Allosteric activation of VCP, an AAA unfoldase, by small molecule mimicry.

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry.

The GenoVA study: Equitable implementation of a pragmatic randomized trial of polygenic-risk scoring in primary care.

Polygenic risk scores (PRSs) hold promise for disease risk assessment and prevention. The Genomic Medicine at Veterans Affairs (GenoVA) Study is addressing three main challenges to the clinical implementation of PRSs in preventive care: defining and determining their clinical utility, implementing them in time-constrained primary care settings, and countering their potential to exacerbate healthcare disparities. The study processes used to test patients, report their PRS results to them and their primary care providers (PCPs), and promote the use of those results in clinical decision-making are modeled on common practices in primary care. The following diseases were chosen for their prevalence and familiarity to PCPs: coronary artery disease; type 2 diabetes; atrial fibrillation; and breast, colorectal, and prostate cancers. A randomized clinical trial (RCT) design and primary outcome of time-to-new-diagnosis of a target disease bring methodological rigor to the question of the clinical utility of PRS implementation. The study's pragmatic RCT design enhances its relevance to how PRS might reasonably be implemented in primary care. Steps the study has taken to promote health equity include the thoughtful handling of genetic ancestry in PRS construction and reporting and enhanced recruitment strategies to address underrepresentation in research participation. To date, enhanced recruitment efforts have been both necessary and successful: participants of underrepresented race and ethnicity groups have been less likely to enroll in the study than expected but ultimately achieved proportional representation through targeted efforts. The GenoVA Study experience to date offers insights for evaluating the clinical utility of equitable PRS implementation in adult primary care.

Classification of PTEN germline non-truncating variants: a new approach to interpretation.

BACKGROUND: PTEN hamartoma tumour syndrome (PHTS) encompasses distinct syndromes, including Cowden syndrome resulting from PTEN pathogenic variants. Missense variants account for 30% of PHTS cases, but their classification remains challenging. To address these difficulties, guidelines were published by the Clinical Genome Resource PTEN Variant Curation Expert Panel. METHODS: Between 2010 and 2020, the Bergonie Institute reference laboratory identified 76 different non-truncating PTEN variants in 166 patients, 17 of which have not previously been reported. Variants were initially classified following the current guidelines. Subsequently, a new classification method was developed based on four main criteria: functional exploration, phenotypic features and familial segregation, in silico modelling, and allelic frequency. RESULTS: This new method of classification is more discriminative and reclassifies 25 variants, including 8 variants of unknown significance. CONCLUSION: This report proposes a revision of the current PTEN variant classification criteria which at present rely on functional tests evaluating only the phosphatase activity of PTEN and apply a particularly stringent clinical PHTS score.The classification of non-truncating variants of PTEN is facilitated by taking into consideration protein stability for variants with intact phosphatase activity, clinical and segregation criteria adapted to the phenotypic variability of PHTS and by specifying the allelic frequency of variants in the general population. This novel method of classification remains to be validated in a prospective cohort.

Sodium-glucose cotransporter 2 inhibition does not improve the acute pressure natriuresis response in rats with type 1 diabetes.

NEW FINDINGS: What is the central question of this study? Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular risk in patients with both diabetic and non-diabetic kidney disease: can SGLT2 inhibition improve renal pressure natriuresis (PN), an important mechanism for long-term blood pressure control, which is impaired in type 1 diabetes mellitus (T1DM)? What is the main finding and its importance? The SGLT2 inhibitor dapagliflozin did not enhance the acute in vivo PN response in either healthy or T1DM Sprague-Dawley rats. The data suggest that the mechanism underpinning the clinical benefits of SGLT2 inhibitors on health is unlikely to be due to an enhanced natriuretic response to increased blood pressure. ABSTRACT: Type 1 diabetes mellitus (T1DM) leads to serious complications including premature cardiovascular and kidney disease. Hypertension contributes importantly to these adverse outcomes. The renal pressure natriuresis (PN) response, a key regulator of blood pressure (BP), is impaired in rats with T1DM as tubular sodium reabsorption fails to down-regulate with increasing BP. We hypothesised that sodium-glucose cotransporter 2 (SGLT2) inhibitors, which reduce cardiovascular risk in kidney disease, would augment the PN response in T1DM rats. Non-diabetic or T1DM (35-50 mg/kg streptozotocin i.p.) adult male Sprague-Dawley rats were anaesthetised (thiopental 50 mg/kg i.p.) and randomised to receive either dapagliflozin (1 mg/kg i.v.) or vehicle. Baseline sodium excretion was measured and then BP was increased by sequential arterial ligations to induce the PN response. In non-diabetic animals, the natriuretic and diuretic responses to increasing BP were not augmented by dapagliflozin. Dapagliflozin induced glycosuria, but this was not influenced by BP. In T1DM rats the PN response was impaired. Dapagliflozin again increased urinary glucose excretion but did not enhance PN. Inhibition of SGLT2 does not enhance the PN response in rats, either with or without T1DM. SGLT2 makes only a minor contribution to tubular sodium reabsorption and does not contribute to the impaired PN response in T1DM.

Psychological resilience and neurodegenerative risk: A connectomics-transcriptomics investigation in healthy adolescent and middle-aged females.

Adverse life events can inflict substantial long-term damage, which, paradoxically, has been posited to stem from initially adaptative responses to the challenges encountered in one's environment. Thus, identification of the mechanisms linking resilience against recent stressors to longer-term psychological vulnerability is key to understanding optimal functioning across multiple timescales. To address this issue, our study tested the relevance of neuro-reproductive maturation and senescence, respectively, to both resilience and longer-term risk for pathologies characterised by accelerated brain aging, specifically, Alzheimer's Disease (AD). Graph theoretical and partial least squares analyses were conducted on multimodal imaging, reported biological aging and recent adverse experience data from the Lifespan Human Connectome Project (HCP). Availability of reproductive maturation/senescence measures restricted our investigation to adolescent (N = 178) and middle-aged (N = 146) females. Psychological resilience was linked to age-specific brain senescence patterns suggestive of precocious functional development of somatomotor and control-relevant networks (adolescence) and earlier aging of default mode and salience/ventral attention systems (middle adulthood). Biological aging showed complementary associations with the neural patterns relevant to resilience in adolescence (positive relationship) versus middle-age (negative relationship). Transcriptomic and expression quantitative trait locus data analyses linked the neural aging patterns correlated with psychological resilience in middle adulthood to gene expression patterns suggestive of increased AD risk. Our results imply a partially antagonistic relationship between resilience against proximal stressors and longer-term psychological adjustment in later life. They thus underscore the importance of fine-tuning extant views on successful coping by considering the multiple timescales across which age-specific processes may unfold.

An Empiricist's Guide to Using Ecological Theory.

AbstractA scientific understanding of the biological world arises when ideas about how nature works are formalized, tested, refined, and then tested again. Although the benefits of feedback between theoretical and empirical research are widely acknowledged by ecologists, this link is still not as strong as it could be in ecological research. This is in part because theory, particularly when expressed mathematically, can feel inaccessible to empiricists who may have little formal training in advanced math. To address this persistent barrier, we provide a general and accessible guide that covers the basic, step-by-step process of how to approach, understand, and use ecological theory in empirical work. We first give an overview of how and why mathematical theory is created, then outline four specific ways to use both mathematical and verbal theory to motivate empirical work, and finally present a practical tool kit for reading and understanding the mathematical aspects of ecological theory. We hope that empowering empiricists to embrace theory in their work will help move the field closer to a full integration of theoretical and empirical research.

Males and females differ in the regulation and engagement of, but not requirement for, protein degradation in the amygdala during fear memory formation.

Over the last decade, strong evidence has emerged that protein degradation mediated by the ubiquitin-proteasome system is critical for fear memory formation in the amygdala. However, this work has been done primarily in males, leaving unanswered questions about whether females also require protein degradation during fear memory formation. Here, we found that male and female rats differed in their engagement and regulation of, but not need for, protein degradation in the amygdala during fear memory formation. Male, but not female, rats had increased protein degradation in the nuclei of amygdala cells after fear conditioning. Conversely, females had elevated baseline levels of overall ubiquitin-proteasome activity in amygdala nuclei. Gene expression and DNA methylation analyses identified that females had increased baseline expression of the ubiquitin coding gene Uba52, which had increased DNA 5-hydroxymethylation (5hmc) in its promoter region, indicating a euchromatin state necessary for increased levels of ubiquitin in females. Consistent with this, persistent CRISPR-dCas9 mediated silencing of Uba52 and proteasome subunit Psmd14 in the amygdala reduced baseline protein degradation levels and impaired fear memory in male and female rats, while enhancing baseline protein degradation in the amygdala of both sexes promoted fear memory formation. These results suggest that while both males and females require protein degradation in the amygdala for fear memory formation, they differ in their baseline regulation and engagement of this process following learning. These results have important implications for understanding the etiology of sex-related differences in fear memory formation.

Empirical evidence that metabolic theory describes the temperature dependency of within-host parasite dynamics.

The complexity of host-parasite interactions makes it difficult to predict how host-parasite systems will respond to climate change. In particular, host and parasite traits such as survival and virulence may have distinct temperature dependencies that must be integrated into models of disease dynamics. Using experimental data from Daphnia magna and a microsporidian parasite, we fitted a mechanistic model of the within-host parasite population dynamics. Model parameters comprising host aging and mortality, as well as parasite growth, virulence, and equilibrium abundance, were specified by relationships arising from the metabolic theory of ecology. The model effectively predicts host survival, parasite growth, and the cost of infection across temperature while using less than half the parameters compared to modeling temperatures discretely. Our results serve as a proof of concept that linking simple metabolic models with a mechanistic host-parasite framework can be used to predict temperature responses of parasite population dynamics at the within-host level.

Longitudinal study of the housing and mental health outcomes of tenants appearing in eviction court.

PURPOSE: Millions of people are evicted from rental properties in the U.S. annually, but little is known about them and their mental health. This study followed a cohort of eviction court participants over time and assessed their housing and mental health outcomes. METHODS: One hundred and twenty-one tenants were recruited from an eviction court in New Haven, Connecticut, and their housing, mental health, and psychosocial status were assessed at baseline, 1, 3, 6, and 9 months following their encounter with the court. Inverse probability weighting was used for missing data. RESULTS: At baseline, 42% of participants had appeared in eviction court before, 28% had experienced eviction, and 44% had been previously homeless. In addition, 39% screened positive for generalized anxiety disorder, 37% for posttraumatic stress disorder, 33% for major depressive disorder, and 17% reported suicidal ideation. At follow-up, participants experienced increased days of housing instability and homelessness over time with some persistent mental health symptoms. Less than one-quarter of participants received any mental health treatment during the 9-month follow-up period. About 54% of participants followed reported that they had to change their residence after their court appearance consistent with court records. Participants who had an eviction-related move experienced greater housing instability over time than participants who did not. CONCLUSION: Together, these findings suggest that there is a sizable subgroup of adults who present to eviction court with persistent housing and mental health issues who do not receive adequate assistance in addressing these issues.

Experimental evidence of warming-induced disease emergence and its prediction by a trait-based mechanistic model.

Predicting the effects of seasonality and climate change on the emergence and spread of infectious disease remains difficult, in part because of poorly understood connections between warming and the mechanisms driving disease. Trait-based mechanistic models combined with thermal performance curves arising from the metabolic theory of ecology (MTE) have been highlighted as a promising approach going forward; however, this framework has not been tested under controlled experimental conditions that isolate the role of gradual temporal warming on disease dynamics and emergence. Here, we provide experimental evidence that a slowly warming host-parasite system can be pushed through a critical transition into an epidemic state. We then show that a trait-based mechanistic model with MTE functional forms can predict the critical temperature for disease emergence, subsequent disease dynamics through time and final infection prevalence in an experimentally warmed system of Daphnia and a microsporidian parasite. Our results serve as a proof of principle that trait-based mechanistic models using MTE subfunctions can predict warming-induced disease emergence in data-rich systems-a critical step towards generalizing the approach to other systems.

Sierra Nevada mountain lake microbial communities are structured by temperature, resources and geographic location.

Warming, eutrophication (nutrient fertilization) and brownification (increased loading of allochthonous organic matter) are three global trends impacting lake ecosystems. However, the independent and synergistic effects of resource addition and warming on autotrophic and heterotrophic microorganisms are largely unknown. In this study, we investigate the independent and interactive effects of temperature, dissolved organic carbon (DOC, both allochthonous and autochthonous) and nitrogen (N) supply, in addition to the effect of spatial variables, on the composition, richness, and evenness of prokaryotic and eukaryotic microbial communities in lakes across elevation and N deposition gradients in the Sierra Nevada mountains of California, USA. We found that both prokaryotic and eukaryotic communities are structured by temperature, terrestrial (allochthonous) DOC and latitude. Prokaryotic communities are also influenced by total and aquatic (autochthonous) DOC, while eukaryotic communities are also structured by nitrate. Additionally, increasing N availability was associated with reduced richness of prokaryotic communities, and both lower richness and evenness of eukaryotes. We did not detect any synergistic or antagonistic effects as there were no interactions among temperature and resource variables. Together, our results suggest that (a) organic and inorganic resources, temperature, and geographic location (based on latitude and longitude) independently influence lake microbial communities; and (b) increasing N supply due to atmospheric N deposition may reduce richness of both prokaryotic and eukaryotic microbes, probably by reducing niche dimensionality. Our study provides insight into abiotic processes structuring microbial communities across environmental gradients and their potential roles in material and energy fluxes within and between ecosystems.

Predators drive community reorganization during experimental range shifts.

Increased global temperatures caused by climate change are causing species to shift their ranges and colonize new sites, creating novel assemblages that have historically not interacted. Species interactions play a central role in the response of ecosystems to climate change, but the role of trophic interactions in facilitating or preventing range expansions is largely unknown. The goal of our study was to understand how predators influence the ability of range-shifting prey to successfully establish in newly available habitat following climate warming. We hypothesized that fish predation facilitates the establishment of colonizing zooplankton populations, because fish preferentially consume larger species that would otherwise competitively exclude smaller-bodied colonists. We conducted a mesocosm experiment with zooplankton communities and their fish predators from lakes of the Sierra Nevada Mountains in California, USA. We tested the effect of fish predation on the establishment and persistence of a zooplankton community when introduced in the presence of higher- and lower-elevation communities at two experimental temperatures in field mesocosms. We found that predators reduce the abundance of larger-bodied residents from the alpine and facilitate the establishment of new lower-elevation species. In addition, fish predation and warming independently reduced the average body size of zooplankton by up to 30%. This reduction in body size offset the direct effect of warming-induced increases in population growth rates, leading to no net change in zooplankton biomass or trophic cascade strength. We found support for a shift to smaller species with climate change through two mechanisms: (a) the direct effects of warming on developmental rates and (b) size-selective predation that altered the identity of species' that could colonize new higher elevation habitat. Our results suggest that predators can amplify the rate of range shifts by consuming larger-bodied residents and facilitating the establishment of new species. However, the effects of climate warming were dampened by reducing the average body size of community members, leading to no net change in ecosystem function, despite higher growth rates. This work suggests that trophic interactions play a role in the reorganization of regional communities under climate warming.

Optimization of a series of potent, selective and orally bioavailable SYK inhibitors.

Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.

"We're, Like, the Most Unhealthy People in the Country": Using an Equity Lens to Reduce Barriers to Healthy Food Access in Rural Appalachia.

INTRODUCTION: Obesity disproportionately affects rural communities, and Appalachia has some of the highest obesity rates in the nation. Successful policy, systems, and environmental (PSE) interventions to reduce obesity must reflect the circumstances of the population. We used a health equity lens to identify barriers and facilitators for healthy food access in Martin County, Kentucky, to design interventions responsive to social, cultural, and historical contexts. METHODS: We conducted 5 focus groups in Martin County, Kentucky, in fall 2019 to obtain perspectives on the local food system and gauge acceptability of PSE interventions. We used grounded theory to identify perceived barriers and facilitators for healthy eating. RESULTS: Thirty-four adults (27 women; median age, 46 years) participated in 5 groups. One prominent theme was declining interest in farming; many participants believed this decline was generational. One participant noted, "Most of my adult male relatives worked in the coal mines, and they worked 6 days a week. . . . My grandpa had the garden, but then my dad's generation is the one quit gardening." Another shared, "You would probably have to have someone to teach [gardening]." Instead of enhancing farmers markets, participants suggested building community capacity for home gardens to increase vegetable consumption. CONCLUSION: Our findings demonstrate the importance of obtaining community input on the development of PSE interventions to mitigate inequities in obesity. Although farmers market interventions were deemed not feasible, other solutions to enhance access to produce were identified. Developers of community-responsive PSE interventions to improve healthy eating in rural, food-insecure locations should consider using an equity-oriented prevention framework to ensure acceptable interventions.

Job Burnout Among Mental Health Staff at a Veterans Affairs Psychosocial Rehabilitation Center.

Mental health providers who serve clients with severe mental illness may be particularly prone to job burnout given the nature of the work. This study examined levels of job burnout among mental health providers who serve clients with severe mental illness. Forty-two mental health staff at a Veterans Affairs psychosocial rehabilitation center completed an online survey that assessed burnout and work-life balance. Maslach Burnout Inventory (MBI) scores were compared to published scores of workers in other professions. Participants reported moderate MBI Emotional Exhaustion, Depersonalization, and Personal Accomplishment scores and overall had lower burnout scores than other healthcare providers and service workers. Being younger and white were associated with higher MBI Emotional Exhaustion scores. These findings suggest job burnout among mental health staff is a concern that should be closely monitored even among staff who express a sense of personal accomplishment from the work.

Glucocorticoid receptor activation stimulates the sodium-chloride cotransporter and influences the diurnal rhythm of its phosphorylation.

The sodium-chloride cotransporter (NCC) in the distal convoluted tubule contributes importantly to sodium balance and blood pressure (BP) regulation. NCC phosphorylation determines transport activity and has a diurnal rhythm influenced by glucocorticoids. Disturbing this rhythm induces "nondipping" BP, an abnormality that increases cardiovascular risk. The receptor through which glucocorticoids regulate NCC is not known. In this study, we found that acute administration of corticosterone to male C57BL6 mice doubled NCC phosphorylation without affecting total NCC abundance in both adrenalectomized and adrenal-intact mice. Corticosterone also increased the whole kidney expression of canonical clock genes: period circadian protein homolog 1 (Per1), Per2, cryptochrome 1, and aryl hydrocarbon receptor nuclear translocator-like protein 1. In adrenal-intact mice, chronic blockade of glucocorticoid receptor (GR) with RU486 did not change total NCC but prevented corticosterone-induced NCC phosphorylation and activation of clock genes. Blockade of mineralocorticoid receptor (MR) with spironolactone reduced the total pool of NCC but did not affect stimulation by corticosterone. The diurnal rhythm of NCC phosphorylation, measured at 6-h intervals, was blunted by chronic GR blockade, and a similar dampening of diurnal variation was seen in GR heterozygous null mice. These effects on NCC phosphorylation did not reflect altered rhythmicity of plasma corticosterone or serum and glucocorticoid-induced kinase 1 activity. Both mineralocorticoids and glucocorticoids emerge as regulators of NCC, acting via distinct receptor pathways. MR activation provides maintenance of the NCC protein pool; GR activation dynamically regulates NCC phosphorylation and establishes the diurnal rhythm of NCC activity. This study has implications for circadian BP homeostasis, particularly in individuals with abnormal glucocorticoid signaling as is found in chronic stress and corticosteroid therapy.

Development of a quantification method for adenosine in tumors by LC-MS/MS with dansyl chloride derivatization.

Adenosine is known to be an important signaling molecule in many physiological processes and has recently been shown to be an important molecule in oncology. A fit for purpose method has been developed for the quantification of adenosine in murine tumor samples using pre-column derivatization and liquid chromatography-mass spectrometry (LC-MS/MS). To overcome adenosine quantification challenges, derivatization with dansyl chloride was employed. This derivatization technique, following protein precipitation and liquid-liquid extraction, improved the sensitivity and selectivity of adenosine in tumor samples through the reduction of endogenous interference and matrix effects. This method utilizes a mouse plasma calibration curve, qualified over a range of 0.019 µM-37 µM. The 15 min derivatization incubation time and 1 min chromatographic run time allow for higher throughput. The following established method overcomes challenges associated with the quantification of low molecular weight, polar, endogenous molecules, such as adenosine, using derivatization and LC-MS/MS. With the additional analysis of murine tumors, this method will contribute to the understanding of the impact adenosine plays in the tumor microenvironment and the bearing it has on targeted cancer therapies.

Experiences with interferon-free hepatitis C therapies: addressing barriers to adherence and optimizing treatment outcomes.

BACKGROUND: Millions of Americans are living with hepatitis C, the leading cause of liver disease in the United States. Medication treatment can cure hepatitis C. We sought to understand factors that contribute to hepatitis C treatment completion from the perspectives of patients and providers. METHODS: We conducted semi-structured interviews at three Veterans Affairs Medical Centers. Patients were asked about their experiences with hepatitis C treatments and perspectives on care. Providers were asked about observations regarding patient responses to medications and perspectives about factors resulting in treatment completion. Transcripts were analyzed using a grounded thematic approach-an inductive analysis that lets themes emerge from the data. RESULTS: Contributors to treatment completion included Experience with Older Treatments, Hope for Improvement, Symptom Relief, Tailored Organized Routines, and Positive Patient-Provider Relationship. Corresponding barriers also emerged, including pill burden and skepticism about treatment effectiveness and safety. CONCLUSION: Despite the improved side-effect profile of newer HCV medications, multiple barriers to treatment completion remain. However, providers and patients were able to identify avenues for addressing such barriers.

The LOTUS: A Journey to Value-Based, Patient-Centered Care.

In response to the merger of our 248-bed community hospital with a new health system, a multidisciplinary team began a journey of holistic transformation via the evolution of a new rounding process called Leadership, Ownership, Transformation, Unity, and Sustainability (LOTUS) in the 20-bed ICU. Morphing from a hierarchical practice structure with limited engagement of multidisciplinary members, the LOTUS initiative (named for the blossom whose petals surround its core, the patient) afforded each discipline (petal) an equal voice and allowed a once-fragmented team to work cohesively, collaboratively, and at the highest level of the scope of practice for each discipline, thus affording expert guidance during care planning while providing a method to collect quality metrics. LOTUS allows us to view our patients in a new way as we refocused goal determination on patients and their families. The restructuring and evolution into a high-functioning team was targeted with the goal of enhancing quality critical care for patients, which, in the literature, has correlated with improved patient safety and decreased mortality and ICU length of stay.

Pulsatility of glucocorticoid hormones in pregnancy: Changes with gestation and obesity.

OBJECTIVE: Hypothalamic-pituitary-adrenal axis (HPA) activity is decreased in obese pregnancy and associates with increased foetal size. Pulsatile release of glucocorticoid hormones regulates their action in target tissues. Glucocorticoids are essential for normal foetal growth, but little is known about glucocorticoid pulsatility in pregnancy. We aimed to investigate the ultradian rhythm of glucocorticoid secretion during obese and lean pregnancy and nonpregnancy. DESIGN: Serum cortisol, cortisone, corticosterone and 11-dehydrocorticosterone were measured by LC-MS/MS from samples obtained at 10-minute intervals between 08.00-11.00 hours and 16.00-19.00 hours, from 8 lean (BMI <25 kg/m2 ) and 7 obese (BMI > 35 kg/m2 ) pregnant women between 16-24 weeks gestation and again at 30-36 weeks), and nonpregnant controls (lean n = 3, obese n = 4) during the luteal phase of their menstrual cycle. Interstitial fluid cortisol was measured by ELISA, from samples obtained using a portable microdialysis and automated collection device at 20-minute intervals over 24 hours. RESULTS: Serum cortisol AUC, highest peak and lowest trough increased significantly with gestation in lean and obese pregnant compared with nonpregnant subjects. Pulsatility of cortisol was detected in interstitial fluid. In pregnant subjects, interstitial fluid pulse frequency was significantly lower with advancing gestation in obese, but not in lean. CONCLUSIONS: We demonstrate cortisol pulsatility in interstitial fluid. Pulse frequency is altered with increased gestation and BMI. This may be a novel mechanism to explain decreased HPA activity in obese pregnancy.