Micropatterned polyelectrolyte nanofilms promote alignment and myogenic differentiation of C2C12 cells in standard growth media

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro‐patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi‐nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer‐by‐layer (LbL) polyelectrolyte multilayer deposition was combined with a micro‐molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self‐assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4‐styrene‐sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly‐L ‐arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)₅‐coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro. Biotechnol. Bioeng. 2013; 110: 586–596. © 2012 Wiley Periodicals, Inc.     

Design of a novel LOX-1 receptor antagonist mimicking the natural substrate

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is overexpressed in atherosclerotic lesions. LOX-1 specific inhibitors, urgently necessary to reduce the rate of atherosclerotic and inflammation processes, are not yet available. We have designed and synthesized a new modified oxidized phospholipid, named PLAzPC, which plays to small scale the ligand-receptor recognition scheme. Molecular docking simulations confirm that PLAzPC disables the hydrophobic component of the ox-LDL recognition domain and allows the interaction of the l-lysine backbone charged groups with the solvent and with the charged/polar residues located around the edges of the LOX-1 hydrophobic tunnel. Binding assays, in a cell model system expressing human LOX-1 receptors, confirm that PLAzPC markedly inhibits ox-LDL binding to LOX-1 with higher efficacy compared to previously identified inhibitors.     

Microbial biofilm structure and organic matter use in mediterranean streams

River and stream biofilms in mediterranean fluvial ecosystems face both extreme seasonality as well as arrhythmic fluctuations. The hydrological extremes (droughts and floods) impose direct changes in water availability but also in the quantity and quality of organic matter and nutrients that sustain the microbial growth. This review analyzes how these ecological pulses might determine unique properties of biofilms developing in mediterranean streams. The paper brings together data from heterotrophic and autotrophic community structure, and extracellular enzyme activities in biofilms in mediterranean streams. Mediterranean stream biofilms show higher use of peptides during the favorable period for epilithic algae development (spring), and preferential use of cellulose and hemicellulose in autumn as a response to allochthonous input. The drying process causes the reduction in bacterial production and chlorophyll biomass, but the rapid recovery of both autotrophs and heterotrophs with rewetting indicates their adaptability to fluctuations. Bacteria surviving the drought are mainly associated with sediment and leaf litter which serve as “humid refuges”. Some algae and cyanobacteria show resistant strategies to cope with the drought stress. The resistance to these fluctuations is strongly linked to the streambed characteristics (e.g., sediment grain size, organic matter accumulation, nutrient content).     

Phylogeographical pattern of Euplotes nobilii,a protist ciliate with a bipolar biogeographical distribution

Nuclear (18S and ITS) and mitochondrial (16S) ribosomal RNA gene sequences were determined from genetically distinct wild‐type strains of Antarctic (nine strains), Fuegian (four strains), Greenland (nine strains) and Svalbard (three strains) populations of the marine ciliate, Euplotes nobilii, and analysed for their nucleotide polymorphisms. A close genetic homogeneity was found within and between the Antarctic and Fuegian populations, while more significant levels of genetic differentiation were detected within and between the two Arctic populations, as well as between these populations and the Antarctic/Fuegian ones. The phylogeographical pattern that was derived from these data indicates that gene flow is not limited among Arctic populations; it equally connects the Arctic and Antarctic populations either directly, or through the Fuegian population. This indication reinforces previous evidence from laboratory assays of mating interactions between some of the strains analysed in this work that Southern and Northern polar populations of E. nobilii belong to a unique, panmictic population that substantially share the same gene pool.     

A geographic mosaic of evolutionary lineages within the insular endemic newt Euproctus montanus

Islands are hotspots of biodiversity, with a disproportionately high fraction of endemic lineages, often of ancient origin. Nevertheless, intra‐island phylogeographies are surprisingly scarce, leading to a scanty knowledge about the microevolutionary processes induced on island populations by Plio‐Pleistocene climatic oscillations, and the manner in which these processes contributed to shape their current genetic diversity. We investigated the phylogeography, historical demography and species distribution models of the Corsican endemic newt Euproctus montanus (north‐western Mediterranean). As for many island endemics, the continuous distribution of E. montanus throughout its range has hitherto been considered as evidence for a single large population, a belief that also guided the species' categorization for conservation purposes. Instead, we found a geographic mosaic of ancient evolutionary lineages, with five main clades of likely Pliocene origin (2.6–5.8 My), all but one restricted to northern Corsica. Moreover, the copresence between main lineages in the same population was limited to a single case. As also suggested by growing literature on intra‐island phylogeographic variation, it seems that the extensive use of simplifying assumption on the population structure and historical demography of island populations—both in theoretical and applicative studies—should be carefully reconsidered, a claim that is well exemplified by the case presented here.     

Extra-virgin olive oil diet and mild physical activity prevent cartilage degeneration in an osteoarthritis model: an in vivo and in vitro study on lubricin expression

Mediterranean diet includes a relatively high fat consumption mostly from monounsaturated fatty acids mainly provided by olive oil, the principal source of culinary and dressing fat. The beneficial effects of olive oil have been widely studied and could be due to its phytochemicals, which have been shown to possess anti-inflammatory properties. Lubricin is a chondroprotective glycoprotein and it serves as a critical boundary lubricant between opposing cartilage surfaces. A joint injury causes an initial flare of cytokines, which decreases lubricin expression and predisposes to cartilage degeneration such as osteoarthritis. The aim of this study was to evaluate the role of extra-virgin olive oil diet and physical activity on inflammation and expression of lubricin in articular cartilage of rats after injury. In this study we used histomorphometric, histological, immunocytochemical, immunohistochemical, western blot and biochemical analysis for lubricin and interleukin-1 evaluations in the cartilage and in the synovial fluid. We report the beneficial effect of physical activity (treadmill training) and extra-virgin olive oil supplementation, on the articular cartilage. The effects of anterior cruciate ligament transection decrease drastically the expression of lubricin and increase the expression of interleukin-1 in rats, while after physical activity and extra-virgin olive oil supplemented diet, the values return to a normal level compared to the control group. With our results we can confirm the importance of the physical activity in conjunction with extra-virgin olive oil diet in medical therapy to prevent osteoarthritis disease in order to preserve the articular cartilage and then the entire joint.     

Ciliate–adenovirus interactions in experimental co-cultures of Euplotes octocarinatus and in wastewater environment

The aim of the present work was to study the dynamics of the interactions between human adenovirus and ciliates under both experimental and field conditions. Experimental co-cultures of the ciliated protozoan Euplotes octocarinatus and human adenovirus (HAdV) type 2 were established and virus internalization was investigated using nested PCR and direct immunofluorescence (IF). In addition, to study protozoa-virus interactions in the field, wild ciliates were isolated from active sludges of a wastewater treatment plant and analyzed for the presence of adenovirus using direct IF. In vitro experiments revealed HAdV type 2 inside Euplotes cells after 15 min of contact and its persistence until at least 35 days post infection. In addition, our results showed the adsorption of adenovirus on the surface of wild ciliates. We conclude that HAdV is taken up by ciliates, however more studies are necessary in order to better investigate the mechanisms, the infectivity of internalized virus and the protective effects of internalization against disinfection.