Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay

Well-controlled studies of the structural and functional interactions between cardiomyocytes and other cells are essential for understanding heart pathophysiology and for the further development of safe and efficient cell therapies. We established a novel in vitro assay composed of a large number of individual micropatterned cell pairs with reproducible shape, size, and region of cell-cell contact. This assay was applied to quantify and compare the frequency of expression and distribution of electrical (connexin43) and mechanical (N-cadherin) coupling proteins in 5,000 cell pairs made of cardiomyocytes (CMs), cardiac fibroblasts (CFs), skeletal myoblasts (SKMs), and mesenchymal stem cells (MSCs). We found that for all cell pair types, side-side contacts between two cells formed 4.5-14.3 times more often than end-end contacts. Both connexin43 and N-cadherin were expressed in all homotypic CM pairs but in only 13.4-91.6% of pairs containing noncardiomyocytes, where expression was either junctional (at the site of cell-cell contact) or diffuse (inside the cytoplasm). CM expression was exclusively junctional in homotypic pairs but predominantly diffuse in heterotypic pairs. Noncardiomyocyte homotypic pairs exhibited diffuse expression 1.7-8.7 times more often than junctional expression, which was increased 2.6-4.4 times in heterotypic pairs. Junctional connexin43 and N-cadherin expression, respectively, were found in 38.6 +/- 7.3 and 39.6 +/- 6.2% of CM-MSC pairs, 21.9 +/- 5.0 and 13.6 +/- 1.9% of CM-SKM pairs, and in only 3.8-9.6% of CM-CF pairs. Measured frequencies of protein expression and distribution were stable for at least 4 days. Described studies in micropatterned cell pairs shed new light on cellular interactions relevant for cardiac function and cell therapies.     

cAMP-mediated induction of cyclin E sensitizes growth-arrested adipose stem cells to DNA damage-induced apoptosis

The differentiation capacity of mesenchymal stem cells has been extensively studied, but little is known on cell cycle–related events in the proliferation and differentiation phases of these cells. Here, we demonstrate that exposure to cAMP-increasing agents inhibits proliferation of adipose stem cells (ASCs). This antiproliferative effect is associated with both reduced cdk2 activity and pRB phosphorylation. Concomitantly, however, the level of cyclin E markedly increases upon cAMP induction, indicating that cyclin E may have cdk2-independent functions in these cells besides its role as a cdk2 activator. Indeed, we found indications of a cdk2-independent role of cyclin E in DNA damage–induced apoptosis. 8-CPT-cAMP sensitizes ASCs to γ-irradiation–induced apoptosis, an effect abolished by knockdown of cyclin E. Moreover, cAMP induces early activation of ERK, leading to reduced degradation of cyclin E. The cAMP-mediated up-regulation of cyclin E was blocked by knockdown of ERK or by an inhibitor of the ERK kinase MEK. We conclude that cAMP inhibits cdk2 activity and pRB phosphorylation, leading to reduced ASC proliferation. Concomitant with this growth inhibition, however, cyclin E levels are increased in a MEK/ERK-dependent manner. Our results suggest that cyclin E plays an important, cdk2-independent role in genotoxic stress–induced apoptosis in mesenchymal stem cells.     

Protective effects of Helicobacter pylori against gastroesophageal reflux disease may be due to a neuroimmunological anti-inflammatory mechanism

There is some evidence that Helicobacter pylori infection has a protective effect against gastroesophageal reflux disease (GORD) and its complications such as Barrett's oesophagus and oesophageal adenocarcinoma. In this paper, we propose that a neuroimmunological mechanism is responsible for the protective effect of H. pylori on GORD. H. pylori infection of the gastric mucosa induces a T helper1-like immune response and production of pro-inflammatory cytokines. These cytokines can inhibit local sympathetic tone, whereas they increase systemic sympathetic tone. Increased sympathetic tone can induce an anti-inflammatory milieu, which in turn can inhibit inflammation in the oesophagus and lower oesophageal sphincter (LOS). Furthermore, H. pylori infection may stimulate the cholinergic anti-inflammatory pathway. It has been suggested that reflux-induced oesophageal inflammation plays an important role in the pathogenesis of reflux oesophagitis. Reduction of oesophageal inflammation by increased systemic sympathetic tone and vagal activity may lead to a decrease in reflux-induced oesophageal injury and LOS dysfunction in GORD.     

Pathways mediating the nuclear import of histones H3 and H4 in yeast.

The correct assembly of chromatin is necessary for the maintenance of genomic stability in eukaryotic cells. A critical step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. Here we demonstrate that the nuclear import pathway of histones H3 and H4 is mediated by at least two karyopherins/importins, Kap123p and Kap121p. Cytosolic H4 is found associated with Kap123p and H3. Kap121p is also present in the H4-PrA-associated fractions, albeit in lesser amounts than Kap123p, suggesting that this Kap serves as an additional import receptor. We further demonstrate that cytosolic Kap123p is associated with acetylated H3 and H4. H3 and H4 each contain a nuclear localization signal (NLS) in their amino-terminal domains. These amino-terminal domains were found to be essential for the nuclear accumulation of H3 and H4-green fluorescent protein reporters. Each NLS mediated direct binding to Kap123p and Kap121p, and decreased nuclear accumulation of H3 and H4 NLS-green fluorescent protein reporters was observed in specific kap mutant strains. H3 and H4 are the first histones to be assembled onto DNA, and these results show that their import is mediated by at least two import pathways.     

Genomic dynamics of brown trout populations released to a novel environment

Population translocations occur for a variety of reasons, from displacement due to climate change to human‐induced transfers. Such actions have adverse effects on genetic variation and understanding their microevolutionary consequences requires monitoring. Here, we return to an experimental release of brown trout (Salmo trutta) in order to monitor the genomic effects of population translocations. In 1979, fish from each of two genetically (F _ST = 0.16) and ecologically separate populations were simultaneously released, at one point in time, to a lake system previously void of brown trout. Here, whole‐genome sequencing of pooled DNA (Pool‐seq) is used to characterize diversity within and divergence between the introduced populations and fish inhabiting two lakes downstream of the release sites, sampled 30 years later (c. 5 generations). Present results suggest that while extensive hybridization has occurred, the two introduced populations are unequally represented in the lakes downstream of the release sites. One population, which is ecologically resident in its original habitat, mainly contributes to the lake closest to the release site. The other population, migratory in its natal habitat, is genetically more represented in the lake further downstream. Genomic regions putatively under directional selection in the new habitat are identified, where allele frequencies in both established populations are more similar to the introduced population stemming from a resident population than the migratory one. Results suggest that the microevolutionary consequences of population translocations, for example, hybridization and adaptation, can be rapid and that Pool‐seq can be used as an initial tool to monitor genome‐wide effects.     

Hybrid chitosan–ß‐glycerol phosphate–gelatin nano‐/micro fibrous scaffolds with suitable mechanical and biological properties for tissue engineering

Scaffold‐based tissue engineering is considered as a promising approach in the regenerative medicine. Graft instability of collagen, by causing poor mechanical properties and rapid degradation, and their hard handling remains major challenges to be addressed. In this research, a composite structured nano‐/microfibrous scaffold, made from a mixture of chitosan–ß‐glycerol phosphate–gelatin (chitosan–GP–gelatin) using a standard electrospinning set‐up was developed. Gelatin–acid acetic and chitosan ß‐glycerol phosphate–HCL solutions were prepared at ratios of 30/70, 50/50, 70/30 (w/w) and their mechanical and biological properties were engineered. Furthermore, the pore structure of the fabricated nanofibrous scaffolds was investigated and predicted using a theoretical model. Higher gelatin concentrations in the polymer blend resulted in significant increase in mean pore size and its distribution. Interaction between the scaffold and the contained cells was also monitored and compared in the test and control groups. Scaffolds with higher chitosan concentrations showed higher rate of cell attachment with better proliferation property, compared with gelatin‐only scaffolds. The fabricated scaffolds, unlike many other natural polymers, also exhibit non‐toxic and biodegradable properties in the grafted tissues. In conclusion, the data clearly showed that the fabricated biomaterial is a biologically compatible scaffold with potential to serve as a proper platform for retaining the cultured cells for further application in cell‐based tissue engineering, especially in wound healing practices. These results suggested the potential of using mesoporous composite chitosan–GP–gelatin fibrous scaffolds for engineering three‐dimensional tissues with different inherent cell characteristics. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 163–175, 2016.     

eRepo-ORP: Exploring the Opportunity Space to Combat Orphan Diseases with Existing Drugs

About 7000 rare, or orphan, diseases affect more than 350 million people worldwide. Although these conditions collectively pose significant health care problems, drug companies seldom develop drugs for orphan diseases due to extremely limited individual markets. Consequently, developing new treatments for often life-threatening orphan diseases is primarily contingent on financial incentives from governments, special research grants, and private philanthropy. Computer-aided drug repositioning is a cheaper and faster alternative to traditional drug discovery offering a promising venue for orphan drug research. Here, we present eRepo-ORP, a comprehensive resource constructed by a large-scale repositioning of existing drugs to orphan diseases with a collection of structural bioinformatics tools, including eThread, eFindSite, and eMatchSite. Specifically, a systematic exploration of 320,856 possible links between known drugs in DrugBank and orphan proteins obtained from Orphanet reveals as many as 18,145 candidates for repurposing. In order to illustrate how potential therapeutics for rare diseases can be identified with eRepo-ORP, we discuss the repositioning of a kinase inhibitor for Ras-associated autoimmune leukoproliferative disease. The eRepo-ORP data set is available through the Open Science Framework at https://osf.io/qdjup/.     

Adipose tissue‐derived mesenchymal stem cells and keratinocytes co‐culture on gelatin/chitosan/β‐glycerol phosphate nanoscaffold in skin regeneration

Using cell‐based engineered skin is an emerging strategy for treating difficult‐to‐heal wounds. To date, much endeavor has been devoted to the fabrication of appropriate scaffolds with suitable biomechanical properties to support cell viability and growth in the microenvironment of a wound. The aim of this research was to assess the impact of adipose tissue‐derived mesenchymal stem cells (AD‐MSCs) and keratinocytes on gelatin/chitosan/β‐glycerol phosphate (GCGP) nanoscaffold in full‐thickness excisional skin wound healing of rats. For this purpose, AD‐MSCs and keratinocytes were isolated from rats and GCGP nanoscaffolds were electrospun. Through an in vivo study, the percentage of wound closure was assessed on days 7, 14, and 21 after wound induction. Samples were taken from the wound sites in order to evaluate the density of collagen fibers and vessels at 7 and 14 days. Moreover, sampling was done on days 7 and 14 from wound sites to assess the density of collagen fibers and vessels. The wound closure rate was significantly increased in the keratinocytes‐AD‐MSCs‐scaffold (KMS) group compared with other groups. The expressions of vascular endothelial growth factor, collagen type 1, and CD34 were also significantly higher in the KMS group compared with the other groups. These results suggest that the combination of AD‐MSCs and keratinocytes seeded onto GCGP nanoscaffold provides a promising treatment for wound healing.     

Integration of Lyoplate Based Flow Cytometry and Computational Analysis for Standardized Immunological Biomarker Discovery

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.