Agarose Spot as a Comparative Method for in situ Analysis of Simultaneous Chemotactic Responses to Multiple Chemokines

yesWe describe a novel protocol to quantitatively and simultaneously compare the chemotactic responses of cells towards different chemokines. In this protocol, droplets of agarose gel containing different chemokines are applied onto the surface of a Petri dish, and then immersed under culture medium in which cells are suspended. As chemokine molecules diffuse away from the spot, a transient chemoattractant gradient is established across the spots. Cells expressing the corresponding cognate chemokine receptors migrate against this gradient by crawling under the agarose spots towards their centre. We show that this migration is chemokine-specific; meaning that only cells that express the cognate chemokine cell surface receptor, migrate under the spot containing its corresponding chemokine ligand. Furthermore, we show that migration under the agarose spot can be modulated by selective small molecule antagonists present in the cell culture medium

Apelin Enhances Directed Cardiac Differentiation of Mouse and Human Embryonic Stem Cells

Apelin is a peptide ligand for an orphan G-protein coupled receptor (APJ receptor) and serves as a critical gradient for migration of mesodermal cells fated to contribute to the myocardial lineage. The present study was designed to establish a robust cardiac differentiation protocol, specifically, to evaluate the effect of apelin on directed differentiation of mouse and human embryonic stem cells (mESCs and hESCs) into cardiac lineage. Different concentrations of apelin (50, 100, 500 nM) were evaluated to determine its differentiation potential. The optimized dose of apelin was then combined with mesodermal differentiation factors, including BMP-4, activin-A, and bFGF, in a developmentally specific temporal sequence to examine the synergistic effects on cardiac differentiation. Cellular, molecular, and physiologic characteristics of the apelin-induced contractile embryoid bodies (EBs) were analyzed. It was found that 100 nM apelin resulted in highest percentage of contractile EB for mESCs while 500 nM had the highest effects on hESCs. Functionally, the contractile frequency of mESCs-derived EBs (mEBs) responded appropriately to increasing concentration of isoprenaline and diltiazem. Positive phenotype of cardiac specific markers was confirmed in the apelin-treated groups. The protocol, consisting of apelin and mesodermal differentiation factors, induced contractility in significantly higher percentage of hESC-derived EBs (hEBs), up-regulated cardiac-specific genes and cell surface markers, and increased the contractile force. In conclusion, we have demonstrated that the treatment of apelin enhanced cardiac differentiation of mouse and human ESCs and exhibited synergistic effects with mesodermal differentiation factors

Competitive Interactions between Invasive Nile Tilapia and Native Fish: The Potential for Altered Trophic Exchange and Modification of Food Webs

Recent studies have highlighted both the positive and negative impacts of species invasions. Most of these studies have been conducted on either immobile invasive plants or sessile fauna found at the base of food webs. Fewer studies have examined the impacts of vagile invasive consumers on native competitors. This is an issue of some importance given the controlling influence that consumers have on lower order plants and animals. Here, we present results of laboratory experiments designed to assess the impacts of unintended aquaculture releases of the Nile tilapia (Oreochromis niloticus), in estuaries of the Gulf of Mexico, on the functionally similar redspotted sunfish (Lepomis miniatus). Laboratory choice tests showed that tilapia prefer the same structured habitat that native sunfish prefer. In subsequent interspecific competition experiments, agonistic tilapia displaced sunfish from their preferred structured habitats. When a piscivore (largemouth bass) was present in the tank with both species, the survival of sunfish decreased. Based on these findings, if left unchecked, we predict that the proliferation of tilapia (and perhaps other aggressive aquaculture fishes) will have important detrimental effects on the structure of native food webs in shallow, structured coastal habitats. While it is likely that the impacts of higher trophic level invasive competitors will vary among species, these results show that consequences of unintended releases of invasive higher order consumers can be important

Interferon Tau Alleviates Obesity-Induced Adipose Tissue Inflammation and Insulin Resistance by Regulating Macrophage Polarization

Chronic adipose tissue inflammation is a hallmark of obesity-induced insulin resistance and anti-inflammatory agents can benefit patients with obesity-associated syndromes. Currently available type I interferons for therapeutic immunomodulation are accompanied by high cytotoxicity and therefore in this study we have examined anti-inflammatory effects of interferon tau (IFNT), a member of the type I interferon family with low cellular toxicity even at high doses. Using a diet-induced obesity mouse model, we observed enhanced insulin sensitivity in obese mice administered IFNT compared to control mice, which was accompanied by a significant decrease in secretion of proinflammatory cytokines and elevated anti-inflammatory macrophages (M2) in adipose tissue. Further investigations revealed that IFNT is a potent regulator of macrophage activation that favors anti-inflammatory responses as evidenced by activation of associated surface antigens, production of anti-inflammatory cytokines, and activation of selective cell signaling pathways. Thus, our study demonstrates, for the first time, that IFNT can significantly mitigate obesity-associated systemic insulin resistance and tissue inflammation by controlling macrophage polarization, and thus IFNT can be a novel bio-therapeutic agent for treating obesity-associated syndromes and type 2 diabetes

A participatory space beyond the "autonomy versus property" dichotomy

Within the normative puzzles that have formed around the biobanking of human biological materials (HBMs) and information, two strategies have been adopted in the US and in Europe; these are distinguished by their focus on two main legal concepts: individual property rights and individual autonomy \u2013 mostly interpreted as the right to privacy. The US addressed the question of proprietary interests in the human body materials explicitly and directly. In fact, the U.S. justice system has always explicitly defined the question in terms of property, removing privacy from the equation essentially as a way to \u201cavert the danger\u201d of the proprietary issue. The tendency has always been to negate the uncertain \u201cproprietary interests\u201d associated with the bodies of private citizens, and to use the much stronger and more consolidated "proprietary rights", meaning intellectual property; or else to justify the acquisition of control over the HBMs by research and market structures through a presumption of donation and subsequent abandonment of human tissues by the sources of those tissues. In Europe, the approach went instead in the direction of removing the corporeity of HBMs, seeing them instead as symbolically representing the most secure limits of human dignity, according to which the body is \u201cpriceless.\u201d The opaqueness and unworkability of this is made clear by the fact that negation of the marketability of the body went hand in hand with the institutionalization of the European tissue market. There are two inconvenient principles inherent in these two perspectives. The first is the reduction of the means of control of HBMs to one proprietary, operative model, where all powers are allocated to the owner-subject and the object in itself lacks any ultimate value, be it individual or even social. This model has demonstrable limits which can be seen, for example, if it is applied to the environment, which has several affinities with the theme of tissues. The second principle concerns the misleading prevalence of language of individual autonomy, when the actual values in question instead reflect public space, collective dimensions, and a sense of scientific citizenship \u2013 which are taken as recognition of the rights of citizens to participate, even if it is epistemic, in public decisions

Hematopoietic knockdown of PPARδ reduces atherosclerosis in LDLR−/− mice

PPAR(δ) (peroxisome proliferator-activated receptor δ) mediates inflammation in response to lipid accumulation. Systemic administration of a PPAR(δ) agonist can ameliorate atherosclerosis. Paradoxically, genetic deletion of PPAR(δ) in hematopoietic cells led to a reduction of atherosclerosis in murine models, suggesting that downregulation of PPAR(δ) expression in these cells may mitigate atherogenesis. To advance this finding forward to potential clinical translation through hematopoietic stem cell transplantation-based gene therapy, we employed a microRNA (miRNA) approach to knock down PPAR(δ) expression in bone marrow cells followed by transplantation of the cells into LDLR −/− mice. We found that knockdown of PPAR(δ) expression in the hematopoietic system caused a dramatic reduction in aortic atherosclerotic lesions. In macrophages, a key component in atherogenesis, knockdown of PPAR(δ) led to decreased expression of multiple pro-inflammatory factors, including monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β and IL-6. Expression of CCR2, a receptor for MCP-1, was also decreased. The downregulation of pro-inflammatory factors is consistent with significant reduction of macrophage presence in the lesions, which may also be attributable to elevation of ABCA1 (ATP-binding cassette, subfamily A, member 1) and depression of adipocyte differentiate-related protein. Furthermore, the abundance of both MCP-1 and matrix metalloproteinase-9 proteins was reduced in plaque areas. Our results demonstrate that miRNA-mediated PPAR(δ) knockdown in hematopoietic cells is able to ameliorate atherosclerosis