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.     

Protection of Cells against Oxidative Stress by Nanomolar Levels of Hydroxyflavones Indicates a New Type of Intracellular Antioxidant Mechanism

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.     

Coupling Demographic and Genetic Variability from Archived Collections of European Anchovy (Engraulis encrasicolus)

It is well known that temporal fluctuations in small populations deeply influence evolutionary potential. Less well known is whether fluctuations can influence the evolutionary potentials of species with large census sizes. Here, we estimated genetic population parameters from as survey of polymorphic microsatellite DNA loci in archived otoliths from Adriatic European anchovy (Engraulis encrasicolus), a fish with large census sizes that supports numerous local fisheries. Stocks have fluctuated greatly over the past few decades, and the Adriatic fishery collapsed in 1987. Our results show a significant reduction of mean genetic parameters as a consequence of the population collapse. In addition, estimates of effective population size (N_e) are much smaller than those expected in a fishes with large population census sizes (N_c). Estimates of N_e indicate low effective population sizes, even before the population collapse. The ratio N_e/N_e ranged between 10⁻⁶ and 10⁻⁸, indicating a large discrepancy between the anchovy gene pool and population census size. Therefore, anchovy populations may be more vulnerable to fishery effort and environmental change than previously thought.     

Effective gene flow in a historically fragmented area at the southern edge of silver fir (<Emphasis Type="Italic">Abies alba</Emphasis> Mill.) distribution

Fragmented populations at the edges of a species’ distribution can be highly exposed to the loss of genetic variation, unless sufficient gene flow maintains their genetic connectivity. Gene movements leading to successful establishment of external gametes (i.e. effective gene flow) into fragmented populations can solely be assessed by investigating the origin of natural regeneration. This study is focused on studying gene flow patterns in two silver fir stands in Central Apennines, where the species has a highly fragmented distribution. By using nuclear and chloroplast microsatellite markers, we investigated genetic variation, fine-scale spatial genetic structure, effective gene flow rates and large-scale connectivity characterizing both stands. Similar levels of genetic variation and low genetic differentiation between stands (F _ST = 0.005) and across generations were found, coupled with low inbreeding and weak to absent SGS in the adult cohort (Sp < 0.003). On the other hand, substantial differences between the two stands in terms of gene flow rates were observed. Irrespective of the parentage approach used, higher gene flow rates were found in the stand located at the upper silver fir altitudinal limit, especially for seed-mediated gene flow (0.79 in the upper stand vs. 0.48 in the lower stand). Conversely, the lower stand was characterized by a higher reproductive dominance of local adults. Our findings suggest that, despite similar levels of genetic variation and generally high gene flow rates, different processes may be acting on the two stands, reflecting varying ecological conditions.     

Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons

Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory.     

Cerebral Blood Volume ASPECTS Is the Best Predictor of Clinical Outcome in Acute Ischemic Stroke: A Retrospective,Combined Semi-Quantitative and Quantitative Assessment

Introduction

The capability of CT perfusion (CTP) Alberta Stroke Program Early CT Score (ASPECTS) to predict outcome and identify ischemia severity in acute ischemic stroke (AIS) patients is still questioned.

Methods

62 patients with AIS were imaged within 8 hours of symptom onset by non-contrast CT, CT angiography and CTP scans at admission and 24 hours. CTP ASPECTS was calculated on the affected hemisphere using cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) maps by subtracting 1 point for any abnormalities visually detected or measured within multiple cortical circular regions of interest according to previously established thresholds. MTT-CBV ASPECTS was considered as CTP ASPECTS mismatch. Hemorrhagic transformation (HT), recanalization status and reperfusion grade at 24 hours, final infarct volume at 7 days and modified Rankin scale (mRS) at 3 months after onset were recorded.

Results

Semi-quantitative and quantitative CTP ASPECTS were highly correlated (p<0.00001). CBF, CBV and MTT ASPECTS were higher in patients with no HT and mRS≤2 and inversely associated with final infarct volume and mRS (p values: from p<0.05 to p<0.00001). CTP ASPECTS mismatch was slightly associated with radiological and clinical outcomes (p values: from p<0.05 to p<0.02) only if evaluated quantitatively. A CBV ASPECTS of 9 was the optimal semi-quantitative value for predicting outcome.

Conclusions

Our findings suggest that visual inspection of CTP ASPECTS recognizes infarct and ischemic absolute values. Semi-quantitative CBV ASPECTS, but not CTP ASPECTS mismatch, represents a strong prognostic indicator, implying that core extent is the main determinant of outcome, irrespective of penumbra size.