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Familial partial lipodystrophy (FPLD) is a heterogenous group of syndromes associated with a high prevalence of cardiometabolic diseases. Prior work has proposed DEXA-derived fat mass ratio (FMR), defined as trunk fat percentage divided by leg fat percentage, as a biomarker of FPLD, but this metric has not previously been characterized in large cohort studies. We set out to 1) understand the cardiometabolic burden of individuals with high FMR in up to 40,796 participants in the UK Biobank and 9,408 participants in the Fenland study, 2) characterize the common variant genetic underpinnings of FMR, and 3) build and test a polygenic predictor for FMR. Participants with high FMR were at higher risk for type 2 diabetes (odds ratio [OR] 2.30, P = 3.5 × 10-41) and metabolic dysfunction-associated liver disease or steatohepatitis (OR 2.55, P = 4.9 × 10-7) in UK Biobank and had higher fasting insulin (difference 19.8 pmol/L, P = 5.7 × 10-36) and fasting triglycerides (difference 36.1 mg/dL, P = 2.5 × 10-28) in the Fenland study. Across FMR and its component traits, 61 conditionally independent variant-trait pairs were discovered, including 13 newly identified pairs. A polygenic score for FMR was associated with an increased risk of cardiometabolic diseases. This work establishes the cardiometabolic significance of high FMR, a biomarker for FPLD, in two large cohort studies and may prove useful in increasing diagnosis rates of patients with metabolically unhealthy fat distribution to enable treatment or a preventive therapy., (© 2024 by the American Diabetes Association.)

Background: Many adverse events are identified as nursing-sensitive indicators (NSIs) and have evidence-based care bundles known to reduce risk of occurrence. Kamishibai cards are a tool from the manufacturing industry used for practice auditing and improvements. Use of Kamishibai cards is believed to be common in the healthcare setting, but true evidence-based guidelines do not yet exist to guide their implementation., Aims: The aim of this integrative review was to identify best practices around the implementation of Kamishibai cards in the healthcare setting for improvement in NSI-associated outcomes., Methods: Eleven nurses at three facilities worked through the evidence using the Johns Hopkins Evidence-Based Practice Model., Results: Ten articles were included for this review. Broad themes included direct observation with non-punitive and timely feedback, clearly visualized results, use of evidence-based care bundles, pre-implementation education, and both leadership and frontline-staff involvement. All facilities showed improvement in NSI-associated outcomes after the implementation of K-cards., Linking Action to Action: In health care, K-cards can be implemented and designed with additional focus on the bundles of care they are intended to audit and staff support, but further evidence would better define guidelines around implementation., (© 2024 Sigma Theta Tau International.)

Adaptations to infectious and dietary pressures shape mammalian physiology and disease risk. How such adaptations affect sex-biased diseases remains insufficiently studied. In this study, we show that sex-dependent hepatic gene programs confer a robust (~300%) survival advantage for male mice during lethal bacterial infection. The transcription factor B cell lymphoma 6 (BCL6), which masculinizes hepatic gene expression at puberty, is essential for this advantage. However, protection by BCL6 protein comes at a cost during conditions of dietary excess, which result in overt fatty liver and glucose intolerance in males. Deleting hepatic BCL6 reverses these phenotypes but markedly lowers male survival during infection, thus establishing a sex-dependent trade-off between host defense and metabolic systems. Our findings offer strong evidence that some current sex-biased diseases are rooted in ancient evolutionary trade-offs between immunity and metabolism.

Objective: This study examined whether body shape and composition obtained by three-dimensional optical (3DO) scanning improved the prediction of metabolic syndrome (MetS) prevalence compared with BMI and demographics., Methods: A diverse ambulatory adult population underwent whole-body 3DO scanning, blood tests, manual anthropometrics, and blood pressure assessment in the Shape Up! Adults study. MetS prevalence was evaluated based on 2005 National Cholesterol Education Program criteria, and prediction of MetS involved logistic regression to assess (1) BMI, (2) demographics-adjusted BMI, (3) 85 3DO anthropometry and body composition measures, and (4) BMI + 3DO + demographics models. Receiver operating characteristic area under the curve (AUC) values were generated for each predictive model., Results: A total of 501 participants (280 female) were recruited, with 87 meeting the criteria for MetS. Compared with the BMI model (AUC = 0.819), inclusion of age, sex, and race increased the AUC to 0.861, and inclusion of 3DO measures further increased the AUC to 0.917. The overall integrated discrimination improvement between the 3DO + demographics and the BMI model was 0.290 (p < 0.0001) with a net reclassification improvement of 0.214 (p < 0.0001)., Conclusions: Body shape measures from an accessible 3DO scan, adjusted for demographics, predicted MetS better than demographics and/or BMI alone. Risk classification in this population increased by 29% when using 3DO scanning., (© 2022 The Obesity Society.)

Background: Low-carbohydrate ketogenic diets are a viable method to lose weight that have regained popularity in recent years. Technology in the form of mobile health (mHealth) apps allows for scalable and remote delivery of such dietary interventions and are increasingly being used by the general population without direct medical supervision. However, it is currently unknown which factors related to app use and user behavior are associated with successful weight loss., Objective: First, to describe and characterize user behavior, we aim to examine characteristics and user behaviors over time of participants who were enrolled in a remotely delivered clinical weight loss trial that tested an mHealth ketogenic diet app paired with a breath acetone biofeedback device. Second, to identify variables of importance to weight loss at 12 weeks that may offer insight for future development of dietary mHealth interventions, we aim to explore which app- and adherence-related user behaviors characterized successful weight loss., Methods: We analyzed app use and self-reported questionnaire data from 75 adults with overweight or obesity who participated in the intervention arm of a previous weight loss study. We examined data patterns over time through linear mixed models and performed correlation, linear regression, and causal mediation analyses to characterize diet-, weight-, and app-related user behavior associated with weight loss., Results: In the context of a low-carbohydrate ketogenic diet intervention delivered remotely through an mHealth app paired with a breath acetone biofeedback device, self-reported dietary adherence seemed to be the most important factor to predict weight loss (β=-.31; t 54 =-2.366; P=.02). Furthermore, self-reported adherence mediated the relationship between greater app engagement (from c=-0.008, 95% CI -0.014 to -0.0019 to c'=-0.0035, 95% CI -0.0094 to 0.0024) or higher breath acetone levels (from c=-1.34, 95% CI -2.28 to -0.40 to c'=-0.40, 95% CI -1.42 to 0.62) and greater weight loss, explaining a total of 27.8% and 28.8% of the variance in weight loss, respectively. User behavior (compliance with weight measurements and app engagement) and adherence-related aspects (breath acetone values and self-reported dietary adherence) over time differed between individuals who achieved a clinically significant weight loss of >5% and those who did not., Conclusions: Our in-depth examination of app- and adherence-related user behaviors offers insight into factors associated with successful weight loss in the context of mHealth interventions. In particular, our finding that self-reported dietary adherence was the most important metric predicting weight loss may aid in the development of future mHealth dietary interventions., Trial Registration: ClinicalTrials.gov NCT04165707; https://clinicaltrials.gov/ct2/show/NCT04165707., International Registered Report Identifier (irrid): RR2-10.2196/19053., (©Kaja Falkenhain, Sean R Locke, Dylan A Lowe, Terry Lee, Joel Singer, Ethan J Weiss, Jonathan P Little. Originally published in JMIR mHealth and uHealth (https://mhealth.jmir.org), 14.03.2022.)

Background: The accurate assessment of total body and regional body circumferences, volumes, and compositions are critical to monitor physical activity and dietary interventions, as well as accurate disease classifications including obesity, metabolic syndrome, sarcopenia, and lymphedema. We assessed body composition and anthropometry estimates provided by a commercial 3-dimensional optical (3DO) imaging system compared to criterion measures., Methods: Participants of the Shape Up! Adults study were recruited for similar sized stratifications by sex, age (18-40, 40-60, >60 years), BMI (under, normal, overweight, obese), and across five ethnicities (non-Hispanic [NH] Black, NH White, Hispanic, Asian, Native Hawaiian/Pacific Islander). All participants received manual anthropometry assessments, duplicate whole-body 3DO (Styku S100), and dual-energy X-ray absorptiometry (DXA) scans. 3DO estimates provided by the manufacturer for anthropometry and body composition were compared to the criterion measures using concordance correlation coefficient (CCC) and Bland-Altman analysis. Test-retest precision was assessed by root mean square error (RMSE) and coefficient of variation., Results: A total of 188 (102 female) participants were included. The overall fat free mass (FFM) as measured by DXA (54.1 ± 15.2 kg) and 3DO (55.3 ± 15.0 kg) showed a small mean difference of 1.2 ± 3.4 kg (95% limits of agreement -7.0 to +5.6) and the CCC was 0.97 (95% CI: 0.96-0.98). The CCC for FM was 0.95 (95% CI: 0.94-0.97) and the mean difference of 1.3 ± 3.4 kg (95% CI: -5.5 to +8.1) reflected the difference in FFM measures. 3DO anthropometry and body composition measurements showed high test-retest precision for whole body volume (1.1 L), fat mass (0.41 kg), percent fat (0.60%), arm and leg volumes, (0.11 and 0.21 L, respectively), and waist and hip circumferences (all <0.60 cm). No group differences were observed when stratified by body mass index, sex, or race/ethnicity., Conclusions: The anthropometric and body composition estimates provided by the 3DO scanner are precise and accurate to criterion methods if offsets are considered. This method offers a rapid, broadly available, and automated method of body composition assessment regardless of body size. Further studies are recommended to examine the relationship between measurements obtained by 3DO scans and metabolic health in healthy and clinical populations., Competing Interests: Conflict of Interest All authors report no conflict of interest., (Published by Elsevier Ltd.)

Background: LDL particle size and number (LDL-P) are emerging lipid risk factors. Nonsystematic reviews have suggested that diets lower in carbohydrates and higher in fats may result in increased LDL particle size when compared with higher-carbohydrate diets., Objectives: This study aimed to systematically review available evidence and conduct meta-analyses of studies addressing the association of carbohydrate restriction with LDL particle size and LDL-P., Methods: We searched 6 electronic databases on 4 January, 2021 for randomized trials of any length that reported on dietary carbohydrate restriction (intervention) compared with higher carbohydrate intake (control). We calculated standardized mean differences (SMDs) in LDL particle size and LDL-P between the intervention and control groups of eligible studies, and pooled effect sizes using random-effects models. We performed prespecified subgroup analyses and examined the effect of potential explanatory factors. Internal validity and publication bias were assessed using Cochrane's risk-of-bias tool and funnel plots, respectively. Studies that could not be meta-analyzed were summarized qualitatively., Results: This review summarizes findings from 38 randomized trials including a total of 1785 participants. Carbohydrate-restricted dietary interventions were associated with an increase in LDL peak particle size (SMD = 0.50; 95% CI: 0.15, 0.86; P < 0.01) and a reduction in LDL-P (SMD = -0.24; 95% CI: -0.43, -0.06; P = 0.02). The effect of carbohydrate-restricted dietary interventions on LDL peak particle size appeared to be partially explained by differences in weight loss between intervention groups and exploratory analysis revealed a shift from small dense to larger LDL subclasses. No statistically significant association was found between carbohydrate-restricted dietary interventions and mean LDL particle size (SMD = 0.20; 95% CI: -0.29, 0.69; P = 0.37)., Conclusions: The available evidence indicates that dietary interventions restricted in carbohydrates increase LDL peak particle size and decrease the numbers of total and small LDL particles.This review was registered at www.crd.york.ac.uk/prospero/ as CRD42020188745., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Objective: The aim of this study was to determine whether a Mediterranean-style, ketogenic diet mobile health application (app) with breath acetone biofeedback is superior to a calorie-restricted, low-fat diet app in promoting weight loss., Methods: Participants (n = 155) with overweight/obesity (mean [SD]: age 41 [11] years, BMI = 34 [5] kg/m 2 , 71% female) were randomized to one of the interventions delivered entirely via app. Participants received a wireless scale and were instructed to take daily weight measurements. A third-party laboratory collected blood samples at baseline and 12 weeks., Results: Weight loss at 12 weeks was greater in the ketogenic (-5.6 kg; 95% CI: -6.7 kg to -4.5 kg) compared with the low-fat group (-2.5 kg; 95% CI: -3.6 kg to -1.4 kg) (between-group difference: -3.1 kg; 95% CI: -4.6 kg to -1.5 kg; p < 0.001). Weight loss at 24 weeks indicated durability of the effect (between-group difference: -5.5 kg; 95% CI: -8.3 kg to -2.8 kg; p < 0.001). Secondary/exploratory outcomes of hemoglobin A1c and liver enzymes were improved to a greater extent in the ketogenic diet group (p < 0.01)., Conclusions: Among adults with overweight/obesity, a ketogenic diet app with breath acetone biofeedback was superior to a calorie-restricted diet app at promoting weight loss in a real-world setting., (© 2021 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society (TOS).)

Background: Infants with cystic fibrosis (CF) suffer from gastrointestinal (GI) complications, including pancreatic insufficiency and intestinal inflammation, which have been associated with impaired nutrition and growth. Recent evidence identified altered fecal microbiota taxonomic compositions in infants with CF relative to healthy infants that were characterized by differences in the abundances of taxa associated with GI health and nutrition. Furthermore, these taxonomic differences were more pronounced in low length infants with CF, suggesting a potential link to linear growth failure. We hypothesized that these differences would entail shifts in the microbiome's functional capacities that could contribute to inflammation and nutritional failure in infants with CF., Results: To test this hypothesis, we compared fecal microbial metagenomic content between healthy infants and infants with CF, supplemented with an analysis of fecal metabolomes in infants with CF. We identified notable differences in CF fecal microbial functional capacities, including metabolic and environmental response functions, compared to healthy infants that intensified during the first year of life. A machine learning-based longitudinal metagenomic age analysis of healthy and CF fecal metagenomic functional profiles further demonstrated that these differences are characterized by a CF-associated delay in the development of these functional capacities. Moreover, we found metagenomic differences in functions related to metabolism among infants with CF that were associated with diet and antibiotic exposure, and identified several taxa as potential drivers of these functional differences. An integrated metagenomic and metabolomic analysis further revealed that abundances of several fecal GI metabolites important for nutrient absorption, including three bile acids, correlated with specific microbes in infants with CF., Conclusions: Our results highlight several metagenomic and metabolomic factors, including bile acids and other microbial metabolites, that may impact nutrition, growth, and GI health in infants with CF. These factors could serve as promising avenues for novel microbiome-based therapeutics to improve health outcomes in these infants., (© 2021. The Author(s).)

Oxysterols are oxidized derivatives of cholesterol that play regulatory roles in lipid biosynthesis and homeostasis. How oxysterol signaling coordinates different lipid classes such as sterols and triglycerides remains incompletely understood. Here, we show that 4β-hydroxycholesterol (HC) (4β-HC), a liver and serum abundant oxysterol of poorly defined functions, is a potent and selective inducer of the master lipogenic transcription factor, SREBP1c, but not the related steroidogenic transcription factor SREBP2. By correlating tracing of lipid synthesis with lipogenic gene expression profiling, we found that 4β-HC acts as a putative agonist for the liver X receptor (LXR), a sterol sensor and transcriptional regulator previously linked to SREBP1c activation. Unique among the oxysterol agonists of the LXR, 4β-HC induced expression of the lipogenic program downstream of SREBP1c and triggered de novo lipogenesis both in primary hepatocytes and in the mouse liver. In addition, 4β-HC acted in parallel to insulin-PI3K-dependent signaling to stimulate triglyceride synthesis and lipid-droplet accumulation. Thus, 4β-HC is an endogenous regulator of de novo lipogenesis through the LXR-SREBP1c axis., Competing Interests: Conflict of interest R. Z. is cofounder, scientific advisor, and stockholder with Frontier Medicines Corp. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021. Published by Elsevier Inc.)

Importance: The efficacy and safety of time-restricted eating have not been explored in large randomized clinical trials., Objective: To determine the effect of 16:8-hour time-restricted eating on weight loss and metabolic risk markers., Interventions: Participants were randomized such that the consistent meal timing (CMT) group was instructed to eat 3 structured meals per day, and the time-restricted eating (TRE) group was instructed to eat ad libitum from 12:00 pm until 8:00 pm and completely abstain from caloric intake from 8:00 pm until 12:00 pm the following day., Design, Setting, and Participants: This 12-week randomized clinical trial including men and women aged 18 to 64 years with a body mass index (BMI, calculated as weight in kilograms divided by height in meters squared) of 27 to 43 was conducted on a custom mobile study application. Participants received a Bluetooth scale. Participants lived anywhere in the United States, with a subset of 50 participants living near San Francisco, California, who underwent in-person testing., Main Outcomes and Measures: The primary outcome was weight loss. Secondary outcomes from the in-person cohort included changes in weight, fat mass, lean mass, fasting insulin, fasting glucose, hemoglobin A1c levels, estimated energy intake, total energy expenditure, and resting energy expenditure., Results: Overall, 116 participants (mean [SD] age, 46.5 [10.5] years; 70 [60.3%] men) were included in the study. There was a significant decrease in weight in the TRE (-0.94 kg; 95% CI, -1.68 to -0.20; P = .01), but no significant change in the CMT group (-0.68 kg; 95% CI, -1.41 to 0.05, P = .07) or between groups (-0.26 kg; 95% CI, -1.30 to 0.78; P = .63). In the in-person cohort (n = 25 TRE, n = 25 CMT), there was a significant within-group decrease in weight in the TRE group (-1.70 kg; 95% CI, -2.56 to -0.83; P < .001). There was also a significant difference in appendicular lean mass index between groups (-0.16 kg/m2; 95% CI, -0.27 to -0.05; P = .005). There were no significant changes in any of the other secondary outcomes within or between groups. There were no differences in estimated energy intake between groups., Conclusions and Relevance: Time-restricted eating, in the absence of other interventions, is not more effective in weight loss than eating throughout the day., Trial Registration: ClinicalTrials.gov Identifiers: NCT03393195 and NCT03637855.

Rationale: The most common antibiotic used to treat people with cystic fibrosis (PWCF) is inhaled tobramycin, administered as maintenance therapy for chronic Pseudomonas aeruginosa lung infections. While the effects of inhaled tobramycin on P. aeruginosa abundance and lung function diminish with continued therapy, this maintenance treatment is known to improve long-term outcomes, underscoring how little is known about why antibiotics work in CF infections, what their effects are on complex CF sputum microbiomes and how to improve these treatments., Objectives: To rigorously define the effect of maintenance tobramycin on CF sputum microbiome characteristics., Methods and Measurements: We collected sputum from 30 PWCF at standardised times before, during and after a single month-long course of maintenance inhaled tobramycin. We used traditional culture, quantitative PCR and metagenomic sequencing to define the dynamic effects of this treatment on sputum microbiomes, including abundance changes in both clinically targeted and untargeted bacteria, as well as functional gene categories., Main Results: CF sputum microbiota changed most markedly by 1 week of antibiotic therapy and plateaued thereafter, and this shift was largely driven by changes in non-dominant taxa. The genetically conferred functional capacities (ie, metagenomes) of subjects' sputum communities changed little with antibiotic perturbation, despite taxonomic shifts, suggesting functional redundancy within the CF sputum microbiome., Conclusions: Maintenance treatment with inhaled tobramycin, an antibiotic with demonstrated long-term mortality benefit, primarily impacted clinically untargeted bacteria in CF sputum, highlighting the importance of monitoring the non-canonical effects of antibiotics and other treatments to accurately define and improve their clinical impact., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Obesity and being overweight are major contributing factors for many diseases. Calorie restricted diets often fail to result in sustained long-term weight loss. Very low-carbohydrate, high-fat ketogenic diets have been suggested to have superior metabolic and weight loss effects. Keyto is a low-cost, highly scalable mobile health (mHealth) app paired with a noninvasive biofeedback tool aimed at facilitating weight loss through a personalized healthy and predominantly plant- and fish-based ketogenic diet., Objective: This protocol describes a randomized trial comparing the efficacy of the Keyto mHealth app and device intervention to that of Weight Watchers' WW app in individuals who are overweight or obese. The primary outcome is weight loss after 12 weeks. Secondary and exploratory outcomes, including metabolic and cardiovascular risk factors, will be assessed at 12, 24, and 48 weeks., Methods: A total of 144 participants will be recruited and randomized to either the Keyto program or Weight Watchers program. Study participants will be guided through the study via video conference or phone calls and will undergo a fasting blood analysis performed by a third-party diagnostic lab at weeks 0 and 12 to assess metabolic and cardiovascular risk markers. All participants will be asked to weigh themselves daily on a study-provided Bluetooth-enabled scale. Participants randomized to the Keyto arm will also be asked to measure their breath acetone levels, a measure of ketosis, with the Keyto device 3 times per day., Results: Recruitment started in December 2019. Rolling recruitment is expected to be completed by July 2020. Data collection and analysis of the primary intervention phase is expected to be completed in October 2020. The 24- and 48-week follow-ups are expected to be completed in January 2021 and July 2021, respectively., Conclusions: This trial will provide high-quality evidence regarding the efficacy of the Keyto weight loss program in individuals who are overweight and obese in a free-living condition. This study also fills a gap by examining the impact of a ketogenic diet emphasizing plant- and fish-based fats on blood lipid profile and cardiovascular disease risk., Trial Registration: ClinicalTrials.gov NCT04165707; https://clinicaltrials.gov/ct2/show/NCT04165707., International Registered Report Identifier (irrid): DERR1-10.2196/19053., (©Sean R Locke, Kaja Falkenhain, Dylan A Lowe, Terry Lee, Joel Singer, Ethan J Weiss, Jonathan P Little. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 17.08.2020.)

Background: Visceral adiposity, more so than overall adiposity, is associated with chronic disease and mortality. There has been, to our knowledge, little research exploring the association between diet quality and visceral adipose tissue (VAT) among a mulitethnic population aged 18-80 y., Objective: The primary objective of this cross-sectional analysis was to examine the association between diet quality [Healthy Eating Index-2010 (HEI-2010) scores] and VAT among a multiethnic population of young, middle, and older aged adults in the United States. Secondary objectives were to repeat these analyses with overall adiposity and blood-based biomarkers for type 2 diabetes and cardiovascular disease risk as outcome measures., Methods: A total of 540 adults (dropped out: n  = 4; age: 18-40 y, n  = 220; 40-60 y, n  = 183; 60-80 y, n  = 133) were recruited across 3 sites (Honolulu County, San Francisco, and Baton Rouge) for the Shape Up! Adults study. Whole-body DXA, anthropometry, fasting blood draw, and questionnaires (food frequency, physical activity, and demographic characteristics) were completed. Linear regression was used to assess the associations between HEI-2010 tertiles and VAT and secondary outcome measures among all participants and age-specific strata, while adjusting for known confounders., Results: VAT, BMI (kg/m 2 ), body fat percentage, total body fat, trunk fat, insulin, and insulin resistance were inversely related to diet quality (all P values < 0.004). When stratified by age, diet quality was inversely associated with VAT among participants aged 60-80 y ( P < 0.006) and VAT/subcutaneous adipose tissue (SAT) among participants aged 40-60 y ( P < 0.008)., Conclusions: Higher-quality diet was associated with lower VAT, overall adiposity, and insulin resistance among this multiethnic population of young, middle, and older aged adults with ages ranging from 18 to 80 y. More specifically, adherence to a high-quality diet may minimize VAT accumulation in adults aged 60-80 y and preferentially promote storage of SAT compared with VAT in adults aged 40-60 y.This study was registered at clinicaltrials.gov as NCT03637855., (Copyright © The Author(s) on behalf of the American Society for Nutrition 2020.)

Most infants with cystic fibrosis (CF) have pancreatic exocrine insufficiency that results in nutrient malabsorption and requires oral pancreatic enzyme replacement. Newborn screening for CF has enabled earlier diagnosis, nutritional intervention and enzyme replacement for these infants, allowing most infants with CF to achieve their weight goals by 12 months of age 1 . Nevertheless, most infants with CF continue to have poor linear growth during their first year of life 1 . Although this early linear growth failure is associated with worse long-term respiratory function and survival 2,3 , the determinants of body length in infants with CF have not been defined. Several characteristics of the CF gastrointestinal (GI) tract, including inflammation, maldigestion and malabsorption, may promote intestinal dysbiosis 4,5 . As GI microbiome activities are known to affect endocrine functions 6,7 , the intestinal microbiome of infants with CF may also impact growth. We identified an early, progressive fecal dysbiosis that distinguished infants with CF and low length from infants with CF and normal length. This dysbiosis included altered abundances of taxa that perform functions that are important for GI health, nutrient harvest and growth hormone signaling, including decreased abundance of Bacteroidetes and increased abundance of Proteobacteria. Thus, the GI microbiota represent a potential therapeutic target for the correction of low linear growth in infants with CF.

Patients with cystic fibrosis (CF) have altered fecal microbiomes compared to those of healthy controls. The magnitude of this dysbiosis correlates with measures of CF gastrointestinal (GI) disease, including GI inflammation and nutrient malabsorption. However, whether this dysbiosis is caused by mutations in the CFTR gene, the underlying defect in CF, or whether CF-associated dysbiosis augments GI disease was not clear. To test the relationships between CFTR dysfunction, microbes, and intestinal health, we established a germ-free (GF) CF mouse model and demonstrated that CFTR gene mutations are sufficient to alter the GI microbiome. Furthermore, flow cytometric analysis demonstrated that colonized CF mice have increased mesenteric lymph node and spleen TH17+ cells compared with non-CF mice, suggesting that CFTR defects alter adaptive immune responses. Our findings demonstrate that CFTR mutations modulate both the host adaptive immune response and the intestinal microbiome., Competing Interests: The authors have declared that no competing interests exist.

Ruthie Weiss was born with white hair, but her parents did not consider the possibility of there being more to the story until they noticed that she was not visually tracking when she was just a month old. Thus began a long and continuing story of the discovery of Ruthie's albinism, her significant visual impairment, but also her courage and determination to do anything and everything her peers do, if not more. But this story is really about how her parents grew to embrace the impact Ruthie (and importantly Ruthie's disability) had on their lives and the lives of everyone with whom Ruthie interacted. The experience of raising Ruthie ultimately led her parents to think about a world where she might not exist, or at least might not exist with albinism. But it also led them to ponder a future in which children with genetic differences like albinism are gene edited using technologies like CRISPR-Cas9.

Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are liver manifestations of the metabolic syndrome and can progress to hepatocellular carcinoma (HCC). Loss of Growth Hormone (GH) signaling is reported to predispose to NAFLD and NASH through direct actions on the liver. Here, we report that aged mice lacking hepatocyte Jak2 (JAK2L), an obligate transducer of Growth Hormone (GH) signaling, spontaneously develop the full spectrum of phenotypes found in patients with metabolic liver disease, beginning with insulin resistance and lipodystrophy and manifesting as NAFLD, NASH and even HCC, independent of dietary intervention. Remarkably, insulin resistance, metabolic liver disease, and carcinogenesis are prevented in JAK2L mice via concomitant deletion of adipocyte Jak2 (JAK2LA). Further, we demonstrate that GH increases hepatic lipid burden but does so indirectly via signaling through adipocyte JAK2. Collectively, these data establish adipocytes as the mediator of GH-induced metabolic liver disease and carcinogenesis. In addition, we report a new spontaneous model of NAFLD, NASH, and HCC that recapitulates the natural sequelae of human insulin resistance-associated disease progression. The work presented here suggests a attention be paid towards inhibition of adipocyte GH signaling as a therapeutic target of metabolic liver disease.

Metagenomic sequencing is a promising approach for identifying and characterizing organisms and their functional characteristics in complex, polymicrobial infections, such as airway infections in people with cystic fibrosis. These analyses are often hampered, however, by overwhelming quantities of human DNA, yielding only a small proportion of microbial reads for analysis. In addition, many abundant microbes in respiratory samples can produce large quantities of extracellular bacterial DNA originating either from biofilms or dead cells. We describe a method for simultaneously depleting DNA from intact human cells and extracellular DNA (human and bacterial) in sputum, using selective lysis of eukaryotic cells and endonuclease digestion. We show that this method increases microbial sequencing depth and, consequently, both the number of taxa detected and coverage of individual genes such as those involved in antibiotic resistance. This finding underscores the substantial impact of DNA from sources other than live bacteria in microbiological analyses of complex, chronic infection specimens., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mature human gut microbiota is established during the first years of life, and altered intestinal microbiomes have been associated with several human health disorders. Escherichia coli usually represents less than 1% of the human intestinal microbiome, whereas in cystic fibrosis (CF), greater than 50% relative abundance is common and correlates with intestinal inflammation and fecal fat malabsorption. Despite the proliferation of E. coli and other Proteobacteria in conditions involving chronic gastrointestinal tract inflammation, little is known about adaptation of specific characteristics associated with microbiota clonal expansion. We show that E. coli isolated from fecal samples of young children with CF has adapted to growth on glycerol, a major component of fecal fat. E. coli isolates from different CF patients demonstrate an increased growth rate in the presence of glycerol compared with E. coli from healthy controls, and unrelated CF E. coli strains have independently acquired this growth trait. Furthermore, CF and control E. coli isolates have differential gene expression when grown in minimal media with glycerol as the sole carbon source. While CF isolates display a growth-promoting transcriptional profile, control isolates engage stress and stationary-phase programs, which likely results in slower growth rates. Our results indicate that there is selection of unique characteristics within the microbiome of individuals with CF, which could contribute to individual disease outcomes., Competing Interests: The authors declare no conflict of interest.

Disruption of hepatocyte growth hormone (GH) signaling through disruption of Jak2 (JAK2L) leads to fatty liver. Previously, we demonstrated that development of fatty liver depends on adipocyte GH signaling. We sought to determine the individual roles of hepatocyte and adipocyte Jak2 on whole-body and tissue insulin sensitivity and liver metabolism. On chow, JAK2L mice had hepatic steatosis and severe whole-body and hepatic insulin resistance. However, concomitant deletion of Jak2 in hepatocytes and adipocytes (JAK2LA) completely normalized insulin sensitivity while reducing liver lipid content. On high-fat diet, JAK2L mice had hepatic steatosis and insulin resistance despite protection from diet-induced obesity. JAK2LA mice had higher liver lipid content and no protection from obesity but retained exquisite hepatic insulin sensitivity. AKT activity was selectively attenuated in JAK2L adipose tissue, whereas hepatic insulin signaling remained intact despite profound hepatic insulin resistance. Therefore, JAK2 in adipose tissue is epistatic to liver with regard to insulin sensitivity and responsiveness, despite fatty liver and obesity. However, hepatocyte autonomous JAK2 signaling regulates liver lipid deposition under conditions of excess dietary fat. This work demonstrates how various tissues integrate JAK2 signals to regulate insulin/glucose and lipid metabolism., (© 2017 by the American Diabetes Association.)

Background: Growth hormone (GH) is known to affect insulin and glucose metabolism.  Blocking its effects in acromegalic patients improves diabetes and glucose metabolism. We aimed to determine the effect of pegvisomant, a GH receptor antagonist, on insulin resistance, endogenous glucose production (EGP) and lipolysis in insulin resistant non-diabetic men.  Methods: Four men between the ages of 18-62 with a BMI of 18-35kg/m 2 , with insulin resistance as defined by a HOMA-IR > 2.77, were treated for four weeks with pegvisomant 20 mg daily.  Inpatient metabolic assessments were performed before and after treatment. The main outcome measurements were: change after pegvisomant therapy in insulin sensitivity as measured by hyperinsulinemic euglycemic clamp; and EGP and lipolysis assessed by stable isotope tracer techniques. Results: Insulin like growth factor-1 (IGF-1) concentrations decreased from 134.0 ± 41.5 (mean ± SD) to 72.0 ± 11.7 ng/mL (p = 0.04) after 4 weeks of therapy. Whole body insulin sensitivity index (M/I 3.2 ± 1.3 vs. 3.4 ± 2.4; P = 0.82), as well as suppression of EGP (89.7 ± 26.9 vs. 83.5 ± 21.6%; p = 0.10) and Ra glycerol (59.4 ± 22.1% vs. 61.2 ± 14.4%; p = 0.67) during the clamp were not changed significantly with pegvisomant treatment. Conclusions: Blockade of the GH receptor with pegvisomant for four weeks had no significant effect on insulin/glucose metabolism in a small phase II pilot study of non-diabetic insulin resistant participants without acromegaly., Competing Interests: Competing interests: This study was generously funded by Pfizer pharmaceuticals, who additionally provided drug. The authors have nothing else to disclose.

For nearly 100 years, growth hormone (GH) has been known to affect insulin sensitivity and risk of diabetes. However, the tissue governing the effects of GH signaling on insulin and glucose homeostasis remains unknown. Excess GH reduces fat mass and insulin sensitivity. Conversely, GH insensitivity (GHI) is associated with increased adiposity, augmented insulin sensitivity, and protection from diabetes. Here, we induce adipocyte-specific GHI through conditional deletion of Jak2 (JAK2A), an obligate transducer of GH signaling. Similar to whole-body GHI, JAK2A mice had increased adiposity and extreme insulin sensitivity. Loss of adipocyte Jak2 augmented hepatic insulin sensitivity and conferred resistance to diet-induced metabolic stress without overt changes in circulating fatty acids. While GH injections induced hepatic insulin resistance in control mice, the diabetogenic action was absent in JAK2A mice. Adipocyte GH signaling directly impinged on both adipose and hepatic insulin signal transduction. Collectively, our results show that adipose tissue governs the effects of GH on insulin and glucose homeostasis. Further, we show that JAK2 mediates liver insulin sensitivity via an extrahepatic, adipose tissue-dependent mechanism., Competing Interests: The authors have declared that no conflict of interest exists.

Background: Comparative analysis of gut microbiomes in clinical studies of human diseases typically rely on identification and quantification of species or genes. In addition to exploring specific functional characteristics of the microbiome and potential significance of species diversity or expansion, microbiome similarity is also calculated to study change in response to therapies directed at altering the microbiome. Established ecological measures of similarity can be constructed from species abundances, however methods for calculating these commonly used ecological measures of similarity directly from whole genome shotgun (WGS) metagenomic sequence are lacking., Results: We present an alignment-free method for calculating similarity of WGS metagenomic sequences that is analogous to the Bray-Curtis index for species, implemented by the General Utility for Testing Sequence Similarity (GUTSS) software application. This method was applied to intestinal microbiomes of healthy young children to measure developmental changes toward an adult microbiome during the first 3 years of life. We also calculate similarity of donor and recipient microbiomes to measure establishment, or engraftment, of donor microbiota in fecal microbiota transplantation (FMT) studies focused on mild to moderate Crohn's disease. We show how a relative index of similarity to donor can be calculated as a measure of change in a patient's microbiome toward that of the donor in response to FMT., Conclusion: Because clinical efficacy of the transplant procedure cannot be fully evaluated without analysis methods to quantify actual FMT engraftment, we developed a method for detecting change in the gut microbiome that is independent of species identification and database bias, sensitive to changes in relative abundance of the microbial constituents, and can be formulated as an index for correlating engraftment success with clinical measures of disease. More generally, this method may be applied to clinical evaluation of human microbiomes and provide potential diagnostic determination of individuals who may be candidates for specific therapies directed at alteration of the microbiome.

Unlabelled: Salmonella enterica serovar Typhimurium is one of the most common S. enterica serovars associated with U.S. foodborne outbreaks. S. Typhimurium bacteria isolated from humans exhibit wide-ranging virulence phenotypes in inbred mice, leading to speculation that some strains are more virulent in nature. However, it is unclear whether increased virulence in humans is related to organism characteristics or initial treatment failure due to antibiotic resistance. Strain diversity and genetic factors contributing to differential human pathogenicity remain poorly understood. We reconstructed phylogeny, resolved genetic population structure, determined gene content and nucleotide variants, and conducted targeted phenotyping assays for S. Typhimurium strains collected between 1946 and 2012 from humans and animals in the United States and abroad. Strains from recent U.S. salmonellosis cases were associated with five S. Typhimurium lineages distributed within three phylogenetic clades, which are not restricted by geography, year of acquisition, or host. Notably, two U.S. strains and four Mexican strains are more closely related to strains associated with human immunodeficiency virus (HIV)-infected individuals in sub-Saharan Africa than to other North American strains. Phenotyping studies linked variants specific to these strains in hmpA and katE to loss of fitness under nitrosative and oxidative stress, respectively. These results suggest that U.S. salmonellosis is caused by diverse S. Typhimurium strains circulating worldwide. One lineage has mutations in genes affecting fitness related to innate immune system strategies for fighting pathogens and may be adapting to immunocompromised humans by a reduction in virulence capability, possibly due to a lack of selection for its maintenance as a result of the worldwide HIV epidemic., Importance: Nontyphoidal Salmonella bacteria cause an estimated 1.2 million illnesses annually in the United States, 80 million globally, due to ingestion of contaminated food or water. Salmonella Typhimurium is one of the most common serovars associated with foodborne illness, causing self-limiting gastroenteritis and, in approximately 5% of infected patients, systemic infection. Although some S. Typhimurium strains are speculated to be more virulent than others, it is unknown how strain diversity and genetic factors contribute to differential human pathogenicity. Ours is the first study to examine the diversity of S. Typhimurium associated with recent cases of U.S. salmonellosis and to provide some initial correlation between observed genotypes and phenotypes. Definition of specific S. Typhimurium lineages based on such phenotype/genotype correlations may identify strains with greater capability of associating with specific food sources, allowing outbreaks to be more quickly identified. Additionally, defining simple correlates of pathogenesis may have predictive value for patient outcome., (Copyright © 2016 Hayden et al.)

CD36/FAT (fatty acid translocase) is associated with human and murine nonalcoholic fatty liver disease, but it has been unclear whether it is simply a marker or whether it directly contributes to disease pathogenesis. Mice with hepatocyte-specific deletion of Janus kinase 2 (JAK2L mice) have increased circulating free fatty acids (FAs), dramatically increased hepatic CD36 expression and profound fatty liver. To investigate the role of elevated CD36 in the development of fatty liver, we studied two models of hepatic steatosis, a genetic model (JAK2L mice) and a high-fat diet (HFD)-induced steatosis model. We deleted Cd36 specifically in hepatocytes of JAK2L mice to generate double knockouts and from wild-type mice to generate CD36L single-knockout mice. Hepatic Cd36 disruption in JAK2L livers significantly improved steatosis by lowering triglyceride, diacylglycerol, and cholesterol ester content. The largest differences in liver triglycerides were in species comprised of oleic acid (C18:1). Reduction in liver lipids correlated with an improvement in the inflammatory markers that were elevated in JAK2L mice, namely aspartate aminotransferase and alanine transaminase. Cd36 deletion in mice on HFD (CD36L-HFD) reduced liver lipid content and decreased hepatic 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-FA uptake as compared with CON-HFD. Additionally, CD36L-HFD mice had improved whole-body insulin sensitivity and reduced liver and serum inflammatory markers. Therefore, CD36 directly contributes to development of fatty liver under conditions of elevated free FAs by modulating the rate of FA uptake by hepatocytes. In HFD-fed animals, disruption of hepatic Cd36 protects against associated systemic inflammation and insulin resistance.

Unlabelled: Acinetobacter baumannii is a Gram-negative bacterial pathogen notorious for causing serious nosocomial infections that resist antibiotic therapy. Research to identify factors responsible for the pathogen's success has been limited by the resources available for genome-scale experimental studies. This report describes the development of several such resources for A. baumannii strain AB5075, a recently characterized wound isolate that is multidrug resistant and displays robust virulence in animal models. We report the completion and annotation of the genome sequence, the construction of a comprehensive ordered transposon mutant library, the extension of high-coverage transposon mutant pool sequencing (Tn-seq) to the strain, and the identification of the genes essential for growth on nutrient-rich agar. These resources should facilitate large-scale genetic analysis of virulence, resistance, and other clinically relevant traits that make A. baumannii a formidable public health threat., Importance: Acinetobacter baumannii is one of six bacterial pathogens primarily responsible for antibiotic-resistant infections that have become the scourge of health care facilities worldwide. Eliminating such infections requires a deeper understanding of the factors that enable the pathogen to persist in hospital environments, establish infections, and resist antibiotics. We present a set of resources that should accelerate genome-scale genetic characterization of these traits for a reference isolate of A. baumannii that is highly virulent and representative of current outbreak strains., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Shigella dysenteriae type 1 (Sd1) causes recurrent epidemics of dysentery associated with high mortality in many regions of the world. Sd1 infects humans at very low infectious doses (10 CFU), and treatment is complicated by the rapid emergence of antibiotic resistant Sd1 strains. Sd1 is only detected in the context of human infections, and the circumstances under which epidemics emerge and regress remain unknown., Results: Phylogenomic analyses of 56 isolates collected worldwide over the past 60 years indicate that the Sd1 clone responsible for the recent pandemics emerged at the turn of the 20th century, and that the two world wars likely played a pivotal role for its dissemination. Several lineages remain ubiquitous and their phylogeny indicates several recent intercontinental transfers. Our comparative genomics analysis reveals that isolates responsible for separate outbreaks, though closely related to one another, have independently accumulated antibiotic resistance genes, suggesting that there is little or no selection to retain these genes in-between outbreaks. The genomes appear to be subjected to genetic drift that affects a number of functions currently used by diagnostic tools to identify Sd1, which could lead to the potential failure of such tools., Conclusions: Taken together, the Sd1 population structure and pattern of evolution suggest a recent emergence and a possible human carrier state that could play an important role in the epidemic pattern of infections of this human-specific pathogen. This analysis highlights the important role of whole-genome sequencing in studying pathogens for which epidemiological or laboratory investigations are particularly challenging.

Nonalcoholic fatty liver disease (NAFLD) is considered the hepatic expression of the metabolic syndrome, and its prevalence is increasing. The factors that influence the development of fatty liver and its progression to steatohepatitis and cirrhosis are not well understood. The pleiotropic hormone, GH, has been associated with an increased risk of NAFLD in humans and mice. GH is known to have diverse effects on lipid metabolism including decreasing body fat in vivo, presumably through stimulation of lipolysis via an undefined mechanism. Previously we described mice with hepatocyte-specific deletion of the GH signaling mediator, Janus kinase 2 (JAK2L). JAK2L animals have elevated serum GH, reduced body fat, high liver triglyceride content, and increased serum markers of hepatocyte injury (alanine transaminase and aspartate transaminase). We aimed to determine whether the elevation of GH in JAK2L mice contributed to fatty liver by promoting lipolysis directly in adipocytes. We generated mice with adipocyte-specific disruption of JAK2 (JAK2A) and found that GH resistance in adipocytes reduced lipolysis and increased body fat. JAK2A mice were then crossed to JAK2L mice, and the resultant JAK2L/A animals had increased body fat and decreased lipolysis, despite elevated circulating GH. Furthermore, the increased triglyceride content, serum alanine transaminase, and serum aspartate transaminase observed in JAK2L mice were nearly normalized with the additional disruption of JAK2 in adipocytes (JAK2L/A mice). Our results offer novel mechanistic insights into the long-recognized effects of GH on lipid flux and suggest that GH signaling may play an important regulatory role in the development of NAFLD.

Background: Excess risk of cardiovascular disease occurs in effectively treated individuals with human immunodeficiency virus (HIV) infection. Although elevated plasma D-dimer levels are associated with increased morbidity and mortality, the impact of HIV infection on coagulation in vivo has not been well studied., Methods: We measured D-dimers, antithrombin, endogenous thrombin potential (ETP; a functional measure of thrombin generation in vitro), thrombin/antithrombin complexes (TAT; a measure of thrombin generation in vivo), tissue factor, prothrombin fragment 1 + 2 (F1+2), and normalized APC sensitivity ratio (nAPCsr) in 199 HIV-positive men who were receiving antiretroviral therapy and had an undetectable HIV RNA level, in 79 HIV-positive untreated men, and in 39 uninfected controls., Results: Median antithrombin levels were higher while the ETP was lower among HIV-infected adults (treated and untreated), compared with controls. There were few differences between coagulation markers in the 2 HIV groups. Compared with controls, the nAPCsr was lower in treated men and the TAT level was lower in untreated individuals. We observed little difference among measured levels of D-dimer, tissue factor, or F1+2 between HIV-infected individuals and controls. Antiretroviral therapy exposure was associated with a lower antithrombin level, a lower nAPCsr, and a lower ETP, while history of opportunistic infection was associated with a higher nAPCsr., Conclusions: HIV infection is associated with decreased thrombin generation, as measured by the ETP, and an increased antithrombin level. These data suggest that HIV infection may not be associated with increased propensity toward clotting, as has been suggested on the basis of isolated measures of D-dimer levels.

The relative contributions of circulating and locally produced IGF-I in growth remain controversial. The majority of circulating IGF-I is produced by the liver, and numerous mouse models have been developed to study the endocrine actions of IGF-I. A common drawback to these models is that the elimination of circulating IGF-I disrupts a negative feedback pathway, resulting in unregulated GH secretion. We generated a mouse with near total abrogation of circulating IGF-I by disrupting the GH signaling mediator, Janus kinase (JAK)2, in hepatocytes. We then crossed these mice, termed JAK2L, to GH-deficient little mice (Lit). Compound mutant (Lit-JAK2L) and control (Lit-Con) mice were treated with equal amounts of GH such that the only difference between the two groups was hepatic GH signaling. Both groups gained weight in response to GH but there was a reduction in the final weight of GH-treated Lit-JAK2L vs. Lit-Con mice. Similarly, lean mass increased in both groups, but there was a reduction in the final lean mass of Lit-JAK2L vs. Lit-Con mice. There was an equivalent increase in skeletal length in response to GH in Lit-Con and Lit-JAK2L mice. There was an increase in bone mineral density (BMD) in both groups, but Lit-JAK2L had lower BMD than Lit-Con mice. In addition, GH-mediated increases in spleen and kidney mass were absent in Lit-JAK2L mice. Taken together, hepatic GH-dependent production of IGF-I had a significant and nonredundant role in GH-mediated acquisition of lean mass, BMD, spleen mass, and kidney mass; however, skeletal length was dependent upon or compensated for by locally produced IGF-I.

Non-alcoholic fatty liver disease is associated with multiple comorbid conditions, including diabetes, obesity, infection, and malnutrition. Mice with hepatocyte-specific disruption of growth hormone (GH) signaling develop fatty liver (FL), although the precise mechanism underlying this finding remains unknown. Because GH signals through JAK2, we developed mice bearing hepatocyte-specific deletion of JAK2 (referred to herein as JAK2L mice). These mice were lean, but displayed markedly elevated levels of GH, liver triglycerides (TGs), and plasma FFAs. Because GH is known to promote lipolysis, we crossed GH-deficient little mice to JAK2L mice, and this rescued the FL phenotype. Expression of the fatty acid transporter CD36 was dramatically increased in livers of JAK2L mice, as was expression of Pparg. Since GH signaling represses PPARγ expression and Cd36 is a known transcriptional target of PPARγ, we treated JAK2L mice with the PPARγ-specific antagonist GW9662. This resulted in reduced expression of liver Cd36 and decreased liver TG content. These results provide a mechanism for the FL observed in mice with liver-specific disruption in GH signaling and suggest that the development of FL depends on both GH-dependent increases in plasma FFA and increased hepatic uptake of FFA, likely mediated by increased expression of CD36.

Active sensing involves memory retrieval and updating as well as mechanisms that trigger corrections to the ongoing exploratory movement. The present study examined this process in a task where human subjects moved the index fingertip clockwise around the circumference of a virtual sphere created by a robotic device. The fingertip pressed into the sphere during the movement, and the subjects were to report slight differences in sphere size (or surface curvature), which occurred from trial to trial. During each 2- to 3-s trial, subjects gradually adjusted their speed and pressure according to the current surface curvature, achieving a consistent level of contact force in the last half of the exploration. The results demonstrate that subjects were gradually accumulating haptic information about curvature and, at the same time, gradually changing the motor commands for the movement. When subjects encountered an unexpected transition in curvature (from circular to flat), they reacted by abruptly decreasing contact force at a latency of about 50 ms. This short latency indicates that spinally mediated corrections are engaged during this task. The results support the hypothesis that during haptic exploration, the neural comparison between expected and actual somatosensory feedback takes places at multiple levels, including the spinal cord.

To explore the effect(s) of growth hormone signaling on thrombosis, we studied signal transduction and transcription factor 5 (STAT5)-deficient mice and found markedly reduced survival in an in vivo thrombosis model. These findings were not explained by a compensatory increase in growth hormone secretion. There was a modest increase in the activity of several procoagulant factors, but there was no difference in the rate or magnitude of thrombin generation in STAT5-deficient mice relative to control. However, thrombin-triggered clot times were markedly shorter, and fibrin polymerization occurred more rapidly in plasma from STAT5-deficient mice. Fibrinogen depletion and mixing studies indicated that the effect on fibrin polymerization was not due to intrinsic changes in fibrinogen, but resulted from changes in the concentration of a circulating plasma inhibitor. While thrombin-triggered clot times were significantly shorter in STAT5-deficient animals, reptilase-triggered clot times were unchanged. Accordingly, while the rate of thrombin-catalyzed release of fibrinopeptide A was similar, the release of fibrinopeptide B was accelerated in STAT5-deficient plasma versus control. Taken together, these studies demonstrated that the loss of STAT5 resulted in a decrease in the concentration of a plasma inhibitor affecting thrombin-triggered cleavage of fibrinopeptide B. This ultimately resulted in accelerated fibrin polymerization and greater thrombosis susceptibility in STAT5-deficient animals.

Understanding genetic contributions to platelet function could have profound clinical ramifications for personalizing platelet-directed pharmacotherapy, by providing insight into the risks and possible benefits associated with specific genotypes. This article represents an integrated summary of presentations related to genetic regulation of platelet receptor expression and function given at the Fifth Annual Platelet Colloquium in January 2010. It is supplemented with additional highlights from the literature covering (1) approaches to determining and evidence for the associations of genetic variants with platelet hypo- and hyperresponsive phenotypes, (2) the ramifications of these polymorphisms with regard to clinical responses to antiplatelet therapies, and (3) the role of platelet function/genetic testing in guiding antiplatelet therapy.

Sex differences in thrombosis are well described, but their underlying mechanism(s) are not completely understood. Coagulation proteins are synthesized in the liver, and liver gene expression is sex specific and depends on sex differences in growth hormone (GH) secretion--males secrete GH in a pulsatile fashion, while females secrete GH continuously. Accordingly, we tested the hypothesis that sex-specific GH secretion patterns cause sex differences in thrombosis. Male mice were more susceptible to thrombosis than females in the thromboplastin-induced pulmonary embolism model and showed shorter clotting times ex vivo. GH-deficient little (lit) mice were protected from thrombosis, and pulsatile GH given to lit mice restored the male clotting phenotype. Moreover, pulsatile GH administration resulted in a male clotting phenotype in control female mice, while continuous GH caused a female clotting phenotype in control male mice. Expression of the coagulation inhibitors Proc, Serpinc1, Serpind1, and Serpina5 were strongly modulated by sex-specific GH patterns, and GH modulated resistance to activated protein C. These results reveal what we believe to be a novel mechanism whereby sex-specific GH patterns mediate sex differences in thrombosis through coordinated changes in the expression of coagulation inhibitor genes in the liver.

Because humans have limited ability to independently control the many joints of the hand, a wide variety of hand shapes can be characterized as a weighted combination of just two or three main patterns of covariation in joint rotations, or "postural synergies." The present study sought to align muscle synergies with these main postural synergies and to describe the form of membership of motor units in these postural/muscle synergies. Seventeen joint angles and the electromyographic (EMG) activities of several hand muscles (both intrinsic and extrinsic muscles) were recorded while human subjects held the hand statically in 52 specific shapes (i.e., shaping the hand around 26 commonly grasped objects or forming the 26 letter shapes of a manual alphabet). Principal-components analysis revealed several patterns of muscle synergy, some of which represented either coactivation of all hand muscles, or reciprocal patterns of activity (above and below average levels) in the intrinsic index finger and thumb muscles or (to a lesser extent) in the extrinsic four-tendoned extensor and flexor muscles. Single- and multiunit activity was generally a multimodal function of whole hand shape. This implies that motor-unit activation does not align with a single synergy; instead, motor units participate in multiple muscle synergies. Thus it appears that the organization of the global pattern of hand muscle activation is highly distributed. This organization mirrors the highly fractured somatotopy of cortical hand representations and may provide an ideal substrate for motor learning and recovery from injury.

The recent observation that knock-out of protease-activated receptor-4 (PAR4) ablates thrombin signaling in mouse platelets and protects against ferric chloride-induced thrombosis of mouse mesenteric arterioles suggests that thrombin's actions on platelets can play an important role in thrombosis. Complete ablation of thrombin signaling would be difficult to achieve in human beings because human platelets have 2 thrombin receptors that are each capable of mediating transmembrane signaling. However, it is possible that complete ablation of thrombin signaling in platelets is not necessary for an antithrombotic effect. In mouse platelets, PAR3 functions as a cofactor that binds thrombin and promotes productive cleavage of PAR4, and thrombin responses are decreased but not absent in Par3(-/-) platelets. We now report that Par3(-/-) mice were protected against ferric chloride-induced thrombosis of mesenteric arterioles and against thromboplastin-induced pulmonary embolism. Surprisingly, Par3(-/-) and Par4(-/-) mice showed similar degrees of protection in these models and similar prolongation of tail bleeding times. Thus, even a partial decrease in mouse platelet responsiveness to thrombin protected against thrombosis and impaired hemostasis in some settings. These results demonstrate the importance of PAR3's unusual cofactor function and underscore the relative importance of thrombin's actions on platelets in vivo. They also suggest that PAR inhibition might be explored for the prevention or treatment of thrombosis in human beings.

Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.

Protease-activated receptors 1 and 4 (PAR1 and PAR4) mediate thrombin signaling in human platelets. Whether these receptors are redundant, interact, or serve only partially overlapping functions is unknown. We report that PAR1 and PAR4 signal with distinct tempos. In transfected fibroblasts, PAR4 triggered substantially more phosphoinositide hydrolysis per activated receptor than PAR1 and was shut off more slowly than PAR1. Shutoff and internalization of PAR1 depends upon phosphorylation of its carboxyl tail upon receptor activation. In contrast to PAR1, phosphorylation of PAR4 was undetectable, and activation-dependent internalization of PAR4 was much slower than that seen for PAR1. Mutation of potential phosphorylation sites in the carboxyl tail of PAR1 enhanced PAR1 signaling, whereas analogous mutations in PAR4 had no effect. Thus PAR4 signaling is shut off less rapidly than PAR1, probably due to differences in receptor phosphorylation. PAR1 and PAR4 also signaled with distinct tempos in platelets. PAR1 triggered a rapid and transient increase in intracellular calcium, whereas PAR4 triggered a more prolonged response. Together, the tempo of these responses accounted for that triggered by thrombin. Thus differences in the rates at which PAR1 and PAR4 are shut off allow thrombin to trigger intracellular signaling with distinct temporal characteristics.

Thrombin activates protease-activated receptors (PARs) by specific cleavage of their amino-terminal exodomains to unmask a tethered ligand that binds intramolecularly to the body of the receptor to effect transmembrane signaling. Peptides that mimic such ligands are valuable as agonists for probing PAR function, but the tethered ligand peptide for PAR4, GYPGKF, lacks potency and is of limited utility. In a structure-activity analysis of PAR4 peptides, AYPGKF was approximately 10-fold more potent than GYPGKF and, unlike GYPGKF, elicited PAR4-mediated responses comparable in magnitude to those elicited by thrombin. AYPGKF was relatively specific for PAR4 in part due to the tyrosine at position 2; substitution of phenylalanine or p-fluorophenylalanine at this position produced peptides that activated both PAR1 and PAR4. Because human platelets express both PAR1 and PAR4, it might be desirable to inhibit both receptors. Identifying a single agonist for both receptors raises the possibility that a single antagonist for both receptors might be developed. The AYPGKF peptide is a useful new tool for probing PAR4 function. For example, AYPGKF activated and desensitized PAR4 in platelets and, like thrombin, triggered phosphoinositide hydrolysis but not inhibition of adenylyl cyclase in PAR4-expressing cells. The latter shows that, unlike PAR1, PAR4 couples to G(q) and not G(i).

Identification of the mechanisms by which the coagulation protease thrombin activates platelets is critical for understanding haemostasis and thrombosis. Thrombin activates cells at least in part by cleaving protease-activated G-protein-coupled receptors (PARs). PAR3 and PAR4 are thrombin receptors expressed in mouse platelets. Inhibition of thrombin binding to mPAR3 (ref. 4) and knockout of the mPAR3 gene inhibited mouse platelet activation at low but not high concentrations of thrombin. Thus PAR3 is important for thrombin signalling in mouse platelets. Expression of human PAR3 in heterologous expression systems reliably resulted in responsiveness to thrombin. Curiously, despite its importance for the activation of mouse platelets by thrombin, mouse PAR3 (mPAR3) did not lead to thrombin signalling even when overexpressed. We now report that mPAR3 and mPAR4 interact in a novel way: mPAR3 does not itself mediate transmembrane signalling but instead functions as a cofactor for the cleavage and activation of mPAR4 by thrombin. This establishes a paradigm for cofactor-assisted PAR activation and for a G-protein-coupled receptor's acting as an accessory molecule to present ligand to another receptor.

Background: The Pl(A2) polymorphism of GPIIIa has been associated with unstable coronary syndromes in some studies, but the association has remained debated. None of the previous studies have focused on families at high risk. Risk factors tend to cluster within kindreds with high prevalence of premature coronary heart disease (CHD). Therefore, a heightened prevalence of the Pl(A2) polymorphism among siblings of patients with CHD would support the hypothesis that Pl(A2) is linked, directly or indirectly, to CHD., Objectives: To measure the prevalence of the Pl(A2) polymorphism among siblings of patients with CHD before the age of 60 years and to seek an association between the Pl(A2) polymorphism and established atherosclerotic and thrombogenic risk factors., Methods: From January 1994 to April 1996, we genotyped 116 asymptomatic siblings (60 Caucasians, 56 Afro-Caribbeans) of patients with CHD manifestations before the age of 60 years for the Pl(A) polymorphism (also called HPA-1). A control cohort was used for comparison, consisting of individuals that were matched for race and geographic area but were free of CHD (n = 268, 168 Caucasians and 100 Afro-Caribbeans). In addition, we have characterized the sibling cohort for other atherogenic and thrombogenic risk factors., Results: The prevalence of Pl(A2)-positive individuals (Pl(A2)[+], Pl(A1/A2) heterozygotes plus Pl(A2/A2) homozygotes) in the sibling cohort was high: 41.4%. When analyzed separately, the prevalence of Pl(A2)(+) siblings was 53.3% among Caucasians and 28.6% among Afro-Caribbeans. There was no association between Pl(A2) and other established atherogenic or thrombogenic risk factors. Interestingly, the clustering of other risk factors was lesser among Pl(A2)(+) siblings than their Pl(A1) counterparts., Conclusions: This study supports the hypothesis that the prevalence of Pl(A2)(+) individuals is high in kindreds with premature CHD. Hence, like the established risk factors that tend to cluster in families with premature CHD and contribute strongly to the accelerated atherosclerotic process affecting these individuals, the Pl(A2) polymorphism of GPIIIa may represent an inherited risk that promotes the thromboembolic complications of CHD. That these asymptomatic Pl(A2)(+) siblings had overall less established risk factors than their Pl(A1) counterparts might represent an explanation for why they remained asymptomatic despite their Pl(A2) positivity.

Background: Platelet glycoprotein IIb/IIIa is a membrane receptor for fibrinogen and von Willebrand factor, and it has an important role in platelet aggregation. It is known to be involved in the pathogenesis of acute coronary syndromes. Previously, we found a high frequency of a particular polymorphism, PlA2, of the gene encoding glycoprotein IIIa in kindreds with a high prevalence of premature myocardial infarction., Methods: To investigate the relation between the PlA2 polymorphism and acute coronary syndromes, we conducted a case-control study of 71 case patients with myocardial infarction or unstable angina and 68 inpatient controls without known heart disease. The groups were matched for age, race, and sex. We used two methods to determine the PlA genotype: reverse dot blot hybridization and allele-specific restriction digestion., Results: The prevalence of PlA2 was 2.1 times higher among the case patients than among the controls (39.4 percent vs. 19.1 percent, P=0.01). In a subgroup of patients whose disease began before the age of 60 years, the prevalence of PlA2 was 50 percent, a value that was 3.6 times that among control subjects under 60 years of age (13.9 percent, P=0.002). Among subjects with the PlA2 polymorphism, the odds ratio for having a coronary event was 2.8 (95 percent confidence interval, 1.2 to 6.4). In the patients less than 60 years of age at the onset of disease, the odds ratio was 6.2 (95 percent confidence interval, 1.8 to 22.4)., Conclusions: We observed a strong association between the PlA2 polymorphism of the glycoprotein IIIa gene and acute coronary thrombosis, and this association was strongest in patients who had had coronary events before the age of 60 years.