Individual calcium syntillas do not trigger spontaneous exocytosis from nerve terminals of the neurohypophysis

Recently, highly localized Ca(2+) release events, similar to Ca(2+) sparks in muscle, have been observed in neuronal preparations. Specifically, in murine neurohypophysial terminals (NHT), these events, termed Ca(2+) syntillas, emanate from a ryanodine-sensitive intracellular Ca(2+) pool and increase in frequency with depolarization in the absence of Ca(2+) influx. Despite such knowledge of the nature of these Ca(2+) release events, their physiological role in this system has yet to be defined. Such localized Ca(2+) release events, if they occur in the precise location of the final exocytotic event(s), may directly trigger exocytosis. However, directly addressing this hypothesis has not been possible, since no method capable of visualizing individual release events in these CNS terminals has been available. Here, we have adapted an amperometric method for studying vesicle fusion to this system which relies on loading the secretory granules with the false transmitter dopamine, thus allowing, for the first time, the recording of individual exocytotic events from peptidergic NHT. Simultaneous use of this technique along with high-speed Ca(2+) imaging has enabled us to establish that spontaneous neuropeptide release and Ca(2+) syntillas do not display any observable temporal or spatial correlation, confirming similar findings in chromaffin cells. Although these results indicate that syntillas do not play a direct role in eliciting spontaneous release, they do not rule out indirect modulatory effects of syntillas on secretion

miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production.

Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o(-) tracheal, CFBE41o(-) and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases

Finite element investigation of the effect of spina bifida on loading of the vertebral isthmus

Background: Spondylolysis (SL) of the lower lumbar spine is frequently associated with spina bifida occulta (SBO). There has not been any study that has demonstrated biomechanical or genetic predispositions to explain the coexistence of these two pathologies. In axial rotation, the intact vertebral arch allows torsional load to be shared between the facet joints. In SBO, the load cannot be shared across the arch, theoretically increasing the mechanical demand of the vertebral isthmus during combined axial loading and rotation when compared to the normal state. Purpose: To test the hypothesis that fatigue failure limits will be exceeded in the case of a bifid arch, but not in the intact case, when the segment is subjected to complex loading corresponding to normal sporting activities. Study Design: Descriptive Laboratory Study. Methods: Finite element models of natural and SBO (L4-S1) including ligaments were loaded axially to 1kN and were combined with axial rotation of 3°. Bilateral stresses, alternating stresses and shear fatigue failure on intact and SBO L5 isthmus were assessed and compared. Results: Under 1kN axial load, the von Mises stresses observed in SBO and in the intact cases were very similar (differences <5MPa) having a maximum at the ventral end of the isthmus that decreases monotonically to the dorsal end. However, under 1kN axial load and rotation, the maximum von Mises stresses observed in the ipsilateral L5 isthmus in the SBO case (31MPa) was much higher than the intact case (24.2MPa) indicating a lack of load sharing across the vertebral arch in SBO. When assessing the equivalent alternating shear stress amplitude, this was found to be 22.6 MPa for the SBO case and 13.6 MPa for the intact case. From this it is estimated that shear fatigue failure will occur in less than 70,000 cycles, under repetitive axial load & rotation conditions in the SBO case, while for the intact case, fatigue failure will occur only over 10 million cycles. Conclusion: SBO predisposes spondylolysis by generating increased stresses across the inferior isthmus of the inferior articular process, specifically in combined axial rotation and anteroposterior shear. Clinical Relevance: Athletes with SBO who participate in sports that require repetitive lumbar rotation, hyperextension and/or axial loading are at a higher risk of developing spondylolysis compared to athletes with an intact spine

A Tribute to William S. Geimer

A Tribute to William S. Geimer Meredith Susan Palmer* * Associate Dean for Student Affairs and Admissions, Washington and Lee University School of Law, J.D., 1985, Washington and Lee University. Truly, I say to you, as you did it to one of the least of these my brethren, you did it to me.1 Bill Geimer is not a man of material things. But somehow, in the material things that he chose to have around him - the mementos, the odd souvenirs, the offerings from students, clients, friends - I find the story of the man, the teacher, and the lawyer Bill Geimer is to me. In my mind's eye, I see Bill's office here at Washington and Lee as I first saw it, as a hesitant first-year law student approaching the sanctum sanctorum of a faculty member. There is a desktop name plate, regular Army issue, decorated with grenades or something equally militaristic and intimidating. But next to that is a picture of Dr. Martin Luther King, with his stirring words evoking hope for justice and freedom on earth. And next to that, a membership certificate for the Lawyer's Alliance for Nuclear Arms Control, and a drawing of Quijote, lance raised to a windmill. How could this be? While reconciling the seemingly irreconcilable is the everyday task of the law student, no trick of logic could make these disparate pieces fit; one simply had to get to know the man. That one could do so as a law student is itself part of what defines Bill Geimer. The student who came to know Bill heard a story of a kid from the rural south, off to a small state university to play basketball but who, like countless kids before him, spent a bit more time playing than studying. Come Graduation Day, with no game plan, he joined Uncle Sam's family. A grounding in tanks, guns, and tactics preceded duty with the military police, but something else happened during those years. Assigned to assist with an investigation, he finds an aptitude for the law, an appreciation of justice, and a burning sense of the unfairness of justice denied. The young man who returned from service to go on to a distinguished career as a law student at the University of North Carolina was a man with a game plan, and a mission. It was no surprise that Bill's mission as a lawyer was not in the office towers of Atlanta or the corporate boardrooms of Wall Street, but among the plain folks of Fayetteville, North Carolina, and later in the community of migrant farm labor working and living hard under the hot Carolina sun. I see another memento, this time in Bill's home. It's an odd piece of, well, let's call it folk art. Crudely carved, frankly ugly. Taste is a delicate thing, so one observes diplomatically that it is an "interesting" object. There's a story, of course, a story of a client for whom Bill had worked, putting together the paperwork to get a fledgling arts-and-crafts shop off the ground. This client, like many Bill served, claimed not to have the cash to pay. But, the client said, he could give Bill this valuable piece of work. Barter being a fundamentally more honest exchange and thus appealing to Bill, the transaction was concluded. Not until Bill brought his sculpture home did he discover the price tag stuck to the underside, for something substantially less than the agreed-upon fee. He kept the awkward thing, price tag intact, I think as a reminder of his own fallibility, of the ingenuity of the ordinary man, and of the need to forget neither. Another memento, in some ways the most important to this former student, is a large, faded, well-handled poster on the professor's office door. A young man in Carolina Blue goes up for a shot, stretching for the basket, with seconds left on the clock. Anyone who knows Bill Geimer knows that he is devoted to Carolina basketball. In fact, Bill may have spent so much time watching the Tar Heels play that he began to confuse teaching with coaching. As a former student of Bill's, I'm not sure that was a bad thing.

Understanding uncertainty in temperature effects on vector-borne disease: A Bayesian approach

Extrinsic environmental factors influence the distribution and population dynamics of many organisms, including insects that are of concern for human health and agriculture. This is particularly true for vector-borne infectious diseases, like malaria, which is a major source of morbidity and mortality in humans. Understanding the mechanistic links between environment and population processes for these diseases is key to predicting the consequences of climate change on transmission and for developing effective interventions. An important measure of the intensity of disease transmission is the reproductive number $R_0$. However, understanding the mechanisms linking $R_0$ and temperature, an environmental factor driving disease risk, can be challenging because the data available for parameterization are often poor. To address this we show how a Bayesian approach can help identify critical uncertainties in components of $R_0$ and how this uncertainty is propagated into the estimate of $R_0$. Most notably, we find that different parameters dominate the uncertainty at different temperature regimes: bite rate from 15-25$^\circ$ C; fecundity across all temperatures, but especially $\sim$25-32$^\circ$ C; mortality from 20-30$^\circ$ C; parasite development rate at $\sim$15-16$^\circ$C and again at $\sim$33-35$^\circ$C. Focusing empirical studies on these parameters and corresponding temperature ranges would be the most efficient way to improve estimates of $R_0$. While we focus on malaria, our methods apply to improving process-based models more generally, including epidemiological, physiological niche, and species distribution models.Comment: 27 pages, including 1 table and 3 figure

Race differences in interventions and survival after Out-of-Hospital Cardiac Arrest in North Carolina, 2010 to 2014

Background Following the implementation of the HeartRescue project, with interventions in the community, emergency medical services, and hospitals to improve care and outcomes for out‐of‐hospital cardiac arrests (OHCA) in North Carolina, improved bystander and first responder treatments as well as survival were observed. This study aimed to determine whether these improvements were consistent across Black versus White individuals. Methods and Results Using the Cardiac Arrest Registry to Enhance Survival (CARES), we identified OHCA from 16 counties in North Carolina (population 3 million) from 2010 to 2014. Temporal changes in interventions and outcomes were assessed using multilevel multivariable logistic regression, adjusted for patient and socioeconomic neighborhood‐level factors. Of 7091 patients with OHCA, 36.5% were Black and 63.5% were White. Black patients were younger, more females, had more unwitnessed arrests and non‐shockable rhythm (Black: 81.0%; White: 75.4%). From 2010 to 2014, the adjusted probabilities of bystander cardiopulmonary resuscitation (CPR) went from 38.5% to 51.2% in White, P<0.001; and 36.9% to 45.6% in Black, P=0.002, and first‐responder defibrillation went from 13.2% to 17.2% in White, P=0.002; and 14.7% to 17.3% in Black, P=0.16. From 2010 to 2014, survival to discharge only increased in White (8.0% to 11.4%, P=0.004; Black 8.9% to 9.5%, P=0.60), though, in shockable patients the probability of survival to discharge went from 24.8% to 34.6% in White, P=0.02; and 21.7% to 29.0% in Black, P=0. 10. Conclusions After the HeartRescue program, bystander CPR and first‐responder defibrillation increased in both patient groups; however, survival only increased significantly for White patients

The TREAT-NMD advisory committee for therapeutics (TACT): an innovative de-risking model to foster orphan drug development

Despite multiple publications on potential therapies for neuromuscular diseases (NMD) in cell and animal models only a handful reach clinical trials. The ability to prioritise drug development according to objective criteria is particularly critical in rare diseases with large unmet needs and a limited numbers of patients who can be enrolled into clinical trials. TREAT-NMD Advisory Committee for Therapeutics (TACT) was established to provide independent and objective guidance on the preclinical and development pathway of potential therapies (whether novel or repurposed) for NMD. We present our experience in the establishment and operation of the TACT. TACT provides a unique resource of recognized experts from multiple disciplines. The goal of each TACT review is to help the sponsor to position the candidate compound along a realistic and well-informed plan to clinical trials, and eventual registration. The reviews and subsequent recommendations are focused on generating meaningful and rigorous data that can enable clear go/no-go decisions and facilitate longer term funding or partnering opportunities. The review process thereby acts to comment on viability, de-risking the process of proceeding on a development programme. To date TACT has held 10 review meeting and reviewed 29 program applications in several rare neuromuscular diseases: Of the 29 programs reviewed, 19 were from industry and 10 were from academia; 15 were for novel compounds and 14 were for repurposed drugs; 16 were small molecules and 13 were biologics; 14 were preclinical stage applications and 15 were clinical stage applications. 3 had received Orphan drug designation from European Medicines Agency and 3 from Food and Drug Administration. A number of recurrent themes emerged over the course of the reviews and we found that applicants frequently require advice and education on issues concerned with preclinical standard operating procedures, interactions with regulatory agencies, formulation, repurposing, clinical trial design, manufacturing and ethics. Over the 5 years since its establishment TACT has amassed a body of experience that can be extrapolated to other groups of rare diseases to improve the community's chances of successfully bringing new rare disease drugs to registration and ultimately to marke

Tribute to Professor David Bruck

A tribute to Professor David I. Bruck, who served on the faculty of the Washington and Lee University School of Law from 2004 to 2020. Bruck directed W&L\u27s death penalty defense clinic, the Virginia Capital Case Clearinghouse, also known as VC3 . He became Professor of Law, Emeritus in 2020