In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations

Background

Although numerous sequence variants in desmoglein-2 (DSG2) have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), the functional impact of new sequence variations is difficult to estimate.

Methodology/Principal Findings

To test the functional consequences of DSG2-variants, we established an expression system for the extracellular domain and the full-length DSG2 using the human cell line HT1080. We established new tools to investigate ARVC-associated DSG2 variations and compared wild-type proteins and proteins with one of the five selected variations (DSG2-p.R46Q, -p.D154E, -p.D187G, -p.K294E, -p.V392I) with respect to prodomain cleavage, adhesion properties and cellular localisation.

Conclusions/Significance

The ARVC-associated DSG2-p.R46Q variation was predicted to be probably damaging by bioinformatics tools and to concern a conserved proprotein convertase cleavage site. In this study an impaired prodomain cleavage and an influence on the DSG2-properties could be demonstrated for the R46Q-variant leading to the classification of the variant as a potential gain-of-function mutant. In contrast, the variants DSG2-p.K294E and -p.V392I, which have an arguable impact on ARVC pathogenesis and are predicted to be benign, did not show functional differences to the wild-type protein in our study. Notably, the variants DSG2-p.D154E and -p.D187G, which were predicted to be damaging by bioinformatics tools, had no detectable effects on the DSG2 protein properties in our study.     

Stallion spermatozoa selected by single layer centrifugation are capable of fertilization after storage for up to 96 h at 6°C prior to artificial insemination

ABSTRACT: BACKGROUND: One of the challenges faced by equine breeders is ensuring delivery of good quality semen doses for artificial insemination when the mare is due to ovulate. Single Layer Centrifugation (SLC) has been shown to select morphologically normal spermatozoa with intact chromatin and good progressive motility from the rest of the ejaculate, and to prolong the life of these selected spermatozoa in vitro. The objective of the present study was a proof of concept, to determine whether fertilizing ability was retained in SLC-selected spermatozoa during prolonged storage. FINDINGS: Sixteen mares were inseminated with SLC-selected sperm doses that had been cooled and stored at 6°C for 48 h, 72 h or 96 h. Embryos were identified in 11 mares by ultrasound examination 16-18 days after presumed ovulation. CONCLUSION: SLC-selected stallion spermatozoa stored for up to 96 h are capable of fertilization.     

PGC‐1α affects aging‐related changes in muscle and motor function by modulating specific exercise‐mediated changes in old mice

The age‐related impairment in muscle function results in a drastic decline in motor coordination and mobility in elderly individuals. Regular physical activity is the only efficient intervention to prevent and treat this age‐associated degeneration. However, the mechanisms that underlie the therapeutic effect of exercise in this context remain unclear. We assessed whether endurance exercise training in old age is sufficient to affect muscle and motor function. Moreover, as muscle peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) is a key regulatory hub in endurance exercise adaptation with decreased expression in old muscle, we studied the involvement of PGC‐1α in the therapeutic effect of exercise in aging. Intriguingly, PGC‐1α muscle‐specific knockout and overexpression, respectively, precipitated and alleviated specific aspects of aging‐related deterioration of muscle function in old mice, while other muscle dysfunctions remained unchanged upon PGC‐1α modulation. Surprisingly, we discovered that muscle PGC‐1α was not only involved in improving muscle endurance and mitochondrial remodeling, but also phenocopied endurance exercise training in advanced age by contributing to maintaining balance and motor coordination in old animals. Our data therefore suggest that the benefits of exercise, even when performed at old age, extend beyond skeletal muscle and are at least in part mediated by PGC‐1α.     

Einige Feinstrukturbefunde an den Plexus chorioidei von Affen

Zusammenfassung An der lichtmikroskopischen Struktur des plexus chorioideus ventriculi III von Ateles, Cebus, Macaca und Pan fallen deutliche speziesbedingte Unterschiede auf. Bei Macaca wurde die Ultrastruktur der Plexus chorioidei ventriculi III und IV sowie der Plexus chorioidei der Seitenventrikel verglichen. Kuppenzellen überwiegen im elektronen-mikroskopischen Bild der Plexusepithelien des III. und IV. Ventrikels, während unter den Epithelien der Seitenventrikel flachere Zellen dominieren. Diese strukturellen Besonderheiten könnten auf funktionelle Unterschiede in der Liquorbildung hinweisen. Das Problem der extrachorioidalen Liquorproduktion (Curl and Pollay, 1968; Milhorat u. Mitarb., 1971) wird im Zusammenhang mit den regionalen Unterschieden in der Wandstruktur des Aquaeductus cerebri (Sylvii) diskutiert (Merker, 1970). Im Stroma und im Interstitium aller Plexus chorioidei des Rhesusaffen sind makrophagenartige Wanderzellen zu beobachten, deren Strukturbild dem der Epiplexuszellen gleicht; sie werden als ausgewanderte Monozyten gedeutet. Wahrscheinlich gelangen diese Zellen in den Ventrikel und werden dort zu Epiplexuszellen. Einschlüsse vom Typ der Biondi-Körper wurden in den Plexusepithelien der untersuchten Affen nicht beobachtet; die untersuchten Tiere hatten aber noch kein hohes Lebensalter erreicht.

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The fine structure of the choroid plexuses in monkeys

Summary The light microscopic structure of the choroid plexus of the III^rd ventricle was studied in Ateles, Cebus, Macaca and Pan. These choroid plexuses display distinct species differences. The ultrastructure of the choroid plexuses of the III^rd, IV^th and lateral ventricles was compared in Macaca. Cuboidal cells with dome-like protrusions predominate in the epithelium of the choroid plexuses of the III^rd and the IV^th ventricles, while flattened cells are characteristic of the choroid epithelium of the lateral ventricle. These structural peculiarities may reflect functional differences in the production of the cerebrospinal fluid. The problem of extrachoroidal production of the cerebrospinal fluid (Curl and Pollay, 1968; Milhorat et al., 1971) is discussed in connection with regional differences in the structure of the aqueduct wall (Merker, 1970). Macrophage-like cells which morphologically resemble epiplexus cells and which are very abundant within the connective tissue stroma and also between the epithelial cells of all choroid plexuses of the rhesus monkey, are thought to be monocytes. Probably these cells migrate into the ventricle where they become epiplexus cells. Epithelial inclusions of the type of Biondi bodies were not observed in the simian choroid plexuses. However, the animals investigated were not old.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (Arbeitskreis Prof. Dr. A. Oksche).     

Metabolism of lutein and zeaxanthin in rhesus monkeys: identification of (3R,6'R)- and (3R,6'S)-3'-dehydro-lutein as common metabolites and comparison to humans

Lutein and zeaxanthin are xanthophylls that can be found highly concentrated in the macula of the retina. They are thought to protect the macula through their role as blue-light filters and because of their antioxidant and singlet oxygen quenching properties. Examination of metabolites unique to lutein and zeaxanthin such as 3'-dehydro-lutein, and of their stereochemistry may provide insight to the mechanism by which they are formed and by which they exert protection. To evaluate the formation of such metabolites, eleven monkeys were raised on a xanthophyll-free diet, and supplemented with pure lutein or pure zeaxanthin (2.2 mg/kg body weight/d). The period of supplementation ranged between 12 and 92 weeks. At study start and throughout the study, serum samples were taken and analyzed for xanthophylls using different HPLC systems. Xanthophyll metabolites were identified using UV/VIS and HR-MS detection. Lutein and zeaxanthin metabolites were found in detectable amounts with 3'-dehydro-lutein being a common metabolite of both. Using chiral-phase HPLC, two diastereomers, (3R,6'R)-3'-dehydro-lutein and (3R,6'S)-3'-dehydro-lutein, were identified and shown to be present in nearly equimolar amounts. A pathway for their formation from either lutein or zeaxanthin is proposed. These findings were comparable to results obtained with human plasma.     

Silicon load and the development of diatoms in three river-lake systems in countries surrounding the Baltic Sea

We studied the formation of dissolved silicon loads from rivers to lakes, the development of diatoms in lakes and the role of climatic forcing on the silicon cycle in three river-lake systems in Sweden, Estonia and Northern Germany. We found coherent seasonality in the silicon loads of the two northern rivers, which was probably caused by the common snow-type hydrology of the catchments as distinct from the rain-type hydrology of the catchment, further south. The similarity among lakes in the dynamics of the Si-related variables studied resulted from similarities in mean lake depth and mixing type rather than the climatic regime. Among the variables measured at the three sites, river water discharge responded most coherently to climatic forcing as synchronized by the North Atlantic Oscillation winter index (NAOw). Water discharge and Si load were strongly linked variables and showed coherent patterns among the river systems. We found significant season-specific correlations of the NAOw with either the biomass or the share of diatoms in each lake, but no coherent pattern among the lakes. Our results indicate that processes driven by water discharge are more coherent across regions than in-lake processes.     

Choice of scan-sampling intervals—An example with quantifying neighbours in dairy cows

To date, only a few studies using scan-sampling intervals to record neighbours in groups of animals explain how the interval length was chosen. In this study, we investigated the effects of different scan-sampling intervals, behavioural context, and the definition of neighbours on the accuracy of data as compared to a quasi-continuous observation. The data was collected from dairy cows kept in cubicle housing systems. Dairy herds of 22–43 cows on 6 farms were observed once a minute with an automatic tracking system for 6 days, and both the nearest neighbour and all neighbours within a defined distance of each cow were recorded with references to the activity, feeding and lying area and stored in neighbourship matrices. In the analysis, we used data collected at intervals of 2, 3, 4, …, 30 min, and correlated this simulated data with the data based on the 1-min interval using a specialised correlation coefficient for matrices τ_Kr. This correlation coefficient was then used as the response variable in mixed-effects models. We found that the size of the correlation coefficients generally decreased as interval length increased. This decrease was less pronounced for all neighbours than for the nearest neighbour. Moreover, the decrease was greatest with data from the activity area and lowest with data from the lying area. We concluded that, even with the relatively slow dairy cows in a barn environment, neighbour recordings should be conducted at short scan-sampling intervals in order to achieve a minimum correlation of τ_Kr = 0.8 with the quasi-continuous data. Intervals of every 2, 8 and 17 min are recommended for observation of neighbours in dairy cows for the activity, feeding and lying areas, respectively, and species that move faster may well require even shorter sampling intervals.