Notch signaling in the pigmented epithelium of the anterior eye segment promotes ciliary body development at the expense of iris formation

The ciliary body and iris are pigmented epithelial structures in the anterior eye segment that function to maintain correct intra‐ocular pressure and regulate exposure of the internal eye structures to light, respectively. The cellular and molecular factors that mediate the development of the ciliary body and iris from the ocular pigmented epithelium remain to be fully elucidated. Here, we have investigated the role of Notch signaling during the development of the anterior pigmented epithelium by using genetic loss‐ and gain‐of‐function approaches. Loss of canonical Notch signaling results in normal iris development but absence of the ciliary body. This causes progressive hypotony and over time leads to phthisis bulbi, a condition characterized by shrinkage of the eye and loss of structure/function. Conversely, Notch gain‐of‐function results in aniridia and profound ciliary body hyperplasia, which causes ocular hypertension and glaucoma‐like disease. Collectively, these data indicate that Notch signaling promotes ciliary body development at the expense of iris formation and reveals novel animal models of human ocular pathologies.     

Dendritic cells and hepatocytes use distinct pathways to process protective antigen from plasmodium in vivo

Malaria-protective CD8+ T cells specific for the circumsporozoite (CS) protein are primed by dendritic cells (DCs) after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2K(b) epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d) and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase.     

Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep

Background

Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep.

Methodology/Principal Findings

The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation.

Conclusions/Significance

This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.     

Seeking the true time: Exploring otolith chemistry as an age-determination tool

Fish otoliths' chronometric properties make them useful for age and growth rate estimation in fisheries management. For the Eastern Baltic Sea cod stock (Gadus morhua), unclear seasonal growth zones in otoliths have resulted in unreliable age and growth information. Here, a new age estimation method based on seasonal patterns in trace elemental otolith incorporation was tested for the first time and compared with the traditional method of visually counting growth zones, using otoliths from the Baltic and North seas. Various trace elemental ratios, linked to fish metabolic activity (higher in summer) or external environment (migration to colder, deeper habitats with higher salinity in winter), were tested for age estimation based on assessing their seasonal variations in concentration. Mg:Ca and P:Ca, both proxies for growth and metabolic activity, showed greatest seasonality and therefore have the best potential to be used as chemical clocks. Otolith image readability was significantly lower in the Baltic than in the North Sea. The chemical (novel) method had an overall greater precision and percentage agreement among readers (11.2%, 74.0%) than the visual (traditional) method (23.1%, 51.0%). Visual readers generally selected more highly contrasting zones as annuli whereas the chemical readers identified brighter regions within the first two annuli and darker zones thereafter. Visual estimates produced significantly higher, more variable ages than did the chemical ones. Based on the analyses in our study, we suggest that otolith microchemistry is a promising alternative ageing method for fish populations difficult to age, such as the Eastern Baltic cod.     

Effects of freezing on length and mass measurements of Atlantic cod Gadus morhua in the Baltic Sea

An aggregated sample of 925 Atlantic cod Gadus morhua collected by four countries in different regions of the Baltic Sea during different seasons were measured (total length, L_T = 161–890 mm and weighed (mass, M = 45–6900 g) both before freezing and after defrosting. The cod were found to decrease significantly in both L_T and M following death and frozen storage. There was an average (±SD) change in L_T of −2.91% (±0.05%) following freezing, independent of starting L_T. Total M changed by −2.65% (±0.14%), independent of starting mass. Shrinkage of L_T and M did not differ significantly between 1 and 4 months frozen storage, though L_T shrinkage was significantly greater after 1 or 4 months in the freezer compared with after 5 days. There was significant variation in L_T and M shrinkage between regions of capture. A significant negative relationship between condition of cod and L_T or M change was also observed. Equations to back-calculate fresh L_T and M from thawed L_T, M and standard length (L_S), gutted L_T, gutted L_S and gutted M are provided.     

Control of Insulin Secretion by Cytochrome c and Calcium Signaling in Islets with Impaired Metabolism

The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal.     

Herpesviruses Exploit Several Host Compartments for Envelopment

Enveloped viruses acquire their host‐derived membrane at a variety of intracellular locations. Herpesviruses are complex entities that undergo several budding and fusion events during an infection. All members of this large family are believed to share a similar life cycle. However, they seemingly differ in terms of acquisition of their mature envelope. Herpes simplex virus is often believed to bud into an existing intracellular compartment, while the related cytomegalovirus may acquire its final envelope from a novel virus‐induced assembly compartment. This review focuses on recent advances in the characterization of cellular compartment(s) potentially contributing to herpes virion final envelopment. It also examines the common points between seemingly distinct envelopment pathways and highlights the dynamic nature of intracellular compartments in the context of herpesvirus infections.      

Characterization of the structural and functional changes in the myocardium following focal ischemia-reperfusion injury

High-resolution (11.7 T) cardiac magnetic resonance imaging (MRI) and histological approaches have been employed in tandem to characterize the secondary damage suffered by the murine myocardium following the initial insult caused by ischemia-reperfusion (I/R). I/R-induced changes in the myocardium were examined in five separate groups at the following time points after I/R: 1 h, day 1, day 3, day 7, and day 14. The infarct volume increased from 1 h to day 1 post-I/R. Over time, the loss of myocardial function was observed to be associated with increased infarct volume and worsened regional wall motion. In the infarct region, I/R caused a decrease in end-systolic thickness coupled with small changes in end-diastolic thickness, leading to massive wall thickening abnormalities. In addition, compromised wall thickening was also observed in left ventricular regions adjacent to the infarct region. A tight correlation (r2 = 0.85) between measured MRI and triphenyltetrazolium chloride (TTC) infarct volumes was noted. Our observation that until day 3 post-I/R the infarct size as measured by TTC staining and MRI was much larger than that of the myocyte-silent regions in trichrome- or hematoxylin-eosin-stained sections is consistent with the literature and leads to the conclusion that at such an early phase, the infarct site contains structurally intact myocytes that are functionally compromised. Over time, such affected myocytes were noted to structurally disappear, resulting in consistent infarct sizes obtained from MRI and TTC as well as trichrome and hematoxylin-eosin analyses on day 7 following I/R. Myocardial remodeling following I/R includes secondary myocyte death followed by the loss of cardiac function over time.