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Dr. Davis discusses the case management of a patient with an upper extremity repetitive stress disorder that resulted from median nerve entrapment. The case was successfully treated with a functional approach, using manipulation/mobilization, an office-home-based rehabilitative system, and nutritional intervention. [ABSTRACT FROM AUTHOR]

Competing Interests: Competing interests: Work by all authors was supported by grants from either the US National Institutes of Health (NIH), National Center for Complementary and Integrative Health (NCCIH), the Office of Behavioral and Social Sciences Research (OBSSR) or the US Department of Defense. In all cases, funding was provided to institutions and not to individuals. No other competing interests to declare.

Goal: To document shifts in rural hospital service line offerings between 2010 and 2021 and to assess the resulting impacts on hospital profitability., Methods: We used annual Medicare cost report data for all rural hospitals that did not change payment classifications between 2010 and 2021. We documented changes in the percentages of hospitals offering each of the 37 inpatient or ancillary service lines included in the data. We then used panel event studies to assess effects on hospital operating margin for specific service lines that changed most prominently during this period., Principal Findings: Twelve service lines changed by more than 5% during our period of analysis. These are highlighted by hospitals adding rural health clinics (+32%) and CT scans (+20%) and removing delivery rooms (-21%) and skilled nursing facilities (-19%). Panel event studies demonstrated that the addition or subtraction of most services did not have statistically significant impacts on future hospital operating margins. Notable exceptions were the addition of rural health clinics and the removal of delivery services, both of which positively affected future operating margins. The addition of occupational therapy services had a positive effect on operating margin in the near term, but adding MRI services had a negative effect., Practical Applications: The finding that only a select few service line changes resulted in meaningful impacts to hospital operating margins suggests that hospital leaders should be wary of implementing such changes as a means of improving financial viability., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Foundation of the American College of Healthcare Executives.)

Aims: The aim of the present study was to assess the relationship between adolescent IQ and midlife alcohol use and to explore possible mediators of this relationship., Methods: Study data were from 6300 men and women who participated in the Wisconsin Longitudinal Study of high-school students graduating in 1957. IQ scores were collected during the participants' junior year of high school. In 2004, participants reported the number of alcoholic beverages consumed (past 30 days) and the number of binge-drinking episodes. A multinomial logistic regression was conducted to determine the relationship between adolescent IQ and future drinking pattern (abstainer, moderate drinker, or heavy drinker), and Poisson regression was used to examine the number of binge-drinking episodes. Two mediators-income and education-were also explored., Results: Every one-point increase in IQ score was associated with a 1.6% increase in the likelihood of reporting moderate or heavy drinking as compared to abstinence. Those with higher IQ scores also had significantly fewer binge-drinking episodes. Household income, but not education, partially mediated the relationship between IQ and drinking pattern., Conclusions: The present study suggests that higher adolescent IQ may predict a higher likelihood of moderate or heavy drinking in midlife, but fewer binge-drinking episodes. The study also suggests that this relationship is mediated by other psychosocial factors, specifically income, prompting future exploration of mediators in subsequent studies., (© The Author(s) 2024. Medical Council on Alcohol and Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Studies have examined floor-to-stand performance in varied adult populations both quantitatively and qualitatively. Despite an elevated risk of falls and inability to independently return to stand after a fall, few have examined the ability to stand from the floor in patients recovering from stroke. There were 2 objectives of the study: to identify the relationships between floor-to-stand performance using a timed supine-to-stand test (TSS) and physical performance measures of gait, balance, and balance confidence among persons in the subacute phase after stroke; and to analyze descriptive strategies used in the completion of the TSS., Methods: A cross-sectional design was implemented. Fifty-eight adults (mean age = 59.2 [standard deviation (SD) = 13.9] years; 34 [58.6%] men) who were in the subacute phase after ischemic or hemorrhagic stroke and who could stand from the floor with no more than supervision completed the TSS and physical performance assessments., Results: The median time to complete the TSS in our sample was 13.0 (interquartile range = 15.5) seconds. TSS time was significantly correlated with physical performance tests, including the Timed "Up & Go" Test (ρ = 0.70), gait speed (ρ = -0.67), Dynamic Gait Index (ρ = -0.52), and Activities-Specific Balance Confidence Scale (ρ = -0.43). Thirty-two percent of the variance in TSS time was attributed to Timed "Up & Go" Test time and the use of the quadruped position to transition to standing. Participants who used a gait device were more likely to use a chair during rise to stand., Conclusion: The TSS demonstrates concurrent validity with physical performance measures., Impact: Findings serve to improve functional mobility examination after stroke and to formulate effective treatment interventions to improve floor-to-stand performance., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Physical Therapy Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: To determine whether community sociodemographic factors are associated with the survival or closure of rural hospitals at risk of financial distress between 2010 and 2019., Methods: We use a national sample of 985 rural hospitals at risk of financial distress to analyze the relationship between community sociodemographic characteristics and hospital survival or closure. We control for financial distress using the Financial Distress Index developed by the Sheps Center for Health Services Research. Community characteristics are retrieved from the Census and the Robert Wood Johnson Foundation. We first use Wilcoxon rank-sum tests to demonstrate annual sociodemographic differences between rural communities with financially distressed hospitals that closed between 2010 and 2019, and those that remained open. Multilevel Weibull proportional hazards regressions then uncover which sociodemographic factors are significantly associated with survival., Findings: Our initial results confirm that closures of rural hospitals at risk of financial distress disproportionately affect communities with certain sociodemographic characteristics. However, most of these characteristics are not associated with higher rates of closure in the multivariate survival analysis. The final results suggest that financially distressed hospitals are more likely to experience closure if their communities have higher rates of unemployment (Hazard Ratio = 1.36, P < .05) or uninsured residents under 65 (Hazard Ratio = 1.13, P < .05)., Conclusions: Among financially distressed rural hospitals, specific community-level sociodemographic characteristics (unemployment and uninsurance rates) are positively associated with the likelihood of closure. Social policies addressing these issues should emphasize their broader relationship with the local health sector., (© 2022 The Authors. The Journal of Rural Health published by Wiley Periodicals LLC on behalf of National Rural Health Association.)

Nonpharmacological treatments are considered first-line pain management strategies, but they remain clinically underused. For years, pain-focused pragmatic clinical trials (PCTs) have generated evidence for the enhanced use of nonpharmacological interventions in routine clinical settings to help overcome implementation barriers. The Pragmatic Explanatory Continuum Indicator Summary (PRECIS-2) framework describes the degree of pragmatism across 9 key domains. Among these, "flexibility in delivery" and "flexibility in adherence," address a key goal of pragmatic research by tailoring approaches to settings in which people receive routine care. However, to maintain scientific and ethical rigor, PCTs must ensure that flexibility features do not compromise delivery of interventions as designed, such that the results are ethically and scientifically sound. Key principles of achieving this balance include clear definitions of intervention core components, intervention monitoring and documentation that is sufficient but not overly burdensome, provider training that meets the demands of delivering an intervention in real-world settings, and use of an ethical lens to recognize and avoid potential trial futility when necessary and appropriate. PERSPECTIVE: This article presents nuances to be considered when applying the PRECIS-2 framework to describe pragmatic clinical trials. Trials must ensure that patient-centered treatment flexibility does not compromise delivery of interventions as designed, such that measurement and analysis of treatment effects is reliable., (Copyright © 2023 United States Association for the Study of Pain, Inc. All rights reserved.)

Pragmatic clinical trials (PCTs) are well-suited to address unmet healthcare needs, such as those arising from the dual public health crises of chronic pain and opioid misuse, recently exacerbated by the COVID-19 pandemic. These overlapping epidemics have complex, multifactorial etiologies, and PCTs can be used to investigate the effectiveness of integrated therapies that are currently available but underused. Yet individual pragmatic studies can be limited in their reach because of existing structural and cultural barriers to dissemination and implementation. The National Institutes of Health, Department of Defense, and Department of Veterans Affairs formed an interagency research partnership, the Pain Management Collaboratory. The partnership combines pragmatic trial design with collaborative tools and relationship building within a large network to advance the science and impact of nonpharmacological approaches and integrated models of care for the management of pain and common co-occurring conditions. The Pain Management Collaboratory team supports 11 large-scale, multisite PCTs in veteran and military health systems with a focus on team science with the shared aim that the "whole is greater than the sum of the parts." Herein, we describe this integrated approach and lessons learned, including incentivizing all parties; proactively offering frequent opportunities for problem-solving; engaging stakeholders during all stages of research; and navigating competing research priorities. We also articulate several specific strategies and their practical implications for advancing pain management in active clinical, "real-world," settings., (© The Association of Military Surgeons of the United States 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The aim of the study is to examine the relationship between electronic health record (EHR) use/functionality and hospital operating costs (divided into five subcategories), and to compare the results across rural and urban facilities., Methods: We match hospital-level data on EHR use/functionality with operating costs and facility characteristics to perform linear regressions with hospital- and time-fixed effects on a panel of 1,596 U.S. hospitals observed annually from 2016 to 2019. Our dependent variables are the logs of the various hospital operating cost categories, and alternative metrics for EHR use/functionality serve as the primary independent variables of interest. Data on EHR use/functionality are retrieved from the American Hospital Association's (AHA) Annual Survey of Hospitals Information Technology (IT) Supplement, and hospital operating cost and characteristic data are retrieved from the American Hospital Directory. We include only hospitals classified as "general medical and surgical," removing specialty hospitals., Results: Our results suggest, first, that increasing levels of EHR functionality are associated with hospital operating cost reductions. Second, that these significant cost reductions are exclusively seen in urban hospitals, with the associated coefficient suggesting cost savings of 0.14% for each additional EHR function. Third, that urban EHR-related cost reductions are driven by general/ancillary and outpatient costs. Finally, that a wide variety of EHR functions are associated with cost reductions for urban facilities, while no EHR function is associated with significant cost reductions in rural locations., Conclusion: Increasing EHR functionality is associated with significant hospital operating cost reductions in urban locations. These results do not hold across geographies, and policies to promote greater EHR functionality in rural hospitals will likely not lead to short-term cost reductions., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Introduction: Pragmatic clinical trials (PCTs) can overcome implementation challenges for bringing evidence-based therapies to people living with pain and co-occurring conditions, providing actionable information for patients, providers, health systems, and policy makers. All studies, including those conducted within health systems that have a history of advancing equitable care, should make efforts to address justice and equity., Methods: Drawing from collective experience within pragmatic pain clinical trials networks, and synthesizing relevant literature, our multidisciplinary working group examined challenges related to integrating justice and equity into pragmatic pain management research conducted in large, integrated health systems. Our analysis draws from military and veteran health system contexts but offers strategies to consider throughout the lifecycle of pragmatic research more widely., Results: We found that PCTs present a unique opportunity to address major influences on health inequities by occupying a space between research, healthcare delivery, and the complexities of everyday life. We highlight key challenges that require attention to support complementary advancement of justice and equity via pragmatic research, offering several strategies that can be pursued., Conclusions: Efforts are needed to engage diverse stakeholders broadly and creatively in PCTs, such as through dedicated health equity working groups and other collaborative relationships with stakeholders, to support robust and inclusive approaches to research design and implementation across study settings. These considerations, while essential to pain management research, offer important opportunities toward achieving more equitable healthcare and health systems to benefit people living with pain and co-occurring conditions., Competing Interests: The authors declares no conflicts of interest., (© 2021 The Authors. Learning Health Systems published by Wiley Periodicals LLC on behalf of University of Michigan.)

Background: Melasma is a common pigmentary disorder that responds well to treatment with oral and/or locally injected tranexamic acid but less so to topical application. We hypothesized that this may be due to an inability of some topical formulations of tranexamic acid to achieve robust therapeutic concentrations at the viable epidermal target site in the skin., Aims: To measure in vitro human epidermal and dermal skin concentrations of tranexamic acid following topical application of Fairence ® T-Complex, co-enhancer cream and a Japanese branded cream control ("branded") and compare these with estimates of tranexamic acid potency obtained from in vivo human pharmacokinetic and clinical studies and in vitro pharmacodynamic studies on inhibition of fibrinolysis., Methods: Static vertical Franz cells and human abdominal skin were used to measure stratum corneum, viable epidermal, and dermal concentrations of tranexamic acid using HPLC-MS-MS analysis at 6 and 24 hour periods after topical application., Results: Skin concentrations of tranexamic acid following application of the co-enhancer cream were robustly within the concentration range estimated to be required for efficacy at both 6 and 24 hours. Those from the branded cream control were within the lower range at 24 hours., Conclusions: These preclinical results support the benefits of conducting further studies, including a double-blind placebo-controlled clinical study, on Fairence ® T-Complex, co-enhancer cream in patients with melasma. It is hypothesized that a more robust and timely clinical response may be achieved especially in refractory patients., (© 2020 Wiley Periodicals, Inc.)

Despite efforts to promote diversity in the biomedical workforce, there remains a lower rate of funding of National Institutes of Health R01 applications submitted by African-American/black (AA/B) scientists relative to white scientists. To identify underlying causes of this funding gap, we analyzed six stages of the application process from 2011 to 2015 and found that disparate outcomes arise at three of the six: decision to discuss, impact score assignment, and a previously unstudied stage, topic choice. Notably, AA/B applicants tend to propose research on topics with lower award rates. These topics include research at the community and population level, as opposed to more fundamental and mechanistic investigations; the latter tend to have higher award rates. Topic choice alone accounts for over 20% of the funding gap after controlling for multiple variables, including the applicant's prior achievements. Our findings can be used to inform interventions designed to close the funding gap., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Previous research suggests that the endocrine disrupting chemical tolylfluanid (TF) may promote metabolic dysfunction and insulin resistance in humans. The potential impact of TF on skeletal muscle metabolism has yet to be fully investigated. The purpose of this study was to determine whether TF can promote insulin resistance and metabolic dysfunction in mammalian skeletal muscle cells. C2C12 murine skeletal myotubes were exposed to 1 ppm TF for 24 h. To examine the potential effect of cellular fatty acid levels on TF-dependent regulation of mitochondrial metabolism and insulin signaling, we treated skeletal myotubes with 0.25 mM or 1.0 mM oleic acid (OA) during TF exposure trials. Tolylfluanid (1-10 ppm) reduced lipid accumulation by approximately 20% in 0.25 and 1.0 mM OA treated cells. The addition of 0.25 mM OA completely inhibited the TF-dependent reduction in maximal mitochondrial oxygen consumption rate (OCR) while 1.0 mM OA exacerbated the TF-dependent reduction in mitochondrial OCR. Exposing skeletal myotubes to 1 ppm TF promoted an 80% reduction in mitochondrial membrane potential, which was completely inhibited by 0.25 mM OA and partially inhibited by1.0 mM OA. The addition of 0.25 mM OA promoted a TF-dependent increase in insulin-dependent P-Akt (Ser473). In contrast, the addition of 1.0 mM OA promoted a significant reduction in insulin-dependent P-Akt (Ser473). Further, the addition of 1 ppm TF significantly reduced insulin-dependent mTORC1 activity regardless of OA concentration. Finally, TF significantly reduced insulin-dependent protein synthesis in the 1 mM OA treated cells only. Our results demonstrate that the effect of 1 ppm TF on mitochondrial function and insulin-dependent protein synthesis in skeletal myotubes was largely dependent upon cellular fatty acid levels., (Copyright © 2018. Published by Elsevier Inc.)

Context: As today's rural hospitals have struggled with financial sustainability for the past 2 decades, it is critical to understand their value relative to alternatives, such as rural health clinics and private practices., Purpose: To estimate the willingness-to-pay for specific attributes of rural health care facilities in rural Kentucky to determine which services and operational characteristics are most valued by rural residents., Methodology: We fitted choice experiment data from 769 respondents in 10 rural Kentucky counties to a conditional logit model and used the results to estimate willingness-to-pay for attributes in several categories, including hours open, types of insurance accepted, and availability of health care professionals and specialized care., Findings: Acceptance of Medicaid/Medicare with use of a sliding fee scale versus acceptance of only private insurance was the most valued attribute. Presence of full diagnostic services, an emergency room, and 24-hour/7-day-per-week access were also highly valued. Conversely, the presence of specialized care, such as physical therapy, cancer care, or dialysis, was not valued. In total, respondents were willing to pay $225 more annually to support a hospital relative to a rural health clinic., Conclusion: Rural Kentuckians value the services, convenience, and security that rural hospitals offer, though they are not willing to pay more for specialized care that may be available in larger medical treatment centers. The results also inform which attributes might be added to existing rural health facilities to make them more valuable to local residents., (© 2014 National Rural Health Association.)

Our eyes are, both literally and figuratively, windows to the world, and ophthalmic approaches offer a tremendous space for conducting research to learn more. Male/female differences in ocular health and disease are prevalent but we know far too little about root causes to design and implement diagnostic, preventive, and treatment strategies to address sex- and gender-based disparities in eye health. Herein, we discuss several ophthalmic diseases and other conditions with ocular manifestations, with a focus upon those that disproportionately affect women. Because the vast majority of biomedical research in this area comes from studies of mixed-gender populations, or of male-predominant populations, there is a pressing need for sex- and/or gender-based research at various points along the basic to clinical biomedical research continuum. Moreover, the multitude of factors that affect eye health call for a balanced look at the influence of biology, culture, and societal contributors. As clinicians, we owe our patients the best care for their needs, and that care must be derived from research that shows what is effective, for whom, and under what conditions.

Topical aciclovir cream (ACV, Zovirax Cream) containing 40% propylene glycol (PG), the optimum found for skin penetration, is clinically effective in the treatment of recurrent herpes labialis. One hundred and thirty-nine ACV generic creams were analysed and 80% of these contained less than 20% PG. From this, we hypothesised that these generics might be bioinequivalent to the innovator cream. A pilot in vitro skin permeation study compared the innovator cream with two generics containing about 15% PG. Next, 10 generics containing 0-15% PG were tested in an independent laboratory. Finally, a PG dose-ranging study was conducted in Zovirax cream base. In all studies, human skin was used and ACV analysed by LC-MS-MS. In the pilot study, the innovator cream delivered 7.5-fold more ACV than the two generics. Superiority was confirmed in the second study against all 10 ACV generic creams. By grouping the creams according to PG content, a relationship to ACV skin permeation was suggested. The PG dose effect was confirmed in the third study. These studies suggest that not all marketed ACV creams are bioequivalent to the clinically proven innovator. Given the magnitude of the differences seen, there is concern over therapeutic inequivalence of generic ACV creams to the innovator cream.

We hypothesised that the depletion of propylene glycol from topical formulations applied at clinically relevant doses (approximately mg/cm2) would limit its penetration enhancement effect. The in vitro percutaneous permeation of a model drug-loperamide hydrochloride-in formulations containing propylene glycol was therefore investigated under finite dose conditions. The flux of loperamide and propylene glycol across dermatomed human skin was measured. The first study examined the effect of topical loading of a gel containing 12% propylene glycol. The second study investigated the effect of propylene glycol content in creams containing 15 and 40%. Both studies showed a correlation between the amount of propylene glycol dosed on the skin and the amount of drug that had permeated. The substantial permeation of propylene glycol and relatively small permeation of loperamide, strongly suggests, that the time dependent permeation of the drug was due to the depletion of propylene glycol at the skin surface and not to the depletion of the drug itself. As often doses applied in in vitro skin permeation experiments do not match the intended clinical dosage-they are usually much greater-this study suggests that the penetration enhancement effect of propylene glycol can be overestimated in in vitro studies.

The solubility of penciclovir (C(10)N(5)O(3)H(17)) in a novel film formulation designed for the treatment of cold sores was determined using X-ray, thermal, microscopic and release rate techniques. Solubilities of 0.15-0.23, 0.44, 0.53 and 0.42% (w/w) resulted for each procedure. Linear calibration lines were achieved for experimentally and theoretically determined differential scanning calorimetry (DSC) and X-ray powder diffractometry (XRPD) data. Intra- and inter-batch data precision values were determined; intra values were more precise. Microscopy was additionally useful for examining crystal shape, size distribution and homogeneity of drug distribution within the film. Whereas DSC also determined melting point, XRPD identified polymorphs and release data provided relevant kinetics.

A novel diffusion cell has been constructed which allows study of membrane diffusion processes without the need for sampling of the receiver compartment, that is highly sensitive and, being based around a diode array spectrophotometer also allows for continuous, real-time recording of multi-species concentration changes in the receiving compartment. The system is controlled to operate isothermally (via a Peltier control system) at temperatures between 15 and 85 degrees C. To examine the performance of this novel design, the transfer of tetracaine from a preparation in PEG 400 (20% tetracaine in PEG 400) has been studied. The results have been used to determine flux, lag time and related parameters. The performance of the novel cell is compared with results from traditional Franz cell diffusion studies.

Isothermal microcalorimetry may be used to determine kinetic and thermodynamic parameters for chemical reactions. This paper reports rate constants, determined as a function of temperature, and the activation enthalpy for the degradation of solid benzoyl peroxide as determined by isothermal microcalorimetry. Studies were conducted on aqueous suspension phase, solid benzoyl peroxide. In addition, supporting evidence is cited from work carried out in this laboratory on the solution phase degradation of benzoyl peroxide using UV-visible spectrophotometry. The activation energy obtained by microcalorimetry was E(a)=137.8+/-6.6 kJ mol(-1) and the activation energy obtained from UV-visible spectrophotometry was E(a)=112.7+/-4.2 kJ mol(-1).

Recent developments in the analysis of microcalorimetric data output allow the possibility of determining both thermodynamic and kinetic parameters for complex reaction systems. Such experiments routinely take around 50 h, hence qualifying for the description rapid. The methods have earlier been applied to a study of the stability of benzoyl peroxide itself in aqueous suspension. This paper reports the results of isothermal microcalorimetric study of the stability of benzoyl peroxide in the presence of a wide range of excipients and in formulated materials. The results are shown to assist in formulation design, are achieved rapidly and are derived from direct experimental study of the complex systems themselves. That is, no ancillary information is required nor are the studies invasive or destructive.

Permeation of hydrocortisone acetate (HA) from supersaturated solutions was studied across a model silicone membrane. Supersaturated solutions were prepared using the cosolvent technique with propylene glycol and water (or aqueous polymer solutions) as the cosolvents. In the absence of the polymer, the flux of HA was similar at all degrees of saturation and was not significantly different from the value obtained for a saturated solution. Flux enhancement, as a result of supersaturation, was observed with all the polymers. The flux increased with increasing polymer concentration, reached a maximum and decreased at higher polymer percentages. The amount of polymer required for maximum enhancement differed for each polymer. The decrease of flux at high polymer concentrations is attributed to changes in microviscosity and a marginal increase in solubility. The infrared spectroscopic and differential scanning calorimetry data suggest that HA-polymer interactions occurred through hydrogen bonding thus explaining the proposed mechanism of the anti-nucleant properties of the polymers.

The influence of hydroxypropyl methylcellulose (HPMC), methylcellulose (MC), polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG400) on the crystallization of hydrocortisone acetate (HA) was studied. Supersaturation was created by the cosolvent technique. Spontaneous nucleation was observed when no polymer was used as the additive. In the presence of the polymer, nucleation was delayed. The nucleation time decreased with increasing supersaturation at a particular polymer concentration and increased with increasing polymer concentration at a particular supersaturation. Habit modification from a well-defined polar prismatic morphology to a wing-shaped morphology was observed when HPMC was used as the additive. The effect of PVP and PEG400 on the morphology of HA was less pronounced compared to the cellulose polymers. The mechanism of nucleation retardation by the polymers is explained in terms of association of HA with the polymer through hydrogen bonding. The growth may be inhibited by the hydrodynamic boundary layer, in which the polymers accumulate as well as by the adsorption of the polymer onto the crystal surface. The habit modification of HA by HPMC is due to different extents of adsorption on different faces of the crystal, the extent of which is dependent on the hydrogen bonding functional groups that are exposed at each face of the crystal.

A systematic investigation on the influence of two cellulose polymers, methyl cellulose (MC) and hydroxypropyl cellulose (HPMC) on supersaturation and permeation of hydrocortisone acetate (HA) is reported. Diffusion of HA from a 0.5% Carbopol gel across a model silicone membrane was investigated using the Franz-cell technique. At constant polymer concentration, the flux increases proportionally with the degree of saturation up to 4.8x but decreases thereafter. For a particular degree of supersaturation (4.8x), the flux increases with the concentration of polymer up to 1% and decreases at higher concentrations. The behaviour is found to be consistent with crystallisation experiments. The results suggest that optimisation of supersaturation and polymer content is necessary to achieve both high permeation rates and inherent stability.

The synaptic vesicle protein synaptotagmin I binds Ca2+ and is required for efficient neurotransmitter release. Here, we measure the response time of the C2 domains of synaptotagmin to determine whether synaptotagmin is fast enough to function as a Ca2+ sensor for rapid exocytosis. We report that synaptotagmin is "tuned" to sense Ca2+ concentrations that trigger neuronal exocytosis. The speed of response is unique to synaptotagmin I and readily satisfies the kinetic constraints of synaptic vesicle membrane fusion. We further demonstrate that Ca2+ triggers penetration of synaptotagmin into membranes and simultaneously drives assembly of synaptotagmin onto the base of the ternary SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment receptor) complex, near the transmembrane anchor of syntaxin. These data support a molecular model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex.

GABAA receptors, along with the receptors for acetylcholine, glycine, and serotonin, are members of a ligand-gated ion channel superfamily (Ortells and Lunt, 1995). Because of the paucity of crystallographic information for these ligand-gated channels, little is known about the structure of their binding sites or how agonist binding is transduced into channel gating. We used the substituted cysteine accessibility method to obtain secondary structural information about the GABA binding site and to systematically identify residues that line its surface. Each residue from alpha1 Y59 to K70 was mutated to cysteine and expressed with wild-type beta2 subunits in Xenopus oocytes or HEK 293 cells. The sulfhydryl-specific reagent N-biotinylaminoethyl methanethiosulfonate (MTSEA-Biotin) was used to covalently modify the cysteine-substituted residues. Receptors with cysteines substituted at positions alpha1 T60, D62, F64, R66, and S68 reacted with MTSEA-Biotin, and alpha1 F64C, R66C, and S68C were protected from reaction by agonist. We conclude that alpha1 F64, R66, and S68 line part of the GABA binding site. The alternating pattern of accessibility of consecutive engineered cysteines to reaction with MTSEA-Biotin indicates that the region from alpha1 Y59 to S68 is a beta-strand.

Biochemical and genetic studies indicate that synaptotagmin I functions as a Ca2+ sensor during synaptic vesicle exocytosis and as a membrane receptor for the clathrin adaptor complex, AP-2, during endocytosis. These functions involve the interaction of two conserved domains, C2A and C2B, with effector proteins. The C2B domain mediates Ca2+-triggered synaptotagmin oligomerization, binds AP-2 and is important for the interaction of synaptotagmin with Ca2+ channels. Here, we report that these are conserved biochemical properties: Ca2+ promoted the hetero-oligomerization of synaptotagmin I with synaptotagmins III and IV, and all three synaptotagmin isoforms bound the synprint region of the alpha1B subunit of N-type Ca2+ channels. Using chimeric and truncated C2 domains, we defined a common region of C2B that mediates oligomerization and AP-2 binding. Within this region, two adjacent lysine residues were identified that were critical for synaptotagmin oligomerization, AP-2, and synprint binding. Competition experiments demonstrated that the synprint fragment was an effective inhibitor of synaptotagmin oligomerization and also blocked binding of synaptotagmin to AP-2. In a model for the structure of C2B, the common effector binding site localized to a putative Ca2+-binding loop and a concave region formed by two beta-strands. These studies provide the first structural information regarding C2B target protein recognition and provide the means to selectively disrupt synaptotagmin-effector interactions for functional studies.

Synaptotagmin 1 binds Ca2+ and membranes via its C2A-domain and plays an essential role in excitation-secretion coupling. In this study, we sought to identify Ca2+- and membrane-induced local conformational changes in the C2A-domain of synaptotagmin and to delineate the C2A-lipid binding interface. To address these questions native phenylalanine residues were replaced, at each face of the domain, with tryptophan reporters. Changes in tryptophanyl fluorescence indicated that Ca2+ induced long range conformational changes throughout C2A, including regions distant from an established Ca2+-binding site. Addition of liposomes resulted in Ca2+-dependent increases in the fluorescence of tryptophans 193, 231, and 234. Only the tryptophan residues at positions 234 and 231, which lie within a Ca2+-binding loop of C2A, exhibited liposome-induced blue shifts in their emission spectra. Quenching experiments, using membrane-imbedded doxyl spin labels, revealed that tryptophan residues 231 and 234 penetrated lipid bilayers. These data delineate the lipid binding interface of C2A and provide the first evidence for adjacent Ca2+- and lipid-binding sites within a C2-domain. The penetration of C2A into membranes may function to bring components of the fusion machinery into contact with the lipid bilayer to initiate exocytosis.

PO-B was originally characterized as a transcriptional regulatory factor of the pro-opiomelanocortin (POMC) gene; however, it has become increasingly clear that this protein may be active in tissues outside the pituitary, since it is present in diverse cell types, including differentiated HL-60 promyelocytic leukemia cells. We previously showed that PO-B DNA-binding is progressively induced during differentiation of promyelomonocytic leukemic HL-60 cells to the macrophage-like lineage (with phorbol esters). We now report that PO-B DNA-binding in HL-60 cells is similarly induced during differentiation to the granulocytic lineage (with either retinoic acid or dimethylsulfoxide). Either a genetic or pharmacologic blockade of HL-60 differentiation prohibited these inductive effects. These studies have prompted our interest in the dynamics of other transcription factor changes during HL-60 differentiation. Of these, we observed that another transcription factor (AP-1) is also robustly induced at the DNA-binding level during macrophage-like HL-60 differentiation, but not during granulocytic differentiation. Conversely, the DNA-binding of the transcription factor AP-2 was slightly reduced by TPA-induced HL-60 differentiation but unchanged during granulocyte differentiation. From these data, we conclude that the induction of PO-B DNA binding is a general marker of HL-60 myelomonocytic differentiation, but that qualitative aspects of the induction of additional distinct transcription factors, such as AP-1, may contribute to lineage-specific determinants of cell fate.

Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments. A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades. Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication. Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria. In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1-2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication. Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network.

Mitochondria are essential organelles in all eukaryotic cells where cellular ATP is generated through the process of oxidative phosphorylation. Protein components of the respiratory assembly are gene products of both mitochondrial and nuclear genes. The mitochondrial genome itself encodes several protein and nucleic acid components required for such oxidative phosphorylative processes, but the vast majority of genes encoding respiratory chain components are nuclear. Similarly, the processes of replication and transcription of mitochondrial DNA rely exclusively upon RNA and protein species encoded by nuclear genes. We have analyzed two key nuclear-encoded proteins involved in mitochondrial DNA replication and transcription as a function of the presence or absence of mitochondrial DNA. Mitochondrial DNA polymerase (DNA polymerase gamma), the nuclear-encoded enzyme which synthesizes mtDNA, is expressed and translated in cells devoid of mitochondrial DNA itself. In contrast, mitochondrial transcription factor A protein levels are tightly linked to the mtDNA status of the cell. These results demonstrate that the DNA polymerase gamma protein is stable in the absence of mitochondrial DNA, and that there appears to be no regulatory mechanism present in these cells to alter levels of this protein in the complete absence of mitochondrial DNA. Alternatively, it is possible that this enzyme plays an additional, as yet undefined, role in the cell, thereby mandating its continued production.

RNase MRP is a ribonucleoprotein originally identified on the basis of its ability to cleave RNA endonucleolytically from origins of mitochondrial DNA replication, rendering it a likely candidate for a role in priming leading-strand synthesis of mtDNA. In addition, a nuclear role for RNase MRP has been identified in yeast (Saccharomyces cerevisiae) ribosomal RNA processing. Consistent with a duality of function, RNase MRP has been localized to both mitochondria and nucleoli by in situ techniques. The RNA component of this ribonucleoprotein has been characterized from several different species. We previously cloned the gene for Xenopus laevis MRP RNA and showed that RNase MRP RNA is differentially expressed during amphibian development; in addition, the microinjected X. laevis RNase MRP RNA gene is correctly and efficiently transcribed in vivo. This article presents an analysis of the intracellular movement of in vivo-transcribed RNase MRP RNA in microinjected mature X. laevis oocytes. Although X. laevis MRP RNA is assembled into a ribonucleoprotein form and transported in an expected manner, human and mouse MRP RNAs exhibit markedly different transport patterns even though they are highly conserved in primary sequence. Furthermore, the only currently assigned protein (Th autoantigen) binding site in MRP RNA can be deleted without loss of nuclear export capacity. These results indicate that subtle determinants must exist for nucleocytoplasmic partitioning of this RNP and that the conserved Th autoantigen binding region appears unnecessary for the transit of in vivo-transcribed MRP RNA to the cytoplasm of mature X. laevis oocytes.

RNase MRP is a ribonucleoprotein endoribonuclease found predominantly in nucleoli, but which has been associated with mitochondria and mitochondrial RNA processing. In order to analyze the intracellular localization of specific RNA components of ribonucleoproteins of this type, a whole-mount method for in situ hybridization in Xenopus laevis oocytes was employed. Results with specific probes (for both mitochondrial and nonmitochondrial RNAs) indicate that this procedure is generally effective for the detection of a variety of nucleic acids that reside in different cellular compartments. Probes used to detect the endogenous RNA component of RNase MRP (MRP RNA) during X. laevis oogenesis revealed a continuous nuclear signal as well as a possible dual localization of MRP RNA in nucleoli and mitochondria at developmental stages temporally consistent with both ribosomal and mitochondrial biogenesis. Genomic DNA encoding MRP RNA was injected into the nuclei of stage VI oocytes and correctly transcribed. The in vivo-transcribed RNA was properly assembled with at least some of its cognate proteins as demonstrated by immunoprecipitation with specific autoantiserum. In addition, detectable levels of the RNA were exported to the cytoplasm. This whole-mount procedure has permitted us to identify MRP RNA in situ at different developmental time points as well as during transcription of the injected gene, and suggests differential localization of MRP RNA during oogenesis consistent with its proposed function in both mitochondria and nucleoli.