Programmed translational readthrough generates antiangiogenic VEGF-Ax.

Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here, we show that vascular endothelial growth factor A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino-acid C terminus extension. A cis-acting element in the VEGFA 3' UTR serves a dual function, not only encoding the appended peptide but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits antiangiogenic activity in contrast to the proangiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.

A Practical Guide to Rodent Islet Isolation and Assessment Revisited.

Insufficient insulin secretion is a key component of both type 1 and type 2 diabetes. Since insulin is released by the islets of Langerhans, obtaining viable and functional islets is critical for research and transplantation. The effective and efficient isolation of these small islands of endocrine cells from the sea of exocrine tissue that is the rest of the pancreas is not necessarily simple or quick. Choosing and administering the digestive enzyme, separation of the islets from acinar tissue, and culture of islets are all things that must be considered. The purpose of this review is to provide a history of the development of islet isolation procedures and to serve as a practical guide to rodent islet research for newcomers to islet biology. We discuss key elements of mouse islet isolation including choosing collagenase, the digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews techniques for assessing islet viability and function such as visual assessment, glucose-stimulated insulin secretion and intracellular calcium measurements. A detailed protocol is provided that describes a common method our laboratory uses to obtain viable and functional mouse islets for in vitro study. This review thus provides a strong foundation for successful procurement and purification of high-quality mouse islets for research purposes.

Isolation and Assessment of Pancreatic Islets Versus Dispersed Beta Cells: A Straightforward Approach to Examine Cell-Cell Communication.

Islets of Langerhans, found in the pancreas, are microorgans essential for glucose homeostasis within the body. Many cells are found with an islet, such as beta cells (~70%), alpha cells (~20%), delta cells (~5%), F cells (~4%), and epsilon cells (1%), each with its own unique function. To better understand the roles of these cells and how cell communication alters their function, several techniques have been established such as islet isolation and beta cell dispersion. Here we describe how to isolate primary rodent islets, disperse pancreatic islets, measure intracellular calcium, and use immunofluorescent staining to distinguish beta cells and alpha cells.

The Capacity to Secrete Insulin Is Dose-Dependent to Extremely High Glucose Concentrations: A Key Role for Adenylyl Cyclase.

Insulin secretion is widely thought to be maximally stimulated in glucose concentrations of 16.7-to-30 mM (300-to-540 mg/dL). However, insulin secretion is seldom tested in hyperglycemia exceeding these levels despite the Guinness World Record being 147.6 mM (2656 mg/dL). We investigated how islets respond to 1-h exposure to glucose approaching this record. Insulin secretion from human islets at 12 mM glucose intervals dose-dependently increased until at least 72 mM glucose. Murine islets in 84 mM glucose secreted nearly double the insulin as in 24 mM (p < 0.001). Intracellular calcium was maximally stimulated in 24 mM glucose despite a further doubling of insulin secretion in higher glucose, implying that insulin secretion above 24 mM occurs through amplifying pathway(s). Increased osmolarity of 425-mOsm had no effect on insulin secretion (1-h exposure) or viability (48-h exposure) in murine islets. Murine islets in 24 mM glucose treated with a glucokinase activator secreted as much insulin as islets in 84 mM glucose, indicating that glycolytic capacity exists above 24 mM. Using an incretin mimetic and an adenylyl cyclase activator in 24 mM glucose enhanced insulin secretion above that observed in 84 mM glucose while adenylyl cyclase inhibitor reduced stimulatory effects. These results highlight the underestimated ability of islets to secrete insulin proportionally to extreme hyperglycemia through adenylyl cyclase activity.

Characteristics of the traumatic stressors experienced by rural first responders.

First responders routinely experience work-related events that meet the definition of a traumatic stressor. Despite the high exposure to traumatic events, prevalence rates of posttraumatic stress disorder (PTSD) are relatively low. This discrepancy points to the potential value of identifying factors that distinguish those traumatic stressors that produce ongoing traumatic stress symptoms from those that do not. The present study surveyed 181 first responders from rural settings. A repeated-measures design was used to compare characteristics of traumatic stressors that were or were not associated with ongoing PTSD symptoms. A factor analysis revealed that distressing events were characterized by chaos and resource limitations. Consistent with contemporary models, two mediational analyses revealed that each event characteristic predicted peritraumatic dissociation and posttraumatic cognitions, which in turn predicted PTSD symptoms. Moreover, the effect of each event characteristic on PTSD symptoms was partially mediated by these cognitive processes.

Empirical limits for the forensic assessment of PTSD litigants.

This paper discusses the limits of expert opinion on posttraumatic stress disorder (PTSD) in personal injury claims. The construct of PTSD is hampered by several empirical limitations. Multiple reliable measures of PTSD exist, but have not been evaluated sufficiently within litigating samples and are infrequently used by forensic assessors. Common methods for trauma screening appear insensitive. Opinions about causation of PTSD and disability are complicated by retrospective memory biases, as well as the failure of most anxiety disorders to be detected within primary medical care. PTSD appears to have a steep spontaneous remission curve during the first year, but at least 10% of trauma-exposed people suffer chronic distress. Little is known about the course beyond 1 year. Efficacious psychological treatments have been developed for PTSD, but are not in common use limiting claimants' access to rehabilitative treatments. Research on functional disability associated with PTSD is in its infancy, but it seems likely that PTSD will account for only a part of the variance in work disability. We provide suggestions for improving forensic practice, advising the courts about the limitations of forensic opinions, and necessary research.