Probing Molecular Diversity and Ultrastructure of Brain Cells with Fluorescent Aptamers.

Detergent-free immunolabeling has been proven feasible for correlated light and electron microscopy, but its application is restricted by the availability of suitable affinity reagents. Here we introduce CAptVE, a method using slow off-rate modified aptamers for cell fluorescence labeling on ultrastructurally reconstructable electron micrographs. CAptVE provides labeling for a wide range of biomarkers, offering a pathway to integrate molecular analysis into recent approaches to delineate neural circuits via connectomics.

Radiation Injuries.

Radiation-related injuries are rare. Yet the consequences of an event involving a radiation source can be substantial. As with any clinical emergency that rarely occurs, we are typically less prepared to deal with the situation. Compounding the crisis will be the "worried well" population who may believe that they too are contaminated or suffering from radiation poisoning and report to the hospital for evaluation. Identifying and triaging those who are sick or injured, managing the surge of patients, and knowing where resources can be accessed are all essential.

Results From an Evidenced-Based Curriculum Design With Innovative Simulators to Prepare Providers in Caring for Those With Burn Injuries.

Burn care and medical education have undergone dramatic changes. Trauma has over seven courses covering fundamentals, whereas burns has one. Our goal was to develop a course to meet the needs of healthcare professionals requiring more advanced burn management training. A survey was distributed to burn physicians, nurses, therapists, administrators, and survivors, to assess the perceived proficiency of those managing adult and pediatric patients. Procedure simulators were developed, and a course was designed and delivered. An after-course survey of participants captured how this course filled identified knowledge gaps. A total of 188 initial surveys were sent to individuals involved in burn care. A diverse pool of 109 individuals participated (58% response rate). Survey results by providers demonstrated the lowest self-rated proficiency scores at managing large pediatric burns and frostbite. Nonphysicians reported low proficiency in developing wound treatment algorithms, performing escharotomies, and aftercare/reintegration. Following rigorous curriculum development, the course was conducted, and after-course surveys noted students' improved understanding of managing burn injuries, ability to troubleshoot, confidence to manage patients, and their recommending the course to a peer. Providing quality care beyond the initial assessment and stabilization of a burn-injured patient requires additional skills and knowledge. Providers that are uncomfortable or challenged in providing this care may benefit from additional training. Initial data show that a course, such as this one, provides the education necessary to fill the most commonly reported gaps in knowledge and skills. Further work is being invested to develop disaster management skills, assessment components, and further determine course validity.

Pharmacokinetic Properties of DNA Aptamers with Base Modifications.

The addition of novel side chains at the 5-position of uracil is an effective means to increase chemical diversity of aptamers and hence the success rate for discovery of high-affinity ligands to protein targets. Such modifications also increase nuclease resistance, which is useful in a range of applications, especially for therapeutics. In this study, we assess the impact of these side chains on plasma pharmacokinetics of modified aptamers conjugated to a 40 kDa polyethylene glycol. We show that clearance from plasma depends on relative hydrophobicity: side chains with a negative cLogP (more hydrophilic) result in slower plasma clearance compared with side chains with a positive cLogP (more hydrophobic). We show that clearance increases with the number of side chains in sequences of ≥28 synthons, but this effect is dramatically diminished in shorter sequences. These results serve as a guide for the design of new therapeutic aptamers with diversity-enhancing side chains.

Return on investment of advanced practice medical degrees: NPs vs. PAs.

As the United States faces a predicted physician shortage over the next 2 decades, physician assistants (PAs) and NPs are expected to fill the void. At the same time, because education is expensive, student loan and tuition increases have many potential applicants assessing differences in reimbursement and wondering about their return on investment (ROI). An analysis compared PA and NP salaries by incorporating national salary data, federal income tax, and student loans for a comparative analysis of each career pathway. METHODS: Salaries were abstracted from the 2012 Bureau of Labor Statistics database. The net present value (NPV) of PA and NP salaries was calculated with a 5% discount rate. Principal and interest for student loans was calculated at a 6% interest fixed-rate loan over 30 years. NPVs were then compared with projected ROI at retirement age. Relative career values were also given to each career choice, based on a retirement age of 65 years, which translates to about 41 years of employment for both PAs and NPs. RESULTS: PAs' and NPs' educational loans both equalled $129,484 on total repayment. The median annual salary of a PA was $90,930 and $89,960 for an NP. PA data yielded a 5% NPV of $781,323 compared with $764,348 for NPs. Of note, the 5% NPV of a 4-year nursing degree is $728,436. CONCLUSION: PAs have a slightly higher ROI compared with NPs. These findings may change due to adjustments in nursing training models. Many PA programs allow matriculation immediately after obtaining a bachelor's degree. NP schools often require nursing experience before entering their program. Some schools are considering an accelerated NP program, allowing immediate matriculation after obtaining a bachelor's degree. Because many NP programs have become doctoral degrees, the increased duration of training, higher tuition, and fewer years worked before retirement lower the overall NP ROI. A similar reduction in ROI was considered marginal in PAs who attend residency programs-though these programs are not required for PAs to practice. Comparison of an RN with a 4-year degree to an NP shows little increase in ROI. If interest rates rise, it will become fiscally preferable to remain in a nursing position. Other intangible qualities exist and need further research (for example, weighing the financial aspects with lifestyle or professional satisfaction).

Using Continuous Underway Isotope Measurements To Map Water Residence Time in Hydrodynamically Complex Tidal Environments.

Stable isotopes present in water (δ2H, δ18O) have been used extensively to evaluate hydrological processes on the basis of parameters such as evaporation, precipitation, mixing, and residence time. In estuarine aquatic habitats, residence time (τ) is a major driver of biogeochemical processes, affecting trophic subsidies and conditions in fish-spawning habitats. But τ is highly variable in estuaries, owing to constant changes in river inflows, tides, wind, and water height, all of which combine to affect τ in unpredictable ways. It recently became feasible to measure δ2H and δ18O continuously, at a high sampling frequency (1 Hz), using diffusion sample introduction into a cavity ring-down spectrometer. To better understand the relationship of τ to biogeochemical processes in a dynamic estuarine system, we continuously measured δ2H and δ18O, nitrate and water quality parameters, on board a small, high-speed boat (5 to >10 m s-1) fitted with a hull-mounted underwater intake. We then calculated τ as is classically done using the isotopic signals of evaporation. The result was high-resolution (∼10 m) maps of residence time, nitrate, and other parameters that showed strong spatial gradients corresponding to geomorphic attributes of the different channels in the area. The mean measured value of τ was 30.5 d, with a range of 0-50 d. We used the measured spatial gradients in both τ and nitrate to calculate whole-ecosystem uptake rates, and the values ranged from 0.006 to 0.039 d-1. The capability to measure residence time over single tidal cycles in estuaries will be useful for evaluating and further understanding drivers of phytoplankton abundance, resolving differences attributable to mixing and water sources, explicitly calculating biogeochemical rates, and exploring the complex linkages among time-dependent biogeochemical processes in hydrodynamically complex environments such as estuaries.

Advanced Burn Life Support for Day-to-Day Burn Injury Management and Disaster Preparedness: Stakeholder Experiences and Student Perceptions Following 56 Advanced Burn Life Support Courses.

Educational programs for clinicians managing patients with burn injuries represent a critical aspect of burn disaster preparedness. Managing a disaster, which includes a surge of burn-injured patients, remains one of the more challenging aspects of disaster medicine. During a 6-year period that included the development of a burn surge disaster program for one state, a critical gap was recognized as public presentations were conducted across the state. This gap revealed an acute and greater than anticipated need to include burn care education as an integral part of comprehensive burn surge disaster preparedness. Many hospital and prehospital providers expressed concern with managing even a single, burn-injured patient. While multiple programs were considered, Advanced Burn Life Support (ABLS), a national standardized educational program was selected to help address this need. The curriculum includes initial care for the burn-injured patient as well as an overview of the burn centers role in the disaster preparedness community. After 4 years and 56 classes conducted across the state, a survey was developed including a section that measured the perceptions of those who completed the ABLS educational program. The study specifically examines questions including whether clinicians perceived changes in their burn care knowledge, skills and abilities, and burn disaster preparedness following completion of the program? including whether clinicians.

Characterizing solution surface loop conformational flexibility of the GM2 activator protein.

GM2AP has a ß-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.

Inhibitor-induced conformational shifts and ligand-exchange dynamics for HIV-1 protease measured by pulsed EPR and NMR spectroscopy.

Double electron-electron resonance (DEER) spectroscopy was utilized to investigate shifts in conformational sampling induced by nine FDA-approved protease inhibitors (PIs) and a nonhydrolyzable substrate mimic for human immunodeficiency virus type 1 protease (HIV-1 PR) subtype B, subtype C, and CRF_01 A/E. The ligand-bound subtype C protease has broader DEER distance profiles, but trends for inhibitor-induced conformational shifts are comparable to those previously reported for subtype B. Ritonavir, one of the strong-binding inhibitors for subtypes B and C, induces less of the closed conformation in CRF_01 A/E. (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra were acquired for each protease construct titrated with the same set of inhibitors. NMR (1)H-(15)N HSQC titration data show that inhibitor residence time in the protein binding pocket, inferred from resonance exchange broadening, shifting or splitting correlates with the degree of ligand-induced flap closure measured by DEER spectroscopy. These parallel results show that the ligand-induced conformational shifts resulting from protein-ligand interactions characterized by DEER spectroscopy of HIV-1 PR obtained at the cryogenic temperature are consistent with more physiological solution protein-ligand interactions observed by solution NMR spectroscopy.

Amelioration of type 1 diabetes following treatment of non-obese diabetic mice with INGAP and lisofylline.

Type 1 diabetes mellitus results from the autoimmune and inflammatory destruction of insulin-producing islet ß cells, rendering individuals devoid of insulin production. Recent studies suggest that combination therapies consisting of anti-inflammatory agents and islet growth-promoting factors have the potential to cause sustained recovery of ß cell mass, leading to amelioration or reversal of type 1 diabetes in mouse models. In this study, we hypothesized that the combination of the anti-inflammatory agent lisofylline (LSF) with an active peptide fragment of islet neogenesis associated protein (INGAP peptide) would lead to remission of type 1 diabetes in the non-obese diabetic (NOD) mouse. We treated groups of spontaneously diabetic NOD mice with combinations of LSF, INGAP peptide, or control saline parenterally for up to 6 weeks. Our results demonstrate that the mice receiving combined treatment with LSF and INGAP peptide exhibited partial remission of diabetes with increased plasma insulin levels. Histologic assessment of pancreata in mice receiving combined therapy revealed the presence of islet insulin staining, increased ß cell replication, and evidence of Pdx1-positivity in ductal cells. By contrast, diabetic animals showed severe insulitis with no detectible insulin or Pdx1 staining. We conclude that the novel combination treatment with LSF and INGAP peptide has the potential to ameliorate hyperglycemia in the setting of established type 1 diabetes via the recovery of endogenous ß cells and warrant further studies.

Structure-based parallel medicinal chemistry approach to improve metabolic stability of benzopyran COX-2 inhibitors.

Combination of the structure-based design and solid-phase parallel synthesis provided an integrated approach to rapidly develop the structure-activity relationship of benzopyran COX-2 inhibitors. Binding free energies predicted by free energy perturbation theory yielded good agreement with experimental results. New potent and selective lead compounds with improved metabolic properties were identified.

The novel benzopyran class of selective cyclooxygenase-2 inhibitors-part I: the first clinical candidate.

In this manuscript, we report the discovery of the substituted 2-trifluoromethyl-2H-benzopyran-3-carboxylic acids as a novel series of potent and selective cyclooxygenase-2 (COX-2) inhibitors. 5c-(S) (SD-8381) was advanced into clinical studies due to its superior in vivo potency. The high plasma protein binding (>99% bound) of 5c-(S) has resulted in a surprisingly long human half life t(1/2)=360 h.

The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part 2: the second clinical candidate having a shorter and favorable human half-life.

In this Letter, we provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data for a series of selective COX-2 inhibitors. During the course of our structure-based drug design efforts, we discovered two distinct binding modes within the COX-2 active site for differently substituted members of this class. The challenge of a undesirably long human half-life for the first clinical candidate 1t(1/2)=360 h was addressed by multiple strategies, leading to the discovery of 29b-(S) (SC-75416) with t(1/2)=34 h.

The novel benzopyran class of selective cyclooxygenase-2 inhibitors. Part III: the three microdose candidates.

In this manuscript, we report the discovery of the substituted 2-trifluoromethyl-2H-benzopyran-3-carboxylic acids as a novel series of potent and selective cyclooxygenase-2 (COX-2) inhibitors. We provide the structure-activity relationships, optimization of design, testing criteria, and human half-life data. The challenge of a surprisingly long half-life (t(1/2)=360 h) of the first clinical candidate 1 and human t(1/2) had been difficult to predict based on allometric scaling for this class of highly ppb compounds. We used a microdose strategy which led to the discovery of clinical agents 18c-(S), 29b-(S), and 34b-(S) with human half-life of 57, 13, and 11 h.

A general preparation of (Z)-1-fluorostilbene derivatives for the design of conformationally restricted peptidomimetics.

The preparation of (Z)-1-fluoro-2-bromostyrenes provides a general route for the formation of (Z)-1-fluorostilbene derivatives as configurationally stable spacial linkers for the design of conformationally restricted peptidomimetics. Palladium-catalyzed aryl Suzuki and Stille cross-coupling reactions have been surveyed to proceed with complete retention of fluoroalkene geometry, and permit the direct incorporation of a variety of aryl and heteroaromatic substituents.

Interactions of the GM2 activator protein with phosphatidylcholine bilayers: a site-directed spin-labeling power saturation study.

The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.

Evaluation of COX-1/COX-2 selectivity and potency of a new class of COX-2 inhibitors.

A new class of selective cyclooxygenase-2 (COX-2) inhibitors has been identified by high throughput screening. Structurally distinct from previously described selective COX-2 inhibitors, these benzopyrans contain a carboxylic acid function and CF3 functionality. The compound SC-75,416 is a representative of this class. A range if in vitro and in vivo tests were employed to characterize its potency and selectivity. Using human recombinant enzymes, this compound displays a concentration that provides 50% inhibition (IC50) of 0.25 microM for COX-2 and 49.6 microM for COX-1. A mutation of the side pocket residues in COX-2 to COX-1 had little effect on potency suggesting that these inhibitors bind in a unique manner in COX-2 distinct from COX-2 inhibiting diaryl heterocycles. Using rheumatoid arthritic synovial cells stimulated with interleukin-1beta (IL-1beta) and washed platelets the compound displayed IC50 of 3 nM and 400 nM respectively. Potency and selectivity was maintained but predictably right shifted in whole blood with IC50 of 1.4 microM for lipopolysaccharide (LPS) stimulated induction of COX-2 and >200 microM for inhibition of platelet thromboxane production. SC-75,416 is 89% bioavailable and its in vivo half life is sufficient for once a day dosing. In the rat air pouch model of inflammation, the compound inhibited PGE2 production with an effective dose that provides 50% inhibition (ED50) of 0.4 mg/kg, while sparing gastric prostaglandin E2 (PGE2) production with an ED50 of 26.5 mg/kg. In a model of acute inflammation and pain caused by carrageenan injection into the rat paw, the compound reduced edema and hyperalgesia with ED50s of 2.7 and 4 mg/kg respectively. In a chronic model of arthritis the compound demonstrated an ED50 of 0.081 mg/kg and an ED(80) of 0.38 mg/kg. In a model of neuropathic pain, SC-75,416 had good efficacy. This compound's unique chemical structure and effect on COX enzyme binding and activity as well as its potency and selectivity may prove useful in treating pain and inflammation.

Genistein acutely stimulates insulin secretion in pancreatic beta-cells through a cAMP-dependent protein kinase pathway.

Although genistein, a soy isoflavone, has beneficial effects on various tissues, it is unclear whether it plays a role in physiological insulin secretion. Here, we present evidence that genistein increases rapid glucose-stimulated insulin secretion (GSIS) in both insulin-secreting cell lines (INS-1 and MIN6) and mouse pancreatic islets. Genistein elicited a significant effect at a concentration as low as 10 nmol/l with a maximal effect at 5 micromol/l. The effect of genistein on GSIS was not dependent on estrogen receptor and also not related to an inhibition of protein tyrosine kinase (PTK). Consistent with its effect on GSIS, genistein increases intracellular cAMP and activates protein kinase A (PKA) in both cell lines and the islets by a mechanism that does not involve estrogen receptor or PTK. The induced cAMP by genistein, at physiological concentrations, may result primarily from enhanced adenylate cyclase activity. Pharmacological or molecular intervention of PKA activation indicated that the insulinotropic effect of genistein is primarily mediated through PKA. These findings demonstrated that genistein directly acts on pancreatic beta-cells, leading to activation of the cAMP/PKA signaling cascade to exert an insulinotropic effect, thereby providing a novel role of soy isoflavones in the regulation of insulin secretion.

Bidirectional communication between the brain and the immune system: implications for physiological sleep and disorders with disrupted sleep.

This review describes mechanisms of immune-to-brain and brain-to-immune signaling involved in mediating physiological sleep and altered sleep with disease. The central nervous system (CNS) modulates immune function by signaling target cells of the immune system through autonomic and neuroendocrine pathways. Neurotransmitters and hormones produced and released by these pathways interact with immune cells to alter immune functions, including cytokine production. Cytokines produced by cells of the immune and nervous systems regulate sleep. Cytokines released by immune cells, particularly interleukin-1beta and tumor necrosis factor-alpha, signal neuroendocrine, autonomic, limbic and cortical areas of the CNS to affect neural activity and modify behaviors (including sleep), hormone release and autonomic function. In this manner, immune cells function as a sense organ, informing the CNS of peripheral events related to infection and injury. Equally important, homeostatic mechanisms, involving all levels of the neuroaxis, are needed, not only to turn off the immune response after a pathogen is cleared or tissue repair is completed, but also to restore and regulate natural diurnal fluctuations in cytokine production and sleep. The immune system's ability to affect behavior has important implications for understanding normal and pathological sleep. Sleep disorders are commonly associated with chronic inflammatory diseases and chronic age- or stress-related disorders. The best studied are rheumatoid arthritis, fibromyalgia and chronic fatigue syndromes. This article reviews our current understanding of neuroimmune interactions in normal sleep and sleep deprivation, and the influence of these interactions on selected disorders characterized by pathological sleep.