Abstract Ghrelin is a novel appetite-inducing peptide hormone secreted by the stomach. The purpose of this study was first to identify the cDNA encoding sequence for ghrelin in sea bass ( ...Dicentrarchus labrax ). Using molecular cloning techniques we sequenced the cDNA corresponding to sea bass ghrelin mRNA. A total of 798 bases including a 5′-untranslated region (89 bp), an open reading frame (ORF) (324 bp), and a 3′-untranslated region (385 bp) were detected. Nucleotide sequence (ORF) encoded a 108 amino acid prepropeptide that demonstrated complete conservation of the N-terminal “biological active core” (GSSF) of the predicted mature ghrelin peptide. We also analyzed fasting-induced changes in the expression of ghrelin mRNA, using a one-tube two-temperature real-time RT-PCR with which the gene expression can be absolutely quantified using the standard curve method. Our results revealed that ghrelin was highly expressed in the stomach with much lower levels of expression in the proximal intestine and brain. Levels of ghrelin mRNA in the stomach were upregulated under conditions of negative energy balance, such as starvation, and downregulated during positive energy balance, such as refeeding. These findings offer new information about the sea bass ghrelin gene and support a role of this orexigenic hormone in the regulation of food intake in sea bass.
This work is addressed to provide, by in vitro experiments, results on the repercussion that a nanostructured scaffold could have on viability, differentiation and secretion of bioactive factors of ...human adipose-derived stem cells (hASCs) when used in association to promote angiogenesis, a crucial condition to favour tissue regeneration. To achieve this aim, we evaluated cell viability and morphology by MTT (3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) assay and microscopy analysis, respectively. We also investigated the expression of some of those genes involved in angiogenesis and differentiation processes utilizing quantitative polymerase chain reaction (qPCR), whereas the amounts of Vascular Endothelial Growth Factor A, Interleukin 6 and Fatty Acid-Binding Protein 4 secreted in the culture medium, were quantified by enzyme-linked immunosorbent assay (ELISA). Results suggested that, in the presence of the scaffold, cell proliferation and the exocytosis of factors involved in the angiogenesis process are reduced; by contrast, the expression of those genes involved in hASC differentiation appeared enhanced. To guarantee cell survival, the construct dimensions are, generally, smaller than clinically required. Furthermore, being the paracrine event the primary mechanism exerting the beneficial effects on injured tissues, the use of conditioned culture medium instead of cells may be convenient.
► We show protein carbonylation in human endothelial cells exposed to cigarette smoke. ► The main carbonylated proteins were identified by means of redox proteomics. ► The main carbonylated proteins ...are cytoskeletal proteins, glycolytic enzymes, Hsp90. ► Fluorescence microscopy evidenced cytoskeletal alterations. ► TEM showed cytoplasm vacuolisation and alteration of mitochondria.
Cigarette smoke is a significant independent risk factor for vascular diseases and is a leading cause of structural and functional alterations of the vascular endothelium. In this study, we show protein carbonylation in the human umbilical vein endothelial cell line (ECV-304) exposed to whole-phase cigarette smoke extract. The main carbonylated proteins, including cytoskeletal proteins, glycolytic enzymes, xenobiotic metabolizing and antioxidant enzymes, and endoplasmic reticulum proteins, were identified by means of two-dimensional electrophoresis and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry (redox proteomics). Morphological analyses by fluorescence microscopy evidenced alterations in the microtubule cytoskeleton, especially at longer exposure time to cigarette smoke extract. Morphological analyses by transmission electron microscopy showed vacuolisation of the cytoplasm, alteration of mitochondria ultrastructure, and some enlargement of the perinuclear space. The possible role played by protein carbonylation caused by reactive species contained in cigarette smoke in the cigarette smoke-induced endothelial injury is discussed.
Tumor tissue includes cancer cells and normal stromal cells such as vascular endothelial cells, connective tissue cells (cancer associated fibroblast, mesenchymal stem cell), and immune cells ...(tumor‐infiltrating lymphocytes or TIL, dendritic cells, eosinophils, basophils, mast cells, tumor‐associated macrophages or TAM, myeloid‐derived suppressor cells or MDSC). Anti‐tumor activity is mainly mediated by infiltration of NK cells, Th1 and CD8+ T cells, and correlates with expression of NK cell and T cell attracting chemokines. Nevertheless, cancer cells hijack tissue homeostasis through secretion of cytokines and chemokines that mediate not only the induction of an inflamed status that supports cancer cell survival and growth, but also the recruitment and/or activation of immune suppressive cells. CXCL9, CXCL10, and CXCL11 are known for their tumor‐inhibiting properties, but their overexpression in several hematologic and solid tumors correlates with disease severity, suggesting a role in tumor promotion. The dichotomous nature of CXCR3 ligands activity mainly depends on several molecular mechanisms induced by cancer cells themselves able to divert immune responses and to alter the whole local environment. A deep understanding of the nature of such phenomenon may provide a rationale to build up a CXCR3/ligand axis targeting strategy. In this review, we will discuss the role of CXCR3 in cancer progression and in regulation of anti‐tumor immune response and immunotherapy.
Review on how CXCR3 ligands can shape anti‐tumor immune responses and tumor growth and of the promising possibility to target these molecules for cancer immunotherapy.
Two-dimensional difference gel electrophoresis (2D DIGE) was applied to investigate the impact of slaughtering on the postmortem integrity of muscle tissue proteins in European sea bass ...(Dicentrarchus labrax). Three different slaughtering techniques were evaluated: asphyxia in air (AA), asphyxia in ice (AI), and spinal cord severance (SCS). Principal components analysis (PCA) revealed a significant divergence of SCS samples, whereas AA and AI samples, although grouped separately, were less divergent and could be included in a single asphyxia cluster. In terms of single proteins, the most significant impact was seen on nucleoside diphosphate kinase B, which was consistently less affected when fish were slaughtered by SCS as compared to asphyxia. Integrity of the sarcomeric proteins myosin heavy chain and myosin binding protein C and of the cytosolic proteins fructose biphosphate aldolase, glyceraldehyde 3-phosphate dehydrogenase, and enolase 1 was also better preserved upon SCS slaughtering. Most interestingly, the influence on muscle protein integrity could be detected since the early postmortem phase. In conclusion, slaughtering by SCS preserves protein integrity better than death by asphyxia, either in ice or in air. Both asphyxia conditions are comparably more adverse than SCS to muscle protein integrity, although a general trend favoring AI over AA is observed.
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► Fish slaughtering techniques impact differently on muscle protein integrity. ► Immediate death by spinal cord severance preserves muscle proteins. ► Significant differences in protein degradation can be detected early after death. ► NDKB is an early and highly sensitive indicator of fish protein spoilage. ► Adoption of quick slaughtering protocols is envisaged.
The Ostpolitik pursued by the German government of Chancellor Willy Brandt during the early 1970s aimed at normalizing the relations between the Federal Republic of Germany and its Eastern ...neighbours, and at building a new European security system. Nevertheless, the German government felt compelled to gradually reappraise its aspirations in order to obtain the support and the cooperation of the Western Alliance and especially of the United States. This article examines the case of the negotiations for the reduction of the military presence in central Europe, which initially represented a fundamental component of Brandt's projects: Washington's refusal to endorse early negotiations with the Soviet Union on this subject determined a first major reassessment of Ostpolitik in Bonn.
Heparin has been conjugated to Fe3O4, Co3O4, and NiO nanoparticles (NPs) through electrostatic interactions, producing colloidal suspensions of hybrid metal oxide heparin NPs that are stable in ...water. Negative zeta potentials and retention of heparin's ability to capture toluidine blue indicate that heparin's negative charges are exposed on the surface of the coated NPs. IR results confirmed the formation of nanohybrids as did NMR experiments, which were also interpreted on the basis of toluidine blue tests. Transmission electron microscopy results revealed that the heparin coating does not modify the shape or dimension of the NPs. Dynamic light scattering and negative zeta potential measurements confirmed that heparin surface functionalisation is an effective strategy to prevent NP aggregation.
A Novel Highly Accurate Finite-Element Family Bernardini, Giovanni; Cetta, Fabio; Morino, Luigi
International Journal of Aerospace Engineering,
01/2017, Letnik:
2017
Journal Article
Recenzirano
Odprti dostop
A novel Nth order finite element for interior acoustics and structural dynamics is presented, with N arbitrarily large. The element is based upon a three-dimensional extension of the Coons patch ...technique, which combines high-order Lagrange and Hermite interpolation schemes. Numerical applications are presented, which include the evaluation of the natural frequencies and modes of vibration of (1) air inside a cavity (interior acoustics) and (2) finite-thickness beams and plates (structural dynamics). The numerical results presented are assessed through a comparison with analytical and numerical results. They show that the proposed methodology is highly accurate. The main advantages however are (1) its flexibility in obtaining different level of accuracy (p-convergence) simply by increasing the number of nodes, as one would do for h-convergence, (2) the applicability to arbitrarily complex configurations, and (3) the ability to treat beam- and shell-like structures as three-dimensional small-thickness elements.
Chemokines, a large family of inflammatory cytokines, have been shown to play a critical role in the regulation of angiogenesis during several pathophysiologic processes, such as tumor growth, wound ...healing and ischemia. Semiquantitative or quantitative angiogenesis assays are commonly utilized to screen the angiogenic or angiostatic activity of chemokines. These include in vitro endothelial cell activation assays and ex vivo or in vivo models of neovascularization.
Chemokines may exert their regulatory activity on angiogenesis directly or as a consequence of leukocyte infiltration and/or the induction of growth factor expression. The effect of chemokines on endothelium can be assessed by performing in vitro assays on purified endothelial cell populations or by in vivo assays. Nevertheless, each model used to evaluate the angiogenic or angiostatic activity of a discrete factor has advantages and limitations. Thus, in order to avoid under- or overestimating the regulatory effect of chemokines on angiogenesis and to evaluate all aspects of the angiogenic process, multiple assays are usually performed.
This review summarizes past and recent studies on chemokines as modulators of angiogenesis with particular emphasis on the methods currently used for the assessment of chemokine-mediated angiogenic or angiostatic responses.
As the knowledge about the interferences of nanomaterials on human staminal cells are scarce and contradictory, we undertook a comparative multidisciplinary study based on the size effect of ...zero-valent iron, cobalt, and nickel microparticles (MPs) and nanoparticles (NPs) using human adipose stem cells (hASCs) as a model, and evaluating cytotoxicity, morphology, cellular uptake, and gene expression. Our results suggested that the medium did not influence the cell sensitivity but, surprisingly, the iron microparticles (FeMPs) resulted in being toxic. These data were supported by modifications in mRNA expression of some genes implicated in the inflammatory response. Microscopic analysis confirmed that NPs, mainly internalized by endocytosis, persist in the vesicles without any apparent cell damage. Conversely, MPs are not internalized, and the effects on hASCs have to be ascribed to the release of ions in the culture medium, or to the reduced oxygen and nutrient exchange efficiency due to the presence of MP agglomerating around the cells. Notwithstanding the results depicting a heterogeneous scene that does not allow drawing a general conclusion, this work reiterates the importance of comparative investigations on MPs, NPs, and corresponding ions, and the need to continue the thorough verification of NP and MP innocuousness to ensure unaffected stem cell physiology and differentiation.
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