteosarcoma is an aggressive bone tumor affecting childhood. Currently there are a few proteomic studies analyzing secreted proteins in osteosarcoma. To characterize the osteosarcoma secretome, secreted proteins in the conditioned media of three human osteosarcoma cell lines were analyzed. Exosomes and exosome-free supernatants containing soluble proteins were isolated and characterized. Exosomes were analyzed by transmission electron microscopy (TEM) and western blot. Exosomal and soluble proteins (under 70-100 kDa, respectively) were identified by mass spectrometry analysis using nanoLC-MS/MS. Secreted proteins were classified by functional Gene Ontology clustering. We identified a set of 3300 proteins for both fractions. Secretome of all osteosarcoma cell lines exhibeted commun as well unique proteins. Systemathic comparison of proteomes contained in exosomes and exosome-free fractions reveled a diferential enrichment of categories associated to diferent biological funtions and tumor progression properties as angiogenesis and cell adhesion and cell migration, thus suggesting a secretome-based tumor cell behaviour in osteosarcome. This article is protected by copyright. All rights reserved

Beschreibung: Osteoporosis is a common problem in aged people and those with related diseases, such as inflammatory bowel diseases. Deregulation of vitamin D metabolism plays a role in the pathogenesis of osteoporosis. Micro RNA (miR) can regulate cytokine expression in cells. This study test a hypothesis that inflammatory cytokine interleukin (IL)-13 increases miR-19a to compromise cyp27b1 (a vitamin D hydroxylase) in peripheral CD14 + cells. In this study, bone mineral density of L2-L4 was measured in 20 patients with ulcerative colitis (UC) and 20 healthy subjects. Peripheral CD14 + cells were isolated from healthy people and patients with UC. Expression of cyp27b1 by CD14 + cells was analyzed in the presence or absence of IL-13 in the culture. We observed that bone mineral density (BMD) in UC patients was significantly lower than healthy subjects. The BMD is negatively correlated with miR-19a in peripheral CD14 + cells. MiR-19a in peripheral CD14 + cell was correlated with serum IL-13 in UC patients. Expression of cyp27b1 in peripheral CD14 + cells was correlated with miR-19a and serum IL-13 in UC patients. IL-13 suppressed cyp27b1 expression in CD14 + cells. IL-13 increased expression of miR-19a in CD14 + cells. In conclusion, IL-13 suppresses cyp27b1 expression in peripheral CD14 + cells via up regulating miR-19a expression. This article is protected by copyright. All rights reserved

Beschreibung: Tumor necrosis factor alpha (TNF-α)-induced osteoclastogenesis has profound effects in states of inflammatory osteolysis such as rheumatoid arthritis, periprosthetic implant loosening, and periodontitis. However, the exact mechanisms by which TNF-α promotes RANKL-induced osteoclast formation remains poorly understood. B lymphocyte-induced maturation protein-1 (Blimp1) is a transcriptional repressor that plays crucial roles in the differentiation and/or function of various kinds of cells including osteoclasts. A novel mechanism was identified where TNF-α-mediated Blimp1 expression, which contributed to RANKL-induced osteoclastogenesis. It is shown that TNF-α could promote the level of Blimp1 expression during osteoclast differentiation. Silencing of Blimp1 in osteoclast precursor cells obviously attenuated the stimulatory effect of TNF-α on osteoclastogenesis. Mechanistically, TNF-α-induced Blimp1 expression was markedly rescued by blocking the PI3K/Akt signaling pathway, which suggested that PI3K/Akt signaling was involved in the regulation of TNF-α-stimulated Blimp1 expression. Taken together, the results established a molecular mechanism of TNF-α-induced osteoclasts differentiation, and provided insights into the potential contribution of Blimp1 in the regulation of osteoclastogenesis by TNF-α. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modelling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (k actual ) of 3.25min −1 , 3-fold faster than α3 integrin (1.0 min −1 ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min −1 ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min −1 ). Silencing of α3 integrin protein expression in DU145, PC3 and PC3B1 cells resulted in up to a 1.71-fold increase in k actual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8 fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the k actual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. This article is protected by copyright. All rights reserved

Beschreibung: PhLP2 is a small cytosolic protein that belongs to the highly conserved phosducin-like family of proteins. In amniote genomes there are two PhLP2 homologs, PhLP2A and PhLP2B. It has been shown that mammalian PhLP2A modulates the CCT/TRiC chaperonin activity during folding of cytoskeletal proteins. In order to better understand the function of PhLP2A in cellular protein quality control system, in the present study we have searched for its protein targets. Applying immunoprecipitation followed by mass spectrometry analysis we have identified Hsp90 as a partner of PhLP2A. With pull down experiments we have confirmed this interaction in protein lysate and using purified proteins we have shown that PhLP2A interacts directly with Hsp90. Furthermore, the proximity ligation assay (PLA) performed on mIMCD-3 cells has shown that PhLP2A forms complexes with Hsp90 which are mainly localized in the cytoplasm of these cells. Further analysis has indicated that the level of PhLP2A increases after heat shock or radicicol treatment, similarly as the level of Hsp90, and that expression of PhLP2A after heat shock is regulated at the transcriptional level. Moreover, using recombinant luciferase we have shown that PhLP2A stabilizes this enzyme in a folding competent state and prevents its denaturation and aggregation. In addition, overexpression of PhLP2A in HEK-293 cells leads to increased heat stress resistance. Altogether, our results have shown that PhLP2A interacts with Hsp90 and exhibits molecular chaperone activity toward denatured proteins. This article is protected by copyright. All rights reserved

Beschreibung: Cortisol is an essential regulator of neuroendocrine stress responses in teleosts. Cortisol predominantly affects target tissues through the genomic pathway, which involves interacting with cytoplasmic glucocorticoid receptors and, thereby, modulating stress-response gene expressions. Cortisol also produces rapid effects via non-genomic pathways, which do not involve gene transcription. Although cortisol-mediated genomic pathways are well documented in teleosts, non-genomic pathways are not fully understood. Moreover, no studies have focused on the contribution of non-genomic cortisol pathways in compensatory stress responses in fish. In this study, rainbow trout ( Oncorhynchus mykiss ) skeletal myotubes were stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable agent, resulting in an early induction of reactive oxygen species (ROS). This production was not suppressed by transcription or translation inhibitors, suggesting non-genomic pathway involvement. Moreover, myotube preincubation with RU486 and NAC completely suppressed cortisol- and cortisol-BSA-induced ROS production. Subcellular fractionation analysis revealed the presence of cell membrane glucocorticoid receptors. Finally, cortisol-BSA induced a significant increase in ERK1/2 and CREB phosphorylation, as well as in CREB-dependent transcriptional activation of the pgc1a gene expression. The obtained results strongly suggest that cortisol acts through a non-genomic glucocorticoid receptor-mediated pathway to induce ROS production and contribute to ERK/CREB/PGC1-α signaling pathway activation as stress compensation mechanisms. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Recent evidences show that activation of serotonin 2A receptors (5-HT 2A R) by agonists is significant in improving therapeutic activity of disease conditions, such as obsessive-compulsive disorder (OCD). Though the exact molecular mechanism is still not well understood, it is thought to involve agonist-driven, enhanced expression of 5-HT 2A R in certain areas of brain, such as the pre-frontal cortex (PFC). Several other reports have also demonstrated association of OCD with lower dopamine receptor (D 2 R) availability, primarily in the striatum of the brain along with dysfunction of 5-HT 2A R-D 2 R heteromer regulation. We thus hypothesised that compound(s) interacting with this molecular mechanism could be developed as drugs for long-term beneficial effects against OCD. In the present study, we have obtained experimental evidence in cultured neuronal cells (CLU213) that aqueous extract (AE, 50 µg/mL, p 〈 0.05) of the Australian cane toad skin significantly increased the levels of 5-HT 2A R and D 2 R protein and mRNA expression. AE was also found to enhance the interaction between 5-HT 2A R and D 2 R and formation of expression of 5-HT 2A R-D 2 R heteromer using co-immunoprecipitation and Western blot. Further investigation showed the involvement of classical signalling pathway (G q/11 -PLCβ) along with c-FOS transcription factor preferentially in 5-HT 2A -mediated agonist activation. These results obtained demonstrated that AE upregulates 5-HT 2A R by a mechanism that appears to involve G q/11 -PLCβ signalling pathway and c-FOS transcription factor activation. We indicate this enhanced 5-HT 2A R and D 2 R expression and their interaction to induce increased 5-HT 2A R-D 2 R heteromer formation by exposure to AE might provide a molecular mechanism to develop potential novel drug candidates to ameliorate OCD symptoms. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT In functional cytometric studies , cultured cells are exposed to effectors ( e.g . drugs), and the heterogeneity of cell responses are studied using cytometry techniques ( e.g . image cytometry). Such studies are difficult to perform on 3D cell cultures. A solution is to disperse 3D clusters and transfer the cells to the 2D state before applying effectors and using cytometry. This approach requires that the lifetime of the 3D phenotype be longer than the duration of the experiment. Here we studied the dynamics of phenotype transformation from 3D to 2D and searched for means of slowing this transformation down in dispersed spheroids of MCF7 cells. We found three functional biomarkers of the 3D phenotype in MCF7 cell spheroids that are absent in the 2D cell culture: (i) the presence of a subpopulation with an elevated drug-expelling capacity, (ii) the presence of a subpopulation with an elevated cytoprotective capacity and (iii) the accumulation of cells in the G1 phase of the cell cycle. Monitoring these biomarkers in cells transferred from the 3D state to the 2D state revealed their gradual extinction. We found that the combined application of an elevated cell density and thiol-containing medium supplements increased the lifetime of the 3D phenotype by several fold to as long as 96 h. Our results suggest that extending the lifetime of the 3D phenotype in the cells transferred from the 3D state to the 2D state can facilitate detailed functional cytometric studies, such as measurements of population heterogeneity of cytotoxicity, chemosensitivity and radiosensitivity. This article is protected by copyright. All rights reserved

Beschreibung: We studied the alterations of Elongation Factor 2 (eEF2) in the pineal gland of aged rats as well as the possible protective role of exogenous melatonin on these changes in young rats treated with cumene hydroperoxide (CH), a compound that promotes lipid peroxidation and inhibits protein synthesis. The study was performed using male Wistar rats of 3 (control), 12 and 24 months and 3 month-old rats treated with CH, melatonin and CH plus melatonin. We found that pineal eEF-2 is affected by aging and CH, these changes being prevented by exogenous melatonin in the case of CH-treated rats. The proteomic studies show that many other proteins are affected by aging and oxidative stress in the pineal gland. The results suggest that one of the possible mechanisms underlying pineal gland dysfunction during aging is the effect of lipid peroxidation on eEF-2, which is a key component of protein synthesis machinery. This article is protected by copyright. All rights reserved

Beschreibung: BMPs have been shown to promote adipocyte differentiation through SMAD-dependent signaling. However, the role of TGF-β-activated kinase 1 (TAK1) in non-canonical BMP signaling in adipocyte differentiation remains unclear. Here, we show that TAK1 inhibition decreases lipid accumulation in C3H10T1/2 mesenchymal stem cells (MSCs) induced to differentiate into adipocytes. TAK1 knockdown by siRNA further confirms that TAK1 is required for adipocyte commitment of MSCs. Additionally, TAK1 knockdown inhibits adipogenesis of 3T3-L1 preadipocytes, indicating that TAK1 is not only needed for adipocyte commitment, but also required for adipocyte terminal differentiation. Furthermore, TAK1 ablation specifically in adipocytes reduced high fat diet-induced weight gaining and improved glucose tolerance. Mechanistically, we demonstrate that TAK1 is required for PPARγ transactivation and promotes PPARγ transcriptional activity synergistically with TAK1 binding protein 1 (TAB1). Collectively, our results demonstrate that TAK1 plays an essential role in BMP-mediated adipocyte differentiation. This article is protected by copyright. All rights reserved

Beschreibung: For decades stem cells have proven to be invaluable to the study of tissue development. More recently, mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) (ESC-MSCs) have emerged as a cell source with great potential for the future of biomedical research due to their enhanced proliferative capability compared to adult tissue-derived MSCs and effectiveness of musculoskeletal lineage-specific cell differentiation compared to ESCs. We have previously compared the properties and differentiation potential of ESC-MSCs to bone marrow-derived MSCs. In this study, we evaluated the potential of TGFβ1 and BMP7 to induce chondrogenic differentiation of ESC-MSCs compared to that of TGFβ1 alone and further investigated the cellular phenotype and intracellular signaling in response to these induction conditions. Our results showed that the expression of cartilage-associated markers in ESC-MSCs induced by the TGFβ1 and BMP7 combination was increased compared to induction with TGFβ1 alone. The TGFβ1 and BMP7 combination upregulated the expression of TGFβ receptor and the production of endogenous TGFβs compared to TGFβ1 induction. The growth factor combination also increasingly activated both of the TGF and BMP signaling pathways, and inhibition of the signaling pathways led to reduced chondrogenesis of ESC-MSCs. Our findings suggest that by adding BMP7 to TGFβ1-supplemented induction medium, ESC-MSC chondrogenesis is upregulated through increased production of endogenous TGFβ and activities of TGFβ and BMP signaling. This article is protected by copyright. All rights reserved

Beschreibung: CA125 is serum tumor marker consisting of an epitope carried by a portion of the extremely large (〉 3 MDa), heavily glycosylated cell surface transmembrane mucin, MUC16. In malignancies, membrane bound mucins lose their polarized distribution, become aberrantly over-expressed and protect tumor cells from the actions of chemotherapeutic agents as well as the immune system. Previously, we described stimulation of MUC16 expression by the proinflammatory cytokines, tumor necrosis factor α (TNFα) and interferon γ (IFNγ), in breast and ovarian cancer cells and tissues. Herein, we show that PPARγ modulates cytokine-stimulated MUC16 in a complex manner: at low concentrations (〈 10 µM) rosiglitazone further potentiates cytokine-driven MUC16 expression while at high concentrations (〉 20 µM) rosiglitazone antagonizes cytokine stimulation. Rosiglitazone actions were fully reversible by the PPARγ antagonist, GW9662. Furthermore, siRNA-mediated PPARγ knockdown also prevented a large portion of high dose rosiglitazone suppression of MUC16 expression indicating that rosiglitazone inhibition is largely PPARγ-dependent. Cytokines greatly (〉75%) suppressed PPARγ expression. Conversely, PPARγ activation by rosiglitazone at either low or high concentrations greatly (〉75%) suppressed NFκB/p65 expression. NFκB/p65 expression was largely preserved in the presence of cytokines at low, but not high, rosiglitazone concentrations accounting for the different concentration dependent effects on MUC16 expression. Collectively, these studies demonstrate that PPARγ is an important modulator of MUC16 expression. The ability to deliver high doses of PPARγ agonists to MUC16-expressing tumors offers an avenue to reduce expression of this protective glycoprotein and increase tumor sensitivity to killing by chemotherapeutic drugs and the immune system. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Spleen tyrosine kinase (Syk), a non-receptor tyrosine kinase, regulates tumor progression, either negatively or positively, depending on the tissue lineage. Information about the role of Syk in colorectal cancers (CRC) is limited; and conflicting reports have been published. We studied Syk expression and its role in differentiation and apoptosis of the colonocytes. Here, we reported for the first time that expression of two transcript variants of Syk is suppressed in colonocytes during butyrate-induced differentiation, which mediates apoptosis of HT-29 cells. Despite being a known HDAC inhibitor, butyrate deacetylates histone3/4 around the transcription start site (TSS) of Syk. Histone deacetylation precludes the binding of RNA Polymerase II to the promoter and inhibits transcription. Since butyrate is a colonic metabolite derived from undigested fibres, our study offers a plausible explanation of the underlying mechanisms of the protective role of butyrate as well as the dietary fibres against CRC through the regulation of Syk. We also report that combined use of butyrate and highly specific Syk inhibitor BAY61-3606 does not enhance differentiation and apoptosis of colonocytes. Instead, BAY completely abolishes butyrate-induced differentiation and apoptosis in a Syk-independent and ERK1/2 dependent manner. While butyrate dephosphorylates ERK1/2 in HT-29 cells, BAY re-phosphorylates it, leading to its activation. This study describes a novel mechanism of butyrate action in CRC and explores the role of Syk in butyrate-induced differentiation and apoptosis. In addition, our study highlights those commercial small molecule inhibitors, although attractive drug candidates should be used with concern because of their frequent off-target effects. This article is protected by copyright. All rights reserved

Beschreibung: Controlling the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) in favor of osteogenesis represents a promising approach for osteoporosis therapy and prevention. Previously, Fibroblast Growth Factor 1 (FGF1) and its subfamily member FGF2 were scored as leading candidates to exercise control over skeletal precursor commitment and lineage decision albeit literature results are highly inconsistent. We show here that FGF1 and 2 strongly prevent the osteogenic commitment and differentiation of hBMSCs. Mineralization of extracellular matrix (ECM) and mRNA expression of osteogenic marker genes Alkaline Phosphatase (ALP), Collagen 1A1 (COL1A1), and Integrin-Binding Sialoprotein (IBSP) were significantly reduced. Furthermore, master regulators of osteogenic commitment like Runt-Related Transcription Factor 2 (RUNX2) and Bone Morphogenetic Protein 4 (BMP4) were downregulated. When administered under adipogenic culture conditions, canonical FGFs did not support osteogenic marker expression. Moreover despite the presence of osteogenic differentiation factors, FGFs even disabled the pro-osteogenic lineage decision of pre-differentiated adipocytic cells. In contrast to FGF Receptor 2 (FGFR2), FGFR1 was stably expressed throughout osteogenic and adipogenic differentiation and FGF addition. Moreover, FGFR1 and Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) were found to be responsible for underlying signal transduction using respective inhibitors. Taken together, we present new findings indicating that canonical FGFR-ERK1/2 signaling entrapped hBMSCs in a pre-committed state and arrested further maturation of committed precursors. Our results might aid in unraveling and controlling check points relevant for ageing-associated aberrant adipogenesis with consequences for the treatment of degenerative diseases such as osteoporosis and for skeletal tissue engineering strategies. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Isoflavones widely distributed in plants prevent diabetes. This study investigated the in vivo and in vitro effect of 3′,4′-dihydroxy-6″,6″,6‴,6‴-tetramethylbis(pyrano[2″,3″:5,6::2‴,3‴:7,8]isoflavone (bis-pyrano prenyl isoflavone) on glucose homeostasis in hyperglycemic rats. The ethyl acetate fraction from aerial parts of P. molluginifolia that contain isoflavones was assayed on glucose tolerance, on in vitro maltase activity and on protein glycation. The isoflavone bis-pyrano prenyl isolated from this fraction was investigated on glucose homeostasis. The in vivo action of the isoflavone exhibits an anti-hyperglycemic effect by improving glucose tolerance, augmenting the liver glycogen, inhibiting maltase activity, and stimulating glucagon-like peptide-1 (GLP-1) and insulin secretion. The in vitro isoflavone inhibits dipeptidyl peptidase-4 (DPP-4) activity since the glucose tolerance was improved in the presence of the isoflavone as much as sitagliptin, an inhibitor of DPP-4. However, the co-incubation with isoflavone and sitagliptin exhibited an additive anti-hyperglycemic action. The isoflavone increased the GLP-1 faster than the positive hyperglycemic group, which shows that the intestine is a potential target. Thus, to clarify the main site of action in which isoflavone improves glucose balance, the in vitro mechanism of action of this compound was tested in intestine using calcium influx as a trigger for the signal pathways for GLP-1 secretion. The isoflavone stimulates calcium influx in intestine and its mechanism involves voltage-dependent calcium channels, phospholipase C, protein kinase C and stored calcium, contributing for GLP-1 secretion. In conclusion, the isoflavone regulates glycaemia by acting mainly in a serum target, the DPP-4 inhibitor. Furthermore, the long-term effect of isoflavone prevents protein glycation. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT The comet assay is one of the most widely used approaches for detecting DNA damage; generally, it provides information on the cell population-averaged level of DNA damage. Here, we present an automatic technique for easy measurement of standard comet characteristics and an annotation of the cell cycle phase of each comet. The approach includes the modified neutral comet assay and a pipeline for CellProfiler software designed to analyze DNA damage-related characteristics and annotate the cell cycle phase of each comet. Using this technique we have performed cell cycle phase–specific analysis of DNA damage induced by the topoisomerase II poison etoposide and have shown that the sensitivity of cells to this drug dramatically differed according to their cell cycle phase. It became evident from our results that the proposed protocol provides important additional information that often remains hidden in a standard comet analysis of an asynchronous cell population. This article is protected by copyright. All rights reserved

Beschreibung: 17β-Estradiol (E 2 ) protects several non-reproductive tissues from apoptosis, including skeletal muscle. Previously, we showed that E 2 at physiological concentrations prevented apoptosis induced by H 2 O 2 in skeletal myoblasts, reverting PKCδ, JNK and p66Shc activation and exerting a beneficial action over mitochondria. Since genomic actions underlying the regulation of nuclear gene transcription are a common property of this steroid, the present work characterizes the transcriptional activity modulated by E 2 to exert its antiapoptotic effect. We report that E 2 protects skeletal myoblasts against apoptosis induced by H 2 O 2 modulating p53 and FoxO transcription factors and then their target genes Bcl-2, Bim, Puma, PERP and MDM2, without affecting Noxa gene. The results presented in this work support the notion that the transcription factors FoxO and p53 coordinate apoptosis in C2C12 cells, and deepens our knowledge about a putative molecular mechanism by which E 2 exerts beneficial effects against oxidative stress in skeletal myoblasts. This article is protected by copyright. All rights reserved

Beschreibung: Aims The aim of the study was to extend the potential use of human stem cells isolated from amniotic fluid in medical applications by confirming their high homogeneity and quality. Materials and Methods Amniotic fluid samples were collected during amniocentesis from 165 women during pregnancy. The proliferation rate, clonogenicity, karyotype, aging process, pluripotent cell markers, expression of surface markers and the potential to differentiate into adipose, bone and cartilage cells of hAFSCs were analyzed. Results Obtained results revealed that mesenchymal stem cells could be derived successfully from amniotic fluid, which exhibit properties comparable with MSCs of other origins. Conclusion It is the first study, in which such a large group of patients was involved. Comprehensive statistical and biological analysis were conducted some of which clearly being innovative in relation to human amniotic fluid-derived stem cells. This article is protected by copyright. All rights reserved

Beschreibung: Multiple myeloma (MM) is a malignant plasma cell disorder, clinically characterized by osteolytic lesions, immunodeficiency, and renal disease. Over the past decade, MM therapy is significantly improved by the introduction of novel therapeutics such as immunomodulatory agents (thalidomide, lenalidomide, and pomalidomide), proteasome inhibitors (bortezomib, carfilzomib, and ixazomib), monoclonal antibodies (daratumumab and elotuzumab), histone deacetylase (HDAC) inhibitors (Panobinostat). The clinical success of these agents has clearly identified vulnerabilities intrinsic to the MM cell- as well as targets that emanate from the tumor microenvironment. Despite these significant improvements, MM remains incurable due to the development of drug resistance. This perspective will discuss more recent strategies which take advantage of multiple targets within the proteome recycling pathway, chromatin remodeling, and disruption of nuclear export. In addition, we will review the development of strategies designed to block opportunistic survival signaling that occurs between the MM cell and the tumor microenvironment including strategies for inhibiting myeloma-induced immune suppression. It has become clear that MM tumors continue to evolve on therapy leading to drug resistance. It will be important to understand the mechanism and additional vulnerabilities that occur due to the development of clinical resistance. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Erectile dysfunction (ED) is a complication of diabetes, condition responsible for causing endothelial dysfunction (EDys) and hampering repair mechanisms. However, scarce information is available linking vasculogenesis mediated by Endothelial Progenitor Cells (EPCs) and diabetes-associated ED. Furthermore, it remains to be elucidated if glycemic control plays a role on EPCs functions, EPCs modulators and penile vascular health. We evaluated the effects of diabetes and insulin therapy on bone marrow (BM) and circulating EPCs, testosterone and systemic/penile Stromal Derived Factor-1 alpha (SDF-1α) expression. Male Wistar rats were divided into groups: age-matched controls, 8-weeks streptozotocin-induced type 1 diabetics and insulin-treated 8-weeks diabetics. EPCs were identified by flow cytometry for CD34/CD133/VEGFR2/CXCR4 antigens. Systemic SDF-1α and testosterone levels were evaluated by ELISA. Penile SDF-1α protein expression was assessed, in experimental and human diabetic cavernosal samples, by immunohistochemical techniques. Diabetic animals presented a reduction of BM-derived EPCs and an increase in putative circulating endothelial cells (CECs) sloughed from vessels wall. These alterations were rescued by insulin therapy. In addition, glycemic control promoted an increase in systemic testosterone and SDF-1α levels, which were significantly decreased in animals with diabetes. SDF-1α protein expression was reduced in experimental and human cavernosal diabetic samples, an effect prevented by insulin in treated animals. Insulin administration rescued the effects of diabetes on BM function, CECs levels, testosterone and plasmatic/penile SDF-1α protein expression. This emphasizes the importance of glycemic control in the prevention of diabetes-induced systemic and penile EDys, by the amelioration of endothelial damage and increase in protective pathways. This article is protected by copyright. All rights reserved

Beschreibung: Trigger factor (TF) is a key component of prokaryotic chaperone network, which is involved various basic cellular processes such as nascent peptide folding, protein trafficking, ribosome assembly. To better understanding the physiological roles of TF, global transcriptome profiles of a variety of TF deletion mutant strains of Escherichia coli were determined. We found that deletion of the tig gene, encoding TF, led to a dramatic alteration of transcriptome profile, not only affecting the gene expression of members of the chaperone network, but also changing the levels of quite a few RNAs related to metabolism and other cellular processes. Further studies showed that this alteration was only partially recovered by knockin of TF domain-deletion mutants into the endogenous tig locus, indicating that structural integrity is crucial for the biological function of TF. Finally, by combining the transcriptome and phenotype results, a physiological mechanism underlying the impact of TF deletion on the transcriptome profile was proposed. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Cellular stress leads to an upregulation of gene transcription. We asked if there is a specificity in the activation of the stress-responsive transcription factors Nrf2, ATF4, and AP-1/c-Jun, or if activation of these proteins is a redundant cellular answer towards extracellular stressors. Here, we show that oxidative stress, induced by stimulation of the cells with the oxidant arsenite, strongly activated gene transcription via the stress-responsive element (StRE), while phorbol ester or tunicamycin, activators of AP-1/c-Jun or ATF4, respectively, activated AP-1 or nutrient-sensing response element-mediated transcription. Preincubation of the cells with N -acetyl-cysteine or overexpression of thioredoxin selectively attenuated arsenite-induced upregulation of StRE-regulated transcription. Expression of either dominant-negative or constitutively active mutants of Nrf2, ATF4, or c-Jun confirmed that distinct transcription units are regulated by these transcription factors. Physiological stimuli involving the activation of either Gαq-coupled designer receptors or the protein kinases c-Jun N-terminal protein kinase or p38 strongly stimulated transcription via AP-1/c-Jun, with minimal effects on Nrf2 or ATF4-responsive promoters. Thus, activation of transcription by extracellular signaling molecules shows specificity at the level of the chemical nature of the signaling molecule, at the level of the intracellular transduction process, and at the level of signal-responsive transcription factors. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Reprogramming of energy metabolism particularly switching over of cells to aerobic glycolysis leading to accumulation of lactate is a hallmark ofcancer. Lactate can induce angiogenesis, an important process underlying tumor growth and metastasis. VEGF is one of the most important cytokines which regulate this process and the present study was designed to examine if blocking glycolytic pathway in tumor cells can affect its angiogenic potency with respect to VEGF. For this, the expression and biological activity of VEGF synthesized and secreted by tumor derived cell lines in the presence or absence of 2-deoxy glucose (2-DG), an inhibitor of glycolysis was determined. The results suggested that inhibition of glycolysis using sub-lethal doses of 2-DG down-regulated the expression of VEGF and also significantly reduced its biological activity. Further mechanistic studies revealed that the down regulation of VEGF gene expression by 2-DG was due to an increase in SIRT-1 activity and the reduced biological activity was found to be due to an increase in the PARmodification of VEGF. Activity of SIRT-1 and PAR modification of VEGF in turn, was found to be correlated to the cellular NAD + levels. The results presented here therefore suggest that treatment of cancer cells with 2-DG can significantly reduce its overall angiogenic potency through transcriptional and post-translational mechanisms. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Mammalian NUMB is alternatively spliced generating four isoforms NUMB1-NUMB4 that can function as tumor suppressors. NUMB1-NUMB4 proteins, which normally determine how different cell types develop, are reduced in 21% of primary breast tumors. Our previous work has, however, indicated that two novel NUMB isoforms, NUMB5 and NUMB6 have the pro-oncogenic functions. Herein, we address a novel function of human NUMB isoform 6 (NUMB6) in promoting cancer cell migration and invasion. We found that NUMB6 induced expression of embryonic transcription factor Slug, which in turn actively repressed E-cadherin, prompting cells to undergo epithelial-mesenchymal transition (EMT). Low-metastatic breast cancer cells DB-7 stably expressing NUMB6, lost their epithelial phenotype, exhibited migratory and pro-invasive behavior, and ultimately elevated expression of mesenchymal markers. Among these markers, increased vimentin, β-catenin and fibronectin expression elicited metalloproteinase 9 (MMP9) production. Our results revealed that NUMB6-DB-7 cells have significantly increased level of Akt1 and Akt2 phosphorylation. Therefore, antagonizing Akt signaling using a chemical inhibitor LY294002, we found that NUMB6-induced Slug expression was reduced, and ultimately accompanied with decreased cell migration and invasion. In summary, this study identified a novel molecular determinant of breast cancer progression, uncovering a potential oncogenic role for the NUMB6 protein in cancer cell migration and invasion, coupled to the maintenance of mesenchymal-like cells. This article is protected by copyright. All rights reserved

Beschreibung: The infants of mothers with gestational diabetes mellitus (GDM) have an increased risk of metabolic and cardiovascular disease. It has been difficult to study the direct effects of maternal hyperglycemia on the fetus because of inaccessibility of fetal tissues. The development of tissues that simulate the function of fetal organs using stem cell technology provides an unprecedented opportunity to study this disorder. Stem cells in the Wharton's jelly of the umbilical cord (hWJSCs) possess unique properties that are different from other stem cells. They are primitive, present in large numbers, non-tumorigenic, hypoimmunogenic, tumoricidal and carry a genetic signature that represents the fetus. They are multipotent but their differentiation into functional pancreatic and cardiovascular tissues has been challenging. We have been able to reprogram hWJSCs from normal and GDM cords into induced pluripotent stem cells (iPSCs) from which a variety of functional fetal tissues including insulin-producing and cardiovascular tissues could be derived. Such tissues from reprogrammed hWJSCs of normal and GDM cords that physiologically and genetically mimic the fetus of the diabetic or non-diabetic mother are an ideal platform to study the effects of glucose, the Zika virus and other harmful agents on the fetus. The immature stemness phenotype of hWJSCs, easy accessibility, availability in large numbers without the need for propagation and lower risk of accumulation of epigenetic mutations make them the most attractive candidate over other umbilical cord cell types for reprogramming. Additionally, some of their beneficial genes may be retained in memory in the iPSCs derived from them. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT The epigenetics and molecular biology of human embryonic stem cells (hES cells) have received much more attention than their architecture. We present a more complete look at hES cells by electron microscopy, with a special emphasis on the architecture of the nucleus. We propose that there is an ultrastructural signature of pluripotent human cells. hES cell nuclei lack heterochromatin, including the peripheral heterochromatin, that is common in most somatic cell types. The absence of peripheral heterochromatin may be related to the absence of lamins A and C, proteins important for linking chromatin to the nuclear lamina and envelope. Lamin A and C expression and the development of peripheral heterochromatin were early steps in the development of embryoid bodies. While hES cell nuclei had abundant nuclear pores, they also had an abundance of nuclear pores in the cytoplasm in the form of annulate lamellae. These were not a residue of annulate lamellae from germ cells or the early embryos from which hES cells were derived. Subnuclear structures including nucleoli, interchromatin granule clusters, and Cajal bodies were observed in the nuclear interior. The architectural organization of human ES cell nuclei has important implications for cell structure – gene expression relationships and for the maintenance of pluripotency. This article is protected by copyright. All rights reserved

Beschreibung: Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs have the capacity for self-renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor, EGF were found to positively or negatively regulate mammosphere re-generation, organoid formation and multi-lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs were different, with increased FGF2 and EGF self-renewal was promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Oxidative stress (OS) derived from an increase in intracellular reactive oxygen species (ROS) is a major determinant of aging and lifespan. It has also been associated with several age-related disorders, like postmenopausal osteoporosis. Mesenchymal stem cells (MSCs) are the common precursors for osteoblasts and adipocytes; appropriate commitment and differentiation of MSCs into a specific phenotype is modulated, among other factors, by ROS balance. MSCs have shown more resistance to ROS than differentiated cells, and their redox status depends on complex and abundant anti-oxidant mechanisms. The purpose of this work was to analyze in real time, H 2 O 2 signaling in individual h-MSCs, and to compare the kinetic parameters of H 2 O 2 management by cells derived from both control (c-) and osteoporotic (o-) women. For these purposes, cells were infected with a genetically encoded fluorescent biosensor named HyPer, which is specific for detecting H 2 O 2 inside living cells. Subsequently, cells were sequentially challenged with 50 μM and 500 μM H 2 O 2 pulses, and the cellular response was recorded in real time. The results demonstrated adequate expression of the biosensor allowing registering fluorescence from HyPer at a single cell level. Comparison of the response of c- and o-MSCs to the oxidant challenges demonstrated improved antioxidant activity in o-MSCs. This was further corroborated by measuring the relative expression of mRNAs for catalase, superoxide dismutase-1, thioredoxine and peroxiredoxine, as well as by cell-surviving capacity under short-term H 2 O 2 treatment. We conclude that functional differences exist between healthy and osteoporotic human MSCs. The mechanism for these differences requires further study. This article is protected by copyright. All rights reserved

Beschreibung: Radiotherapy is widely used for advanced rectal tumors. However, refractory metastasis has become the major cause of therapy failure in rectal cancer patients. Understanding the molecular mechanism that controls the aggressive cellular response to this treatment is essential for developing new therapeutic applications and improving radiotherapy response in colorectal cancer patients. Using the progeny of cells that were submitted to irradiation, we have demonstrated that the PI3K/AKT, Wnt/β-catenin signaling pathways as well as ERK1/2 downstream of EPHA4 receptor activation, play an important role in the regulation of events related with the EMT development, which may be associated with the therapeutic failure in rectal cancer after radiotherapy. Here, we further discuss about EphA4 receptor as a potential therapeutic target for the treatment of this cancer type. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT Dentin matrix protein 1 (DMP1) is an extracellular matrix protein involved in phosphate metabolism and biomineralization, and its expression markedly increases during the maturation of osteoblasts into osteocytes. We previously reported that an increased level of inorganic phosphate (Pi) in media up-regulated the expression of Dmp1 in primary osteocytes isolated from mouse bones. In the present study, we found that elevated extracellular Pi strongly induced the expression of Dmp1 in osteoblasts and explored its underlying mechanism of action. In an osteoblastic cell line MC3T3-E1, increases in extracellular Pi induced the phosphorylation of ERK1/2 and up-regulated the expression of Dmp1 , fibroblast growth factor 2 (Fgf2) and Fgf receptor 1 (Fgfr1) . A co-treatment with the MEK inhibitor U0126 abolished the increase in the expression of Dmp1 and Fgfr1 by elevated Pi, suggesting the involvement of the MEK/ERK pathway in this up-regulation. Elevated extracellular Pi also resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), which was diminished by knockdown of Slc20a1 encoding Pit1 sodium-phosphate co-transporter. The co-treatment with an inhibitor against FGFR (SU5402) abolished the up-regulation of Dmp1 induced by elevated extracellular Pi. In primary osteoblasts, a treatment with 4 mM Pi transiently increased the expression of early growth response 1 ( Egr1 ) before the up-regulation of Dmp1 . These results indicate that FGFR mediates the direct effects of extracellular Pi on the expression of Dmp1 in osteoblasts and enhance the close relationship between the signaling evoked by elevated extracellular Pi and FGF/FGFR signaling. This article is protected by copyright. All rights reserved

Beschreibung: Osteoblasts and osteoclasts are well orchestrated through different mechanisms of communication during bone remodeling. Previously we found that osteoclast-specific disruption of one of the BMP receptors, Bmpr1a , results in increased osteoblastic bone formation in mice. We hypothesized that BMPR1A signaling in osteoclasts regulates production of either membrane bound proteins or secreted molecules that regulated osteoblast differentiation. In our current study, we co-cultured wild type osteoblasts with either control osteoclasts or osteoclasts lacking BMPR1A signaling activity. We found that loss of Bmpr1a in osteoclasts promoted osteoblast mineralization in vitro . Further, we found that the expression of Cx43 / Gja1 in the mutant osteoclasts was increased, which encoded for one of the gap junction proteins connexin 43/gap junction alpha 1. Knockdown of Gja1 in the mutant osteoclasts for Bmpr1a reduced osteoblastic mineralization when co-cultured. Our findings suggest that GJA1 may be one of the downstream targets of BMPR1A signaling in osteoclasts that mediates osteoclast-osteoblast communication during bone remodeling. This article is protected by copyright. All rights reserved

Beschreibung: Many infants who develop bronchopulmonary dysplasia (BPD) are born with serious respiratory distress syndrome (RDS), which is associated with impaired vascular and alveolar growth. RDS is a breathing disorder that mostly affects preterm infants and occurs in infants whose lungs have not yet been fully developed. The use of surfactant in RDS treatment does not necessarily prevent BPD. Endothelial progenitor cells (EPCs) may contribute to lung angiogenesis for the prevention and treatment of BPD. The aim of this study was to evaluate the therapeutic efficacy of phototherapy for EPC release in preterm infants born with RDS.75 RDS preterm infants were divided into two groups: RDS with phototherapy and RDS without phototherapy. Respiratory indices were recorded for each patient. Circulating EPCs were isolated before and after phototherapy and colony forming efficiency, chemotactic, tubulogenic, proliferative, and functional properties of these cells were analyzed. Our results showed that phototherapy increased the release of EPCs into the circulation in RDS preterm infants, with augmentation of cell proliferation, tubulogenic, chemotactic, and proliferative properties of EPCs. Phototherapy-induced EPC release was associated with improved lung function as was recorded by significantly decrease in continuous positive airway pressure (CPAP) days, CPAP plus ventilator days, and PCO2 along with a significant increase in PO2 and PaO2/FiO2, resulting in markedly decreased rate of BPD occurrence in preterm infants with RDS. Overall, phototherapy is touted as a promising modality for the amelioration of respiratory performance and prohibition of BPD occurrence in RDS preterm infants. This article is protected by copyright. All rights reserved

Beschreibung: Studies on adipogenesis may be important for regulating human and/or animal obesity, which causes several complications such as, type II diabetes, hypertension, and cardiovascular disease, thus giving rise to increased economic burden in many countries. Previous reports revealed that various flavonoids have anti-apoptotic, antioxidant, and cell differentiation-regulating activities with a number of physiological benefits, including protection from cardiovascular disease, cancers, and oxidative stress. As we found that the hydroxylation patterns of the flavonoid B ring are known to play a critical role in their function, we screened several flavonoids containing different numbers and positions of OH substitutions in B ring for their modulatory property on adipogenesis. In this study, we revealed the anti-adipogenic activity of the naturally-derived flavonoid, 3,4′-dihydroxyflavone (3,4′-DHF) in murine 3T3-L1 pre-adipocytes and equine adipose-derived stromal cells (eADSCs). We found that treatment with 3,4′-dihydroxyflavone (3,4′-DHF) led to decreased expression of adipogenic markers and lipid deposition with differential modulation of ROS and kinase signaling pathways. Regulation of ROS generation through the differential modulation of ROS-regulating gene expression was revealed to have an important role in the suppression of adipogenesis and increase of osteogenesis in eADSCs following 3,4′-DHF treatment. These results suggest that the flavonoid 3,4′-DHF can be used to regulate adipogenesis in ADSCs, which has potential therapeutic application in regenerative medicine or health care for humans and many sport or companion animals. This article is protected by copyright. All rights reserved

Beschreibung: ABSTRACT The phenotypic modulation of VSMCs is a key cellular event driving neointimal formation and vascular remodeling. As a multifaceted cytokine of cell-mediated immunity, IFN-γ has been shown to play a critical role in the pathogenesis of vascular proliferative diseases. Although the important function of IFN-γ on regulating VSMC activation is well established, the molecular mechanisms by which elicits VSMC responses are poorly defined. Recent studies have identified HMGB1 as a principal effector to mediate IFN-γ-dependent biological functions in multiple cell types. Moreover, SIRT1 has emerged as a critical regulator of cellular processes through deacetylating multiple substrates, including HMGB1. Thus, we examined the role of IFN-γ on HMGB1 release, SIRT1 expression, and VSMC phenotypic modulation as well as the underlying molecular mechanisms. We show that IFN-γ dose-dependently induces HMGB1 cytoplasmic accumulation and its active release from VSMCs, resulting in enhanced HMGB1 in the medium. Conversely, IFN-γ treatment led to a dramatic decrease in SIRT1 expression. Additionally, pretreatment with resveratrol, a selective SIRT1 activator, abrogated IFN-γ-induced HMGB1 translocation and its release. Moreover, IFN-γ stimulates VSMC phenotypic modulation to an activated synthetic state characterized by the repression of SMC differentiation markers such as SM22α and calponin and the increase in cell motility. In contrast, blocking HMGB1 release or activity by resveratrol and HMGB1-neutralizing antibody prevents IFN-γ-induced phenotypic modulation of VSMCs. Overall, this study provides the first evidence showing that HMGB1 plays a critical role in regulating VSMC phenotypic modulation, suggesting that HMGB1 may be a potential therapeutic target to prevent vascular occlusive diseases. This article is protected by copyright. All rights reserved

Beschreibung: A large number of studies have showed that women reported feeling pain more acutely than men. In support of this hypothesis, many research groups proved that in different animals model of pain the sex hormones regulate the somatic and visceral sensitivity to different noxious stimuli Therefore, in this study we went to evaluate if estrogen hormones by regulating the CGRP levels are implicated during the visceral pain transmission. Toward this aim, we have investigated the effect of 17beta-estradiol in regulating the synthesis and release of CGRP, as well as the expression levels of the opioid receptor of type K. In order to gain information about the potential effects of 17β-estradiol on K-opioid receptor expression and activity, we have cultured F11 cells. Our results revealed that, when F11 cells were short term exposed (30 min) to 17β-estradiol, the expression of the opioid K receptor was not significantly modified. We carried out enzyme immunoassay analysis to evaluate the potential effects of short term exposure to 17β-estradiol (30 min) on the release of CGRP in F11 cells. The results obtained showed that 17β-estradiol at the dose of 100 nM is able to induce the release of CGRP from F11 cells; whereas, a higher dose of 17β-estradiol (200 nM) did not produce significant effects when compared to control. In conclusion, all these findings suggest that the 17β-estradiol-regulated release of CGRP could at least in part provide a rational explanation for the difference of gender in the visceral pain sensitivity. This article is protected by copyright. All rights reserved