Activation Of Signaling Proteins Research Articles

Endothelial damage is aggravated in acute GvHD and could predict its development.

The aim of the present study was to explore whether there is enhanced endothelial dysfunction in patients developing acute GvHD (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT) and to identify biomarkers with predictive and/or diagnostic value. In in vitro experiments, endothelial cells (ECs) were exposed to serum from patients with (aGvHD, n=31) and without (NoGvHD, n=13) aGvHD, to evaluate changes in surface adhesion receptors, the reactivity of the extracellular matrix by measuring the presence of Von Willebrand factor (VWF) and platelet adhesion, and the activation of intracellular signaling proteins. Plasma levels of VWF, ADAMTS-13, TNF receptor 1 (TNFR1), soluble vascular cell adhesion molecule 1 and soluble intercellular adhesion molecule 1 were also measured. In vitro results showed a more marked proinflammatory and prothrombotic phenotype in ECs in association with aGvHD. Regarding circulating biomarkers, levels of VWF and TNFR1 above an optimal cutoff score, taken independently or combined, at day 7 after allo-HCT, would be able to positively predict that around 90% of patients will develop aGvHD. Our results demonstrate that endothelial damage is aggravated in those allo-HCT recipients developing aGvHD, and that VWF and TNFR1 are promising predictive aGvHD biomarkers. These findings could contribute to improve the understanding of the pathophysiology of aGvHD.

Wnt5a induces catabolic signaling and matrix metalloproteinase production in human articular chondrocytes.

Aberrant Wnt signaling may contribute to osteoarthritis (OA) but the Wnt family members involved have not been fully identified. The purpose of this study was to investigate the role of Wnt5a as a potential mediator of cartilage destruction in OA. Immunohistochemistry to detect Wnt5a was performed using normal and OA human articular cartilage. Cultured normal human chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus or recombinant Wnt5a protein with or without pretreatment using a panel of signaling inhibitors. Expression of Wnt5a, anabolic genes and catabolic genes were determined by quantitative real-time PCR. Production of Wnt5a protein and matrix metalloproteinases (MMPs) as well as activation of signaling proteins were analyzed by immunoblotting. Wnt5a was present in human articular cartilage with OA changes and its expression and secretion were increased in FN-f stimulated chondrocytes. FN-f stimulated Wnt5a production through the c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) pathways. Wnt5a reduced aggrecan gene expression after 48h of treatment. Wnt5a seemed to promote MMP1, -3, and -13 expression as well as MMP1 and MMP13 protein production in normal human chondrocytes. Wnt5a inhibitor peptides did not affect FN-f induced MMP production. Wnt5a activated β-catenin independent signaling including calmodulin-dependent protein kinase II (CaMKII), JNK, p38, ERK1/2, p65 and Akt. Inhibition of JNK, p38, ERK, PI-3 kinase and CaMKII by specific signaling inhibitors suppressed Wnt5a mediated MMP1 and MMP13 production. Wnt5a is present in human OA cartilage and can promote chondrocyte catabolic activity through non-canonical Wnt signaling, which suggests a potential role in OA.

Aerobic exercise reduces hippocampal ERK and p38 activation and improves memory of middle-aged rats.

Aging is often accompanied by cognitive decline, memory impairment, and an increased susceptibility to neurodegenerative disorders. Although the physiological processes of aging are not fully understood, these age-related changes have been interpreted by means of various cellular and molecular theories. Among these theories, alterations in the intracellular signaling pathways associated with cell growth, proliferation, and survival have been highlighted. Based on these observations and on recent evidence showing the beneficial effects of exercise on cognitive function in the elderly, we investigated the cell signaling pathways in the hippocampal formation of middle-aged rats (18months old) submitted to treadmill exercise over 10 days. To do this, we evaluated the hippocampal activation of intracellular signaling proteins linked to cell growth, proliferation, and survival, such as Akt, mTOR, p70S6K, ERK, CREB, and p38. We also explored the cognitive performance (inhibitory avoidance) of middle-aged rats. It was found that physical exercise reduces ERK and p38 activation in the hippocampal formation of aged rats, when compared to the control group. The hippocampal activation and expression of Akt, mTOR, p70S6K, and CREB were not statistically different between the groups. It was also observed that aged rats from the exercise group exhibited better cognitive performance in the inhibitory avoidance task (aversive memory) than aged rats from the control group. Our results indicate that physical exercise reduces intracellular signaling pathways linked to inflammation and cell death (i.e., ERK and p38) and improves memory in middle-aged rats.

β-carotene at physiologically attainable concentration induces apoptosis and down-regulates cell survival and antioxidant markers in human breast cancer (MCF-7) cells.

Although β-carotene is known for its anti-carcinogenic and antioxidant properties, a few recent epidemiological and experimental evidence show that at higher concentration it acts as pro-oxidant and induces cancer. Since the global burden of breast cancer exceeds all other types of cancer, and its incidence rates is also in increasing trend, the present study attempted to evaluate the anti-cancer molecular mechanism of β-carotene (at 1µM concentration) isolated from Spinacia oleracea in human breast cancer (MCF-7) cells. The carotenoid was purified by open column chromatography and identified by LC-MS. The anti-proliferative effect of β-carotene at different concentrations was evaluated by WST-1 assay and the changes in cell morphology were examined by microscopic observation. The induction of apoptosis by β-carotene was observed by DAPI staining and colorimetric caspase-3 assay. The expression of cell survival, apoptotic, and antioxidant marker proteins was measured by western blot analysis. Purified β-carotene inhibited the viability of MCF-7 cells in a dose-dependent manner, which was well correlated with changes in cell morphology. Increased apoptotic cells were observed in β-carotene (1µM)-treated cells. This apoptosis induction was associated with increased caspase-3 activity. The protein expression studies showed that β-carotene at 1µM concentration effectively decreases the expression of the anti-apoptotic protein, Bcl-2 and PARP, and survival protein, NF-kB. It also inhibited the activation of intracellular growth signaling proteins, Akt and ERK1/2. The inhibition of Akt activation by β-carotene results in decreased phosphorylation of Bad. Further, it down-regulated antioxidant enzyme, SOD-2, and its transactivation factor (Nrf-2), and endoplasmic reticulum (ER) stress marker, XBP-1, at protein levels. These findings exhibit the key role of β-carotene even at a low physiological concentration in MCF-7 cells which further explains its predominant anti-cancer activity.

The HLA-DR mediated signalling increases the migration and invasion of melanoma cells, the expression and lipid raft recruitment of adhesion receptors, PD-L1 and signal transduction proteins

The constitutive expression of Major Histocompatibility Complex (MHC) class II molecules is restricted to professional Antigen-Presenting Cells (APCs), nevertheless almost 50% of melanomas express constitutively the MHC class II molecules. Therefore, in two MHC class II constitutive expressing melanoma cell lines we studied the signalling mediated by the HLA-DR molecules in the aim to understand the consequence of class II mediated signalling on metastatic dissemination of melanoma. In particular, we reported that the HLA-DR mediated signalling play a new role in melanoma progression, increasing the migration and invasion of melanoma cells. Furthermore, we showed that the HLA-DR mediated signalling increases the expression and the lipid raft localisation of class II molecules, PD-L1 receptor, Integrin and CAM adhesion receptors, FAK, AKT and STAT3 signalling proteins. We also showed that the HLA-DR mediated signalling increases the activation of FAK, AKT, ERK, PKC and STAT3 signalling proteins and the expression of ILK, PAX, BRAF, ERK and PKC. Indeed, the results showed suggest that the HLA-DR mediated signalling provides a platform useful to frustrate an effective anti-tumour response and to increase melanoma migration and metastatic dissemination of this cancer.

Frequent AKT1E17K mutations in skull base meningiomas are associated with mTOR and ERK1/2 activation and reduced time to tumor recurrence.

Skull base meningiomas are considered to be difficult for surgical treatment. We wondered whether genetic alterations recently identified in benign non-NF2-mutated World Health Organization (WHO) grade I meningiomas are related to clinical features of skull base meningiomas and whether druggable signaling pathways are activated. We analyzed 93 skull base meningiomas (82 WHO grade I, 11 WHO grade II) for mutations of hot spots or the most relevant exons of AKT1, KLF4/TRAF7, SMO, PI3K, and the TERT promoter. The AKT1E17K mutation was present in 31% of patients and was related to meningothelial histology. AKT1E17K had a negative effect on the time to tumor recurrence. Analyses of activated signaling proteins revealed among AKT1E17K tumors a significantly higher rate of phospho-mammalian target of rapamycin (mTOR) and phospho-p70S6K+ tumors. AKT1E17K tumors with immunoexpression of phospho-extracellular signal-regulated kinase 1 or 2 (ERK1/2) were characterized by significantly shorter time to tumor recurrence compared with AKT1wt tumors expressing phospho-ERK1/2 (P = .046). KLF4 mutations (K409Q) were present in 11.8% of cases, with significant association to the secretory/transitional subtype (P < .001). The presence of the KLF4 K409Q mutation was associated with favorable outcome. One phosphatidylinositol-3 kinase (PI3K) mutation but no SMO or TERT promoter mutation was found. AKT1E17K mutation is frequent in skull base meningiomas, results in activation of the mTOR and ERK1/2 signaling pathways, and has negative impact on tumor recurrence. Patients with skull base meningiomas with AKT1E17K mutation might benefit from additional treatment targeting the mTOR pathway. Generally, the PI3K-Akt-mTOR axis might be a potential target for kinase inhibitors in these tumors.

Distinct immune and clinical responses to alcohol-soluble vs. salt-soluble wheat proteins in mice

Abstract Food hypersensitivity is a growing global public health problem. Wheat has been linked to distinct types of human hypersensitivities with incompletely understood mechanisms. Although wheat proteins (WP) differ in solubility, it is unknown whether alcohol-soluble (AS) and salt-soluble (SS) WP differ in their immune and disease activation properties. Here we tested the hypothesis that AS and SSWP extracted from soft-wheat will elicit distinct types of immune and clinical responses in inbred mice. Groups of BALB/cJ mice weaned onto a plant-protein free diet received IP injections with AS and SSWP (0.01 mg/mouse) along with alum. The WP-specific (S) IgE, IgG1, IgG2a, IgA and total (T) IgE responses were analyzed. Systemic anaphylaxis and mucosal mast cell degranulation response (MMCR) upon IP challenge were quantified by hypothermia shock response (HSR) and elevation of plasma mMCP-1, respectively. Mice were monitored for dermatitis and alopecia. Spleen tissue extracts were used in measuring immune-activation biomarkers. Whereas the SSWP elicited higher titers of SIgA, the ASWP elicited higher titers of SIgE, SIgG1, SIgG2a and higher TIgE levels. The ASWP elicited significantly higher HSR and MMCR. Only ASWP but not SSWP elicited dorsal neck dermatitis lesions in 44% of mice. Alopecia lesions, without inflammation, were noted in 22% of ASWP- and 33% of SSWP-injected mice. Furthermore, ASWP and SSWP elicited distinct patterns of activation of acute-phase response proteins, chemokines, cytokines, growth factors, matrix proteins and signaling proteins. These data provide the first direct evidence that WP fractions that differ in solubility elicit qualitatively distinct immune and clinical reactions in genetically identical mice.

MP39-10 INTRA-TUMOR HETEROGENEITY IN RENAL CELL CARCINOMA: IMPLICATIONS FOR PROTEOMIC ANALYSIS OF RENAL MASS BIOPSIES

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2017MP39-10 INTRA-TUMOR HETEROGENEITY IN RENAL CELL CARCINOMA: IMPLICATIONS FOR PROTEOMIC ANALYSIS OF RENAL MASS BIOPSIES Rustin Massoudi, Christian Hoerner, Thomas Metzner, Jennifer O'Rourke, Rachael Curtis, Laurel Stell, Chiara Sabatti, James Brooks, Alice Fan, and John Leppert Rustin MassoudiRustin Massoudi More articles by this author , Christian HoernerChristian Hoerner More articles by this author , Thomas MetznerThomas Metzner More articles by this author , Jennifer O'RourkeJennifer O'Rourke More articles by this author , Rachael CurtisRachael Curtis More articles by this author , Laurel StellLaurel Stell More articles by this author , Chiara SabattiChiara Sabatti More articles by this author , James BrooksJames Brooks More articles by this author , Alice FanAlice Fan More articles by this author , and John LeppertJohn Leppert More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1184AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intra-tumor heterogeneity (ITH) is the presence of cell clones with different genetic or histologic phenotypes that occupy distinct spaces within a tumor. Whether genomic ITH translates into functional ITH on the level of protein expression and activity is less well characterized. We hypothesized that variation in expression and activation of critical RCC signaling proteins is less than genomic ITH, resulting in a limited number of functional phenotypes in a tumor. This framework would allow for representative sampling of RCC using renal mass biopsy and advanced proteomic techniques. METHODS We profiled the expression and activation state of extracellular signal-regulated kinase (ERK), a critical effector of the MAPK signaling pathway, in 131 ex vivo biopsies from 39 clear cell RCC tumors and adjacent normal parenchyma after nephrectomy. We performed fine-needle aspirate (FNA) biopsies of grossly representative sections after bivalving the kidney. We performed nano-scale immunoassays (NIA) to quantify the absolute and relative abundances of the phospho-isoforms of ERK1/2 using the Peggy Sue instrument (Protein Simple). ERK1/2 was interrogated with a pan-ERK antibody. Expression levels of ERK levels were compared across samples by normalization to the levels of the ubiquitously expressed protein HSP70. RESULTS ERK1 was infrequently activated, with most samples (69%) demonstrating no phosphorylation of ERK1. In contrast, ERK2 demonstrated phosphorylation in the majority of the samples. Two distinct mono-phosphorylated ERK2 isoforms were detected (pERK2a and pERK2b), with relative abundances ranging from 0-58% and 1-57%, respectively. The relative abundance of dual-phosphorylated ERK2 (ppERK2) ranged from 0-26%. The overall ITH of ERK2 activation was low (average standard deviation [SD] 6%) compared with the difference in ERK2 activation among the 39 tumors (SD 5% - 22%). CONCLUSIONS The abundance and activation of ERK1/2 can be measured from scant FNAs of RCC using novel proteomic methods. We found little ITH of ERK, which suggests that protein ITH is generally less than functional heterogeneity among tumors. These data suggest that a single renal mass biopsy may accurately measure the activity of relevant signaling pathways within a tumor, which may facilitate future precision medicine approaches. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e496-e497 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Rustin Massoudi More articles by this author Christian Hoerner More articles by this author Thomas Metzner More articles by this author Jennifer O'Rourke More articles by this author Rachael Curtis More articles by this author Laurel Stell More articles by this author Chiara Sabatti More articles by this author James Brooks More articles by this author Alice Fan More articles by this author John Leppert More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Inhibitory effect of Patrinia scabiosifolia Link on the development of atopic dermatitis-like lesions in human keratinocytes and NC/Nga mice

Ethnopharmacological relevanceAtopic dermatitis (AD) is a chronic pruritic and inflammatory disease occurring in skin. Patrinia scabiosifolia Link (PS), a member of the Patrinia genus (Caprifoliaceae family), has traditionally been used in folk medicines to treat various inflammatory diseases such as acute appendicitis, ulcerative colitis, and pelvic inflammation in Korea and other parts of East Asia. Aim of the studyThis study investigated the anti-inflammatory effects of PS on AD in vitro and in vivo. Materials and methodsWhole PS plants were dried, powdered, and then underwent extraction with DMSO. Both ELISA and western blotting were performed to evaluate cytokine concentration and the expression and activation of filaggrin and signaling proteins. Five-week‐old female NC/Nga mice were used as an AD-like mouse model by treating them with 2,4-dinitrochlorobenzene (DNCB). ResultsIn human keratinocytic HaCaT cells, PS extract inhibited the production of IL‐8, and TARC, which had been increased by TNF-α and IFN-γ. The TNF-α and IFN-γ suppressed filaggrin expression was associated with phosphorylation of JNK1 and JNK2, and NF-κB translocation. PS recovered the inhibition of filaggrin expression induced by TNF-α and IFN-γ by blocking the activation of JNK1/2, and NF-κB by the IFN-γ and TNF-α treatment. The in vivo experiment results showed that, compared to DNCB treatment PS administration reduced thickening of the epidermis and infiltration of inflammatory cells into the dermis. Moreover, the decrease of filaggrin expression due to DNCB treatment was recovered by PS administration. The serum IgE level was decreased by PS treatment. Additionally, secretions of IL-4, IL-5, IL-13, and eotaxin in splenocytes were lower in the PS-treated group than in the DNCB group. ConclusionPS may attenuate the development of AD‐like lesions by increasing filaggrin expression and lowering IgE and inflammatory cytokine levels. These results indicate the potential for development of a PS-based drug treatment for AD.

Aloesin from Aloe vera accelerates skin wound healing by modulating MAPK/Rho and Smad signaling pathways in vitro and in vivo

BackgroundCutaneous wound healing is a complex process involving various regulatory factors at the molecular level. Aloe vera is widely used for cell rejuvenation, wound healing, and skin moisturizing. Hypothesis/PurposeThis study aimed to investigate the effects of aloesin from Aloe vera on cutaneous wound healing and mechanisms involved therein. Study DesignThis study consisted of both in vitro and in vivo experiments involving skin cell lines and mouse model to demonstrate the wound healing effects of aloesin by taking into account several parameters ranging from cultured cell migration to wound healing in mice. MethodsThe activities of Smad signaling molecules (Smad2 and Smad3), MAPKs (ERK and JNK), and migration-related proteins (Cdc42, Rac1, and α-Pak) were assessed after aloesin treatment in cultured cells (1, 5 and 10µM) and mouse skin (0.1% and 0.5%). We also monitored macrophage recruitment, secretion of cytokines and growth factors, tissue development, and angiogenesis after aloesin treatment using IHC analysis and ELISAs. ResultsAloesin increased cell migration via phosphorylation of Cdc42 and Rac1. Aloesin positively regulated the release of cytokines and growth factors (IL-1β, IL-6, TGF-β1 and TNF-α) from macrophages (RAW264.7) and enhanced angiogenesis in endothelial cells (HUVECs). Aloesin treatment accelerated wound closure rates in hairless mice by inducing angiogenesis, collagen deposition and granulation tissue formation. More importantly, aloesin treatment resulted in the activation of Smad and MAPK signaling proteins that are key players in cell migration, angiogenesis and tissue development. ConclusionAloesin ameliorates each phase of the wound healing process including inflammation, proliferation and remodeling through MAPK/Rho and Smad signaling pathways. These findings indicate that aloesin has the therapeutic potential for treating cutaneous wounds.

Phaleria macrocarpa (Boerl.) fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 human breast cancer cell

Ethnopharmacological relevancePhaleria macrocarpa (Scheff) Boerl, is a well-known folk medicinal plant in Indonesia. Traditionally, P. macrocarpa has been used to control cancer, impotency, hemorrhoids, diabetes mellitus, allergies, liver and hearth disease, kidney disorders, blood diseases, acne, stroke, migraine, and various skin diseases. Aim of the studyThe purpose of this study was to determine the in situ cytotoxicity effect P. macrocarpa fruit ethyl acetate fraction (PMEAF) and the underlying molecular mechanism of cell death. Materials and methodsMDA-MB-231 cells were incubated with PMEAF for 24h. Cell cycle and viability were examined using flow cytometry analysis. Apoptosis was determined using the Annexin V assay and also by fluorescence microscopy. Apoptosis protein profiling was detected by RayBio® Human Apoptosis Array. ResultsThe AO/PI staining and flow cytometric analysis of MDA-MB-231 cells treated with PMEAF were showed apoptotic cell death. The cell cycle analysis by flow cytometry analysis revealed that the accumulation of PMEAF treated MDA-MB-231 cells in G0/G1 and G2/M-phase of the cell cycle. Moreover, the PMEAF exert cytotoxicity by increased the ROS production in MDA-MB-231 cells consistently stimulated the loss of mitochondrial membrane potential (∆Ψm) and induced apoptosis cell death by activation of numerous signalling proteins. The results from apoptosis protein profiling array evidenced that PMEAF stimulated the expression of 9 pro-apoptotic proteins (Bax, Bid, caspase 3, caspase 8, cytochrome c, p21, p27, p53 and SMAC) and suppressed the 4 anti-apoptotic proteins (Bcl-2, Bcl-w, XIAP and survivin) in MDA-MB-231 cells. ConclusionThe results indicated that PMEAF treatment induced apoptosis in MDA-MB-231 cells through intrinsic mitochondrial related pathway with the participation of pro and anti-apoptotic proteins, caspases, G0/G1 and G2/M-phases cell cycle arrest by p53-mediated mechanism.

Defective signaling through plexin-A1 compromises the development of the peripheral olfactory system and neuroendocrine reproductive axis in mice.

The olfacto-genital syndrome (Kallmann syndrome) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. This is a genetically heterogeneous developmental disease with various modes of transmission, including oligogenic inheritance. Previous reports have involved defective cell signaling by semaphorin-3A in the disease pathogenesis. Here, we report that the embryonic phenotype of Plxna1-/- mutant mice lacking plexin-A1 (a major receptor of class 3 semaphorins), though not fully penetrant, resembles that of Kallmann syndrome fetuses. Pathohistological analysis indeed showed a strongly abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the hypothalamic brain region in some of the mutant mice, which resulted in reduced fertility in adult males. We thus screened 250 patients for the presence of mutations in PLXNA1, and identified different nonsynonymous mutations (p.V349L, p.V437L, p.R528W, p.H684Y, p.G720E, p.R740H, p.R813H, p.R840Q, p.A854T, p.R897H, p.L1464V, p.K1618T, p.C1744F), all at heterozygous state, in 15 patients. Most of these mutations are predicted to affect plexin-A1 stability or signaling activity based on predictive algorithms and a structural model of the protein. Moreover, in vitro experiments allowed us to show the existence of deleterious effects of eight mutations (including a transcript splicing defect), none of which are expected to result in a complete loss of protein synthesis, targeting, or signaling activity, though. Our findings indicate that signaling insufficiency through plexin-A1 can contribute to the pathogenesis of Kallmann syndrome, and further substantiate the oligogenic pattern of inheritance in this developmental disorder.

Digoxin-induced retinal degeneration depends on rhodopsin

Na,K-ATPases are energy consuming ion pumps that are required for maintaining ion homeostasis in most cells. In the retina, Na,K-ATPases are especially important to sustain the dark current in photoreceptor cells needed for rapid hyperpolarization of rods and cones in light. Cardiac glycosides like digoxin inhibit the activity of Na,K-ATPases by targeting their catalytic alpha subunits. This leads to a disturbed ion balance, which can affect cellular function and survival. Here we show that the treatment of wild-type mice with digoxin leads to severe retinal degeneration and loss of vision. Digoxin induced cell death specifically in photoreceptor cells with no or only minor effects in other retinal cell types. Photoreceptor-specific cytotoxicity depended on the presence of bleachable rhodopsin. Photoreceptors of Rpe65 knockouts, which have no measurable rhodopsin and photoreceptors of Rpe65R91W mice that have <10% of the rhodopsin found in retinas of wild-type mice were not sensitive to digoxin treatment. Similarly, cones in the all-cone retina of Nrl knockout mice were also not affected. Digoxin induced expression of several genes involved in stress signaling and inflammation. It also activated proteins such as ERK1/2, AKT, STAT1, STAT3 and CASP1 during a period of up to 10 days after treatment. Activation of signaling genes and proteins, as well as the dependency on bleachable rhodopsin resembles mechanisms of light-induced photoreceptor degeneration. Digoxin-mediated photoreceptor cell death may thus be used as an inducible model system to study molecular mechanisms of retinal degeneration.

Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells

Background aimsClinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. MethodsCells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. ResultsDCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. DiscussionThis study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols.

Dynamic Temporal Control of Signaling Activated Gene Regulation

Many signal transduction and gene regulatory pathways are highly dynamic resulting in temporally varying activity of signaling proteins and time varying gene expression profiles. These time varying signaling profiles are important as these can direct different cellular phenotypes. Currently, our biophysical understanding of these temporal signaling profiles is very rudimentary and relies on the genetic manipulation of specific proteins or drug treatment regimes. Although protein numbers are often manipulated over a wide dynamic range using an inducible promoter, the activity of these signaling proteins may only be manipulated by mutations or drugs. One drawback of this approach is that genetic protein modification may result in a significant interference with the function of the cell, which may cause lethal phenotypes. Another drawback is that drugs are often not available for proteins of interest or have considerable off-target effect. In order to avoid these limitations, we developed a time varying perturbation approach that utilizes time varying ligand profiles to investigate the temporal properties of signaling and gene regulatory pathways, without genetic manipulation or drug treatments. To demonstrate the feasibility of this approach, we chose to interrogate an evolutionary conserved stress response pathway in yeast S. Cerevisiae, which enables to manipulate the intensity, duration and temporal activity of the signal transduction pathway in single cells. We are able to quantify signal transduction activation, signal transduction saturation and gene expression activation thresholds at high precision and demonstrate that the signaling dynamics is proportional to the first time derivative of the external perturbation profile. Because this approach is independent of the biological pathway or organism, it presents a general methodology to interrogate and control signaling and gene expression pathways non-invasively without the need for genetic or drug perturbations.

The daunorubicin interplay with mimetic model membranes of cancer cells: A biophysical interpretation

The present work aimed to study the interactions between the anticancer drug daunorubicin and lipid membrane mimetic models of cancer cells composed by their most representative classes of phospholipids, with different degrees of complexity.Regarding these anticancer drug-membrane interactions, several biophysical parameters were assessed using liposomes (LUVs) composed of different molar ratios of DMPC, DOPC, DPPS, DOPE and Chol. In this context, daunorubicin's membrane concentration was determined by calculating its partition coefficient (Kp) between liposomes and water using derivative UV/vis spectrophotometry at 37°C and pH6.3, a typical tumoral microenvironment. Characterization of the zeta potential of such model membranes, in both the absence and presence of the compound, was accomplished through Electrophoretic Light Scattering (ELS). Fluorescence quenching studies, which determine the location of the drug within the bilayer, were carried out using liposomes labelled with DPH and TMA-DPH, fluorescent probes with known membrane position. Temperature dependent steady-state anisotropy assays were also performed to measure the daunorubicin effect on the membranes' microviscosity.The overall results support that daunorubicin permeation depends on the phospholipid membrane composition and causes alterations in the biophysical properties of the bilayers, namely in the membrane fluidity. The interaction of daunorubicin with the studied phospholipids is mainly driven by electrostatic and hydrophobic interactions.These insights demonstrated that not only membranes can affect daunorubicin accumulation in cells but the compound can alter the properties of membranes. The changes produced by daunorubicin on the lipid structure may constitute an additional mechanism of action, which might lead to modifications in the location and, consequently, the activity of membrane signaling proteins.

Resistance Exercise Reduces Seizure Occurrence, Attenuates Memory Deficits and Restores BDNF Signaling in Rats with Chronic Epilepsy.

Epilepsy is a disease characterized by recurrent, unprovoked seizures. Cognitive impairment is an important comorbidity of chronic epilepsy. Human and animal model studies of epilepsy have shown that aerobic exercise induces beneficial structural and functional changes and reduces the number of seizures. However, little is yet understood about the effects of resistance exercise on epilepsy. We evaluated the effects of a resistance exercise program on the number of seizures, long-term memory and expression/activation of signaling proteins in rats with epilepsy. The number of seizures was quantified by video-monitoring and long-term memory was assessed by an inhibitory avoidance test. Using western blotting, multiplex and enzyme-linked immunosorbent assays, we determined the effects of a 4-week resistance exercise program on IGF-1 and BDNF levels and ERK, CREB, mTOR activation in the hippocampus of rats with epilepsy. Rats with epilepsy submitted to resistance exercise showed a decrease in the number of seizures compared to non-exercised epileptic rats. Memory deficits were attenuated by resistance exercise. Rats with epilepsy showed an increase in IGF-1 levels which were restored to control levels by resistance exercise. BDNF levels and ERK and mTOR activation were decreased in rats with epilepsy and resistance exercise restored these to control levels. In conclusion, resistance exercise reduced seizure occurrence and mitigated memory deficits in rats with epilepsy. These resistance exercise-induced beneficial effects can be related to changes in IGF-1 and BDNF levels and its signaling protein activation. Our findings indicate that the resistance exercise might be included as complementary therapeutic strategy for epilepsy treatment.

Proteases Revisited: Roles and Therapeutic Implications in Fibrosis.

Proteases target many substrates, triggering changes in distinct biological processes correlated with cell migration, EMT/EndMT and fibrosis. Extracellular protease activity, demonstrated by secreted and membrane-bound protease forms, leads to ECM degradation, activation of other proteases (i.e., proteolysis of nonactive zymogens), decomposition of cell-cell junctions, release of sequestered growth factors (TGF-β and VEGF), activation of signal proteins and receptors, degradation of inflammatory inhibitors or inflammation-related proteins, and changes in cell mechanosensing and motility. Intracellular proteases, mainly caspases and cathepsins, modulate lysosome activity and signal transduction pathways. Herein, we discuss the current knowledge on the multidimensional impact of proteases on the development of fibrosis.

Directed Evolution Methods to Rewire Signaling Networks.

The ability to sense and process cues about changing environments is fundamental to life. Cells have evolved elaborate signaling pathways in order to respond to both internal and external stimuli appropriately. These pathways combine protein receptors, signal transducers, and effector genes in highly connected networks. The numerous interactions found between signaling proteins are essential to maintain strict regulation and produce a suitable cellular response. As a result, a signaling protein's activity in isolation can differ greatly from its activity in a native context. This is an important consideration when studying or engineering signaling pathways. Fortunately, the difficulty of studying network interactions is fading thanks to advances in library construction and cell sorting. In this chapter, we describe two methods for generating libraries of mutant proteins that exhibit altered network interactions: whole-gene point mutagenesis and domain shuffling. We then provide a protocol for using fluorescence-activated cell sorting to isolate interesting variants in live cells by focusing on the unicellular eukaryotic model organism Saccharomyces cerevisiae, using as an example recent work that we have done on its G protein-coupled receptor Ste2.

Changes in muscle fiber contractility and extracellular matrix production during skeletal muscle hypertrophy.

Skeletal muscle can adapt to increased mechanical loads by undergoing hypertrophy. Transient reductions in whole muscle force production have been reported during the onset of hypertrophy, but contractile changes in individual muscle fibers have not been previously studied. Additionally, the extracellular matrix (ECM) stores and transmits forces from muscle fibers to tendons and bones, and determining how the ECM changes during hypertrophy is important in understanding the adaptation of muscle tissue to mechanical loading. Using the synergist ablation model, we sought to measure changes in muscle fiber contractility, collagen content, and cross-linking, and in the expression of several genes and activation of signaling proteins that regulate critical components of myogenesis and ECM synthesis and remodeling during muscle hypertrophy. Tissues were harvested 3, 7, and 28 days after induction of hypertrophy, and nonoverloaded rats served as controls. Muscle fiber specific force (sFo), which is the maximum isometric force normalized to cross-sectional area, was reduced 3 and 7 days after the onset of mechanical overload, but returned to control levels by 28 days. Collagen abundance displayed a similar pattern of change. Nearly a quarter of the transcriptome changed over the course of overload, as well as the activation of signaling pathways related to hypertrophy and atrophy. Overall, this study provides insight into fundamental mechanisms of muscle and ECM growth, and indicates that although muscle fibers appear to have completed remodeling and regeneration 1 mo after synergist ablation, the ECM continues to be actively remodeling at this time point.NEW & NOTEWORTHY This study utilized a rat synergist ablation model to integrate changes in single muscle fiber contractility, extracellular matrix composition, activation of important signaling pathways in muscle adaption, and corresponding changes in the muscle transcriptome to provide novel insight into the basic biological mechanisms of muscle fiber hypertrophy.