Cellular engineering of ventricular adult rat cardiomyocytes

Objective: Preparation of viable cultured adult cardiomyocytes (vARCs) is a prerequisite for cell-based transplantation and tissue engineering. Ectopic gene expression is important in this context. Here, we present an in vitro cell replating strategy using Accutase™ for cultured vARCs, allowing ectopic gene expression. Methods: Cultured vARCs from 6- to 8-week-old rats were used. Transfections with EGFP (enhanced green fluorescent protein) constructs, Mlc-3f-EGFP or α-actinin-EGFP were performed using adenovirus-enhanced transferrin-mediated infection (AVET). Accutase™ (PAA Laboratories, Linz, Austria) was used for the detachment of cultured cells. Immunohistochemical analysis, together with confocal laser microscopy was used for structural analysis of the cells. Results: Cultured vARCs could be detached with a high yield (40 to 60%) from primary cultures using Accutase™. The cultivation period plays an important role in the yield of viable cells. Resultant replated vARCs (rep-vARCs) rapidly (1-2 h) acquired a rounded up shape without degradation of their contractile apparatus, which is in contrast to the rod-shaped freshly isolated vARCs (fi-vARCs). The detached cells survived passage through a narrow syringe needle. After seeding, detached cells rapidly attached to various substrates, increased their content of the contractile apparatus, and formed cell-cell contacts within 3 days after reseeding. The detached cells survived passage through a narrow syringe needle. The high recovery of cells after replating enabled the use of the AVET system for gene delivery. AVET is free of infectious particles and does not lead to expression of viral proteins. Transfection of vARCs prior to detachment had a small effect on cell recovery and ectopically synthesized proteins were properly localized after replating. Conclusions: Detachment of cultured vARCs using Accutase™ is well compatible with ectopic gene expression and yields a viable transgenic population of vARCs that eventually may be suitable as transgenic cardiomyocyte graft

Cellular engineering of ventricular adult rat cardiomyocytes

ISSN:1755-3245ISSN:0008-636

Reactivation of the mitosis-promoting factor in postmitotic cardiomyocytes

Cardiomyocytes cease to divide shortly after birth and an irreversible cell cycle arrest is evident accompanied by the downregulation of cyclin-dependent kinase activities. To get a better understanding of the cardiac cell cycle and its regulation, the effect of functional recovery of the mitosis-promoting factor (MPF) consisting of cyclin B1 and the cyclin-dependent kinase Cdc2 was assessed in primary cultures of postmitotic ventricular adult rat cardiomyocytes ( ARC). Gene transfer into ARC was achieved using the adenovirus-enhanced transferrinfection system that was characterized by the absence of cytotoxic events. Simultaneous ectopic expression of wild-type versions of cyclin B1 and Cdc2 was sufficient to induce MPF activity. Reestablished MPF resulted in a mitotic phenotype, marked by an abnormal condensation of the nuclei, histone H3 phosphorylation and variable degree of decay of the contractile apparatus. Although a complete cell division was not observed, the results provided conclusive evidence that cell cycle-related events in postmitotic cardiomyocytes could be triggered by genetic intervention downstream of the G1/S checkpoint. This will be of importance to design novel strategies to overcome the proliferation arrest in adult cardiomyocytes

Purification of a factor from human peritoneal fluid that is able to immobilize spermatozoa

Human peritoneal fluid has been claimed to influence sperm motility. This report gives evidence for the presence in mid-cycle peritoneal fluid of a protein-bound, lipidic (hydrophobic) component able to immobilize spermatozoa as a function of time. This component was extracted from molecular weight-sieving and ion-exchange/high pressure liquid chromatography (HPLC)-purified peritoneal fluid fractions by either chloroform/methanol or charcoal treatments; resuspension of the chloroform/methanol extract with BWW-buffer and subsequent testing on spermatozoa resulted in sperm immobilization. Sequential or step-down chromatographic procedures (molecular weight-sieving→cation-exchange→anion-exchange HPLC separations of native peritoneal fluid) and extensive dialysis against double distilled water allowed the purification of the sperm immobilizing factor, as evidenced by the shorter incubation times necessary for sperm immobilization. Furthermore, the active fraction was found to immobilize spermatozoa without affecting its viability. Separation of the chloroform/methanol extracted immobilizing fraction on thin layer chromatography under conditions for phospholipid detection allowed the identification of a characteristic band which, after re-extraction, was found to be the sperm immobilizing substance. This factor does not contain choline, ethanolamine or serine. These results suggest that some lipidic peritoneal fluid components may influence sperm motilit

Increasing variability of body mass and health correlates in Swiss conscripts, a possible role of relaxed natural selection?

Background and objectives The body mass index (BMI) is an established anthropometric index for the development of obesity related conditions. However, little is known about the distribution of BMI within a population, especially about this distribution’s temporal change. Here, we analysed changes in the distribution of height, weight and BMI over the past 140 years based on data of Swiss conscripts and tested for correlations between anthropometric data and standard blood parameters. Methods Height and weight were measured in 59,504 young Swiss males aged 18-19 years during conscription in 1875-79, 1932-36, 1994 and 2010-12. For 65% of conscripts in 2010-12 results of standard blood analysis were available. We calculated descriptive statistics of the distribution of height, weight, and BMI over the four time periods and tested for associations between BMI and metabolic parameters. Results Average and median body height, body weight and BMI increased over time. Height did no longer increase between 1994 and 2010-12, while weight and BMI still increased over these two decades. Variability ranges of weight and BMI increased over time, while variation of body height remained constant. Elevated levels of metabolic and inflammatory blood parameters were found at both ends of BMI distribution. Conclusions and Implications Both overweight and underweight subgroups showed similar changes in inflammation parameters, pointing towards related metabolic deficiencies in both conditions. In addition to environmental influences, our results indicate a potential role of relaxed natural selection on genes affecting metabolism and body composition

Cryoultramicrotomy and Immunocytochemistry in the Analysis of Muscle Fine Structure

Cryoultramicrotomy, which avoids the use of harsh fixation procedures, deleterious dehydration and plastic embedding can be combined with immunocytochemis try to determine the ultra-structural localization of cellular proteins. Our attempts to use the cryosectioning technique in combination with immunolabelling to bridge the gap between light and electron microscopic analysis of muscle morphology have enabled us to obtain new information on fibre typing at the ultrastructural level. Furthermore, we have obtained a marked improvement in the resolution of myofibrillar structures by using semithin cryosections for fluorescence microscopy. Data are also presented on correlated light and electron microscope immunocytochemistry of myocardial intermediate filaments confirming the presence of longitudinally oriented intermediate filaments of desmin in the region of the intercalated discs of mammalian cardiac myocytes, whereas elsewhere in the myocyte the bulk of intermediate filaments of desmin is concentrated in the intermyofibrillar space at the level of the Z disc

Radiological findings in ancient Egyptian canopic jars: comparing three standard clinical imaging modalities (x-rays, CT and MRI)

© The Author(s) 2018 This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Abstract Background The aim of our study was to evaluate the potential and the limitations of standard clinical imaging modalities for the examination of ancient Egyptian canopic jars and the mummified visceral organs (putatively) contained within them. Methods A series of four ancient Egyptian canopic jars was imaged comparing the three standard clinical imaging modalities: x-rays, computed tomography (CT) and magnetic resonance imaging (MRI). Additionally, imaging-data-based volumetric calculations were performed for quantitative assessment of the jar contents. Results The image contrast of the x-ray images was limited by the thickness and high density of the calcite mineral constituting the examined jars. CT scans showed few artefacts and revealed hyperdense structures of organ-specific morphology, surrounded by a hypodense homogeneous material. The image quality of MRI scans was limited by the low amount of water present in the desiccated jar contents. Nevertheless, areas of pronounced signal intensity coincided well with hyperdense structures previously identified on CT scans. CT-based volumetric calculations revealed holding capacities of the jars of 626–1319 cm3 and content volumes of 206–1035 cm3. Conclusions CT is the modality of choice for non-invasive examination of ancient Egyptian canopic jars. However, despite its limitations, x-ray imaging will often remain the only practicable method for on-site investigations. Overall, the presented radiological findings are more compatible with contained small organ fragments rather than entire mummified organs, as originally expected, with consequent implications for envisioned future sampling for chemical and genetic analysis