The key characteristics of cardiotoxicity for the pervasive pollutant phenanthrene.

The key characteristic (KCs) framework has been used previously to assess the carcinogenicity and cardiotoxicity of various chemical and pharmacological agents. Here, the 12 KCs of cardiotoxicity are used to evaluate the previously reported cardiotoxicity of phenanthrene (Phe), a tricyclic polycyclic aromatic hydrocarbon (PAH), and major component of fossil fuel-derived air pollution. Phe is a semi-volatile pollutant existing in both the gas phase and particle phase through adsorption onto or into particulate matter (PM). Phe can translocate across the airways and gastrointestinal tract into the systemic circulation, enabling body-wide effects. Our evaluation based on a comprehensive literature review, indicates Phe exhibits 11 of the 12 KCs for cardiotoxicity. These include adverse effects on cardiac electromechanical performance, the vasculature and endothelium, immunomodulation and oxidative stress, and neuronal and endocrine control. Environmental agents that have similarly damaging effects on the cardiovascular system are heavily regulated and monitored, yet globally there is no air quality regulation specific for PAHs like Phe. Environmental monitoring of Phe is not the international standard with benzo[a]pyrene being frequently used as a proxy despite the two PAH species exhibiting significant differences in sources, concentration variations and toxic effects. The evidence summarised in this evaluation highlights the need to move away from proxied PAH measurements and develop a monitoring network capable of measuring Phe concentration. It also stresses the need to raise awareness amongst the medical community of the potential cardiovascular impact of PAH exposure. This will allow the production of mitigation strategies and possibly the development of new policies for the protection of the societal groups most vulnerable to cardiovascular disease.

Origin of Octafluorocyclopentene Polyelectrophilicity.

Octafluorocyclopentene (OFCP) has found utility as a polyelectrophile in substitution cascades that form complex macrocyclic compounds. The Harran group synthesis of macrocyclic polypeptides depends on OFCP as a linker, combining with four different nucleophilic units of a polypeptide. We report a computational investigation of the origins of OFCP reactivity and a rationale for controlled mono-, di-, tri-, and tetrasubstitution of fluoride ions by heteroatomic nucleophiles. The roles of inductive, negative hyperconjugative, and resonance electron-donation by fluoride substituents are explored for the reaction of OFCP, less-fluorinated analogues, and common electrophilic alkenes with several different nucleophiles.

Look away now! Defensive processing and unrealistic optimism by level of alcohol consumption.

OBJECTIVE: Health risk information is insufficient as a means of reducing alcohol use, particularly when it evokes negative emotional states amongst those for whom it is most personally relevant. Appraisal biases, or 'defensive processing', may be employed to mitigate the psychological discomfort posed by such information. Few studies have evaluated the role of defensive processing in people with different levels of alcohol consumption. DESIGN: Online participants (n = 597) completed measures of defensive processing of a health risk infographic, perceived susceptibility and severity of alcohol use, efficacy for resisting alcohol use, unrealistic optimism, the Alcohol Use Disorder Identification Test - Consumption (AUDIT-C) and demographics. RESULTS: AUDIT-C scores were positively and linearly associated with all defensive processing measures (Pearson's correlation r from.16 to .36), threat and susceptibility (r = .16) and unrealistic optimism (r = .50). AUDIT-C scores were also negatively associated with efficacy for controlling alcohol use (r = -0.48). CONCLUSION: People with alcohol use disorder (AUD) engaged in much more defensive processing of alcohol-related messages, offering an explanation for why such messages are limited at eliciting behaviour change. High levels of unrealistic optimism in people with alcohol use disorder may reflect low problem recognition in order to maintain a problem-free drinking identity.

Behavioral Outcomes and Neurodevelopmental Disorders Among Children of Women With Epilepsy.

Importance: The association of fetal exposure to antiseizure medications (ASMs) with outcomes in childhood are not well delineated. Objective: To examine the association of fetal ASM exposure with subsequent adaptive, behavioral or emotional, and neurodevelopmental disorder outcomes at 2, 3, and 4.5 years of age. Design, Setting, and Participants: The Maternal Outcomes and Neurodevelopmental Effects of Antiepileptic Drugs (MONEAD) study is a prospective, observational cohort study conducted at 20 epilepsy centers in the US. A total of 456 pregnant women with epilepsy or without epilepsy were enrolled from December 19, 2012, to January 13, 2016. Children of enrolled women were followed up with formal assessments at 2, 3, 4.5, and 6 years of age. Statistical analysis took place from August 2022 to May 2023. Exposures: Exposures included mother's epilepsy status as well as mother's ASM blood concentration in the third trimester (for children of women with epilepsy). Women with epilepsy were enrolled regardless of ASM regimen. Main Outcomes and Measures: The primary outcome was the Adaptive Behavior Assessment System, Third Edition (ABAS-3) General Adaptive Composite (GAC) score among children at 4.5 years of age. Children of women with epilepsy and children of women without epilepsy were compared, and the associations of ASM exposures with outcomes among exposed children were assessed. Secondary outcomes involved similar analyses of other related measures. Results: Primary analysis included 302 children of women with epilepsy (143 boys [47.4%]) and 84 children of women without epilepsy (45 boys [53.6%]). Overall adaptive functioning (ABAS-3 GAC score at 4.5 years) did not significantly differ between children of women with epilepsy and children of women without epilepsy (parameter estimate [PE], 0.4 [95% CI, -2.5 to 3.4]; P = .77). However, in adjusted analyses, a significant decrease in functioning was seen with increasing third-trimester maximum ASM blood concentrations (PE, -7.8 [95% CI, -12.6 to -3.1]; P = .001). This decrease in functioning was evident for levetiracetam (PE, -18.9 [95% CI, -26.8 to -10.9]; P < .001) and lamotrigine (PE, -12.0 [95% CI, -23.7 to -0.3]; P = .04), the ASMs with sample sizes large enough for analysis. Results were similar with third-trimester maximum daily dose. Conclusions and Relevance: This study suggests that adaptive functioning of children of women with epilepsy taking commonly used ASMs did not significantly differ from that of children of women without epilepsy, but there was an exposure-dependent association of ASMs with functioning. Thus, psychiatric or psychological screening and referral of women with epilepsy and their offspring are recommended when appropriate. Additional research is needed to confirm these findings.

Genomic Characteristion of Opportunistic Pathogen Kluyvera Reveals a Novel CTX-M Subgroup.

A rising incidence of clinical infections has been caused by Kluyvera, a significant opportunistic pathogen. Meanwhile, Kluyvera acts as an important reservoir of blaCTX-Ms, which are the dominant genes of class A extended-spectrum ß-lactamases (ESBLs). In this work, 60 strains of Kluyvera were subjected to phylogenetic relationship reconstruction, antimicrobial susceptibility testing, and antibiotic resistance genes prediction. All mature blaCTX-Ms were gathered to perform subgroup reclassification. The findings demonstrate that Kluyvera has a large gene pool with significant genetic flexibility. Notably, 25% of strains showed simultaneous detection of ESBLs and carbapenem resistance genes. The genotypes of fourteen novel blaCTX-Ms were identified. A new subgroup classification approach for blaCTX-Ms was defined by using 20 amino acid site variants, which could split blaCTX-Ms into 10 subgroups. The results of the subgroup division were consistent with the phylogenetic clustering. More significantly, we proposed a novel blaCTX-M subgroup, KLUS, that is chromosomally encoded in K. sichuanensis and the new species put forward in this study, showing amino acid differences from the currently known sequences. Cloning and transformation tests demonstrated that the recipient bacteria had a robust phenotype of cefotaxime resistance. Closely related Kluyvera species had blaCTX-Ms in the same subgroup. Our research lays the groundwork for a deeper comprehension of Kluyvera and emphasizes how important a blaCTX-M reservoir it is. We provide an update on blaCTX-M subgroups reclassification from the aspects of phylogenetic relationship, amino acid differences, and the new subgroup KLUS, which needs to be strengthen monitored due to its strong resistance phenotype to cefotaxime.

Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis. Stimulation of exocytosis by AMPK is mediated by Rab10 and the Rab GTPase-activating protein TBC1D4. APEX2 proximity mapping reveals that GLUT4 traverses both PM-proximal and PM-distal compartments in unstimulated muscle cells, further supporting retention of GLUT4 by a constitutive retrieval mechanism. AMPK-stimulated translocation involves GLUT4 redistribution among the same compartments traversed in unstimulated cells, with a significant recruitment of GLUT4 from the Golgi and trans-Golgi network compartments. Our comprehensive proximal protein mapping provides an integrated, high-density, whole-cell accounting of the localization of GLUT4 at a resolution of ∼20 nm that serves as a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in a physiologically relevant cell type.

Neurodevelopmental outcomes in children and adults with Fetal Valproate Spectrum Disorder: A contribution from the ConcePTION project.

AIM: To describe the neurodevelopmental phenotype of older children and adults with a diagnosis of Fetal Valproate Spectrum Disorder (FVSD). METHODS: In this cross-sectional study, 90 caregivers were recruited and completed a series of questionnaires regarding the neurodevelopmental outcomes of 146 individuals aged 7-37 years (M = 18.1), including individuals with a formal diagnosis of FVSD (n = 99), individuals exposed to Valproate but without an FVSD diagnosis (n = 24), and individuals not exposed to Valproate (N = 23). The mean dose of valproate exposure for individuals with an FVSD diagnosis was 1470 mg/day. RESULTS: Individuals with a diagnosis of FVSD showed significantly higher levels of moderate (43.4%) and severe (14.4%) cognitive impairment than other groups (p = 0.003), high levels of required formal educational support (77.6%), and poorer academic competence than individuals not exposed to Valproate (p = 0.001). Overall psychosocial problems (p = 0.02), internalising problems (p = 0.05) and attention problems (p = 0.001), but not externalising problems, were elevated in individuals with a diagnosis of FVSD. Rates of neurodevelopmental disorders, particularly autistic spectrum disorders (62.9%) and sensory problems (80.6%) are particularly central to the FVSD phenotype. There was no evidence of a statistical dose-dependent effect, possibly due to the high mean dose of exposure having a uniformly negative impact across the sample. Individuals with FVSD had required a significant number of health and child development services. INTERPRETATION: Children and young adults with a diagnosis of FVSD are at an increased risk of a range of altered neurodevelopmental outcomes, highlighting the need for a multidisciplinary approach to clinical management across the lifespan.

GWAS of random glucose in 476,326 individuals provide insights into diabetes pathophysiology, complications and treatment stratification.

Conventional measurements of fasting and postprandial blood glucose levels investigated in genome-wide association studies (GWAS) cannot capture the effects of DNA variability on 'around the clock' glucoregulatory processes. Here we show that GWAS meta-analysis of glucose measurements under nonstandardized conditions (random glucose (RG)) in 476,326 individuals of diverse ancestries and without diabetes enables locus discovery and innovative pathophysiological observations. We discovered 120 RG loci represented by 150 distinct signals, including 13 with sex-dimorphic effects, two cross-ancestry and seven rare frequency signals. Of these, 44 loci are new for glycemic traits. Regulatory, glycosylation and metagenomic annotations highlight ileum and colon tissues, indicating an underappreciated role of the gastrointestinal tract in controlling blood glucose. Functional follow-up and molecular dynamics simulations of lower frequency coding variants in glucagon-like peptide-1 receptor (GLP1R), a type 2 diabetes treatment target, reveal that optimal selection of GLP-1R agonist therapy will benefit from tailored genetic stratification. We also provide evidence from Mendelian randomization that lung function is modulated by blood glucose and that pulmonary dysfunction is a diabetes complication. Our investigation yields new insights into the biology of glucose regulation, diabetes complications and pathways for treatment stratification.

Ancestral Origin and Dissemination Dynamics of Reemerging Toxigenic Vibrio cholerae, Haiti.

The 2010 cholera epidemic in Haiti was thought to have ended in 2019, and the Prime Minister of Haiti declared the country cholera-free in February 2022. On September 25, 2022, cholera cases were again identified in Port-au-Prince. We compared genomic data from 42 clinical Vibrio cholerae strains from 2022 with data from 327 other strains from Haiti and 1,824 strains collected worldwide. The 2022 isolates were homogeneous and closely related to clinical and environmental strains circulating in Haiti during 2012-2019. Bayesian hypothesis testing indicated that the 2022 clinical isolates shared their most recent common ancestor with an environmental lineage circulating in Haiti in July 2018. Our findings strongly suggest that toxigenic V. cholerae O1 can persist for years in aquatic environmental reservoirs and ignite new outbreaks. These results highlight the urgent need for improved public health infrastructure and possible periodic vaccination campaigns to maintain population immunity against V. cholerae.

Seafood-Associated Outbreak of ctx-Negative Vibrio mimicus Causing Cholera-Like Illness, Florida, USA.

Vibrio mimicus caused a seafood-associated outbreak in Florida, USA, in which 4 of 6 case-patients were hospitalized; 1 required intensive care for severe diarrhea. Strains were ctx-negative but carried genes for other virulence determinants (hemolysin, proteases, and types I-IV and VI secretion systems). Cholera toxin-negative bacterial strains can cause cholera-like disease.

Vibrio metschnikovii as an emergent pathogen: analyses of phylogeny and O-antigen and identification of possible virulence characteristics.

Vibrio metschnikovii is an emergent pathogen that causes human infections which may be fatal. However, the phylogenetic characteristics and pathogenicity determinants of V. metschnikovii are poorly understood. Here, the whole-genome features of 103 V. metschnikovii strains isolated from different sources are described. On phylogenetic analysis V. metschnikovii populations could be divided into two major lineages, defined as lineage 1 (L1) and 2 (L2), of which L1 was more likely to be associated with human activity. Meanwhile, we defined 29 V. metschnikovii O-genotypes (VMOg, named VMOg1-VMOg29) by analysis of the O-antigen biosynthesis gene clusters (O-AGCs). Most VMOgs (VMOg1 to VMOg28) were assembled by the Wzx/Wzy pathway, while only VMOg29 used the ABC transporter pathway. Based on the sequence variation of the wzx and wzt genes, an in silico O-genotyping system for V. metschnikovii was developed. Furthermore, nineteen virulence-associated factors involving 161 genes were identified within the V. metschnikovii genomes, including genes encoding motility, adherence, toxins, and secretion systems. In particular, V. metschnikovii was found to promote a high level of cytotoxicity through the synergistic action of the lateral flagella and T6SS. The lateral flagellar-associated flhA gene played an important role in the adhesion and colonization of V. metschnikovii during the early stages of infection. Overall, this study provides an enhanced understanding of the genomic evolution, O-AGCs diversity, and potential pathogenic features of V. metschnikovii.

Primary aldosteronism: molecular medicine meets public health.

Primary aldosteronism is the most common single cause of hypertension and is potentially curable when only one adrenal gland is the culprit. The importance of primary aldosteronism to public health derives from its high prevalence but huge under-diagnosis (estimated to be <1% of all affected individuals), despite the consequences of poor blood pressure control by conventional therapy and enhanced cardiovascular risk. This state of affairs is attributable to the fact that the tools used for diagnosis or treatment are still those that originated in the 1970-1990s. Conversely, molecular discoveries have transformed our understanding of adrenal physiology and pathology. Many molecules and processes associated with constant adrenocortical renewal and interzonal metamorphosis also feature in aldosterone-producing adenomas and aldosterone-producing micronodules. The adrenal gland has one of the most significant rates of non-silent somatic mutations, with frequent selection of those driving autonomous aldosterone production, and distinct clinical presentations and outcomes for most genotypes. The disappearance of aldosterone synthesis and cells from most of the adult human zona glomerulosa is the likely driver of the mutational success that causes aldosterone-producing adenomas, but insights into the pathways that lead to constitutive aldosterone production and cell survival may open up opportunities for novel therapies.

Cascade Synthesis of Fluorinated Spiroheterocyclic Scaffolding for Peptidic Macrobicycles.

Octafluorocyclopentene (OFCP) engages linear, unprotected peptides in polysubstitution cascades that generate complex fluorinated polycycles. The reactions occur in a single flask at 0-25 °C and require no catalysts or heavy metals. OFCP can directly polycyclize linear sequences using native functionality, or fluorospiroheterocyclic intermediates can be intercepted with exogenous nucleophiles. The latter tactic generates molecular hybrids composed of peptides, sugars, lipids, and heterocyclic components. The platform can create stereoisomers of both single- and double-looped macrocycles. Calculations indicate that the latter can mimic diverse protein surface loops. Subsets of the molecules have low energy conformers that shield the polar surface area through intramolecular hydrogen bonding. A significant fraction of OFCP-derived macrocycles tested show moderate to high passive permeability in parallel artificial membrane permeability assays.

Chronic UCN2 treatment desensitizes CRHR2 and improves insulin sensitivity.

Urocortin 2 (UCN2) acts as a ligand for the G protein-coupled receptor corticotropin-releasing hormone receptor 2 (CRHR2). UCN2 has been reported to improve or worsen insulin sensitivity and glucose tolerance in vivo. Here we show that acute dosing of UCN2 induces systemic insulin resistance in male mice and skeletal muscle. Inversely, chronic elevation of UCN2 by injection with adenovirus encoding UCN2 resolves metabolic complications, improving glucose tolerance. CRHR2 recruits Gs in response to low concentrations of UCN2, as well as Gi and ß-Arrestin at high concentrations of UCN2. Pre-treating cells and skeletal muscle ex vivo with UCN2 leads to internalization of CRHR2, dampened ligand-dependent increases in cAMP, and blunted reductions in insulin signaling. These results provide mechanistic insights into how UCN2 regulates insulin sensitivity and glucose metabolism in skeletal muscle and in vivo. Importantly, a working model was derived from these results that unifies the contradictory metabolic effects of UCN2.

Cognitive outcomes at age 3 years in children with fetal exposure to antiseizure medications (MONEAD study) in the USA: a prospective, observational cohort study.

BACKGROUND: The neurodevelopmental effects of fetal exposure to most antiseizure medications are unclear. We aimed to investigate the effects of fetal exposure to commonly used antiseizure medications on neuropsychological outcomes at age 3 years. METHODS: The Maternal Outcomes and Neurodevelopmental Effects of Antiepileptic Drugs (MONEAD) study is a prospective, observational, multicentre cohort study at 20 specialty epilepsy centres in the USA. We have investigated pregnancy outcomes in women (aged 14-45 years) with and without epilepsy who were enrolled during pregnancy (≤20 weeks' gestational age), and their children. The primary outcome for children at age 3 years was a blindly assessed Verbal Index score, which was calculated by averaging scores on the Naming Vocabulary and Verbal Comprehension subtests of Differential Ability Scales-II, Expressive Communication and Auditory Comprehension subscales of Preschool Language Scale-5, and Peabody Picture Vocabulary Test-4. Children of women with and without epilepsy were compared, and the associations of medication exposures to outcomes in exposed children were assessed. The MONEAD study is registered with ClinicalTrials.gov, NCT0730170, and is ongoing. FINDINGS: Between Dec 19, 2012, and Jan 13, 2016, 456 pregnant women (351 with epilepsy and 105 without epilepsy) were enrolled into the study. 345 children were born to women with epilepsy and 106 children were born to women without epilepsy. Verbal Index scores at age 3 years did not differ for children of women with epilepsy (n=284; adjusted least-square mean 102·7, 95% CI 101·4 to 103·9) versus those without epilepsy (n=87; 102·3, 99·8 to 104·7). Significant risk factors for reduced Verbal Index scores included maternal intelligence quotient, maternal education, post-birth anxiety, gestational age at enrolment, child's sex, and child's ethnicity. For Verbal Index scores, antiseizure medication exposure effects were not seen for maximum third trimester blood concentrations (n=258; adjusted parameter estimate -2·9, 95% CI -6·7 to 1·0). However, in secondary analyses, exposure-dependent effects were present on multiple cognitive measures, which varied by medication. INTERPRETATION: We found no difference in neurodevelopmental outcomes between children with fetal exposure to newer antiseizure medications compared with unexposed children. However, some exposure-dependent antiseizure medication effects were seen in secondary analyses. The adverse effects of maternal post-birth anxiety emphasise the importance of screening mothers during pregnancy and postpartum and implementing interventions. Additional studies are needed to clarify the exposure-dependent effects. FUNDING: National Institutes of Health, National Institute of Neurological Disorders and Stroke, and National Institute of Child Health and Development.

Mayaro virus detection by integrating sample preparation with isothermal amplification in portable devices.

Mayaro virus (MAYV) is an emerging mosquito-borne alphavirus that causes clinical symptoms similar to those caused by Chikungunya virus (CHIKV), Dengue virus (DENV), and Zika virus (ZIKV). To differentiate MAYV from these viruses diagnostically, we have developed a portable device that integrates sample preparation with real-time, reverse-transcription, loop-mediated isothermal amplification (rRT-LAMP). First, we designed a rRT-LAMP assay targeting MAYV's non-structural protein (NS1) gene and determined the limit of detection of at least 10 viral genome equivalents per reaction. The assay was specific for MAYV, without cross-reactions with CHIKV, DENV, or ZIKV. The rRT-LAMP assay was integrated with a sample preparation device (SPD) wherein virus lysis and RNA enrichment/purification were carried out on the spot, without requiring pipetting, while subsequent real-time amplification device (RAD) enables virus detection at the point of care (POC). The functions of our platform were demonstrated using purified MAYV RNA or blood samples containing viable viruses. We have used the devices for detection of MAYV in as short as 13 min, with limit of detection to as low as 10 GEs/reaction.

Efficacy of Peracetic Acid and Sodium Hypochlorite against SARS-CoV-2 on Contaminated Surfaces.

SARS-CoV-2 is primarily a respiratory virus that can potentially be transmitted through fomites. Sodium hypochlorite (NaOCl) and peracetic acid (PAA) are widely used disinfectants on surfaces in diverse settings such as hospitals and food production facilities. The objectives of this study were to investigate the virucidal efficacy of NaOCl and PAA against SARS-CoV-2 using the ASTM standard methods. In the suspension assay, NaOCl and PAA (5, 50, and 200 ppm) were tested against SARS-CoV-2 in the presence/absence of soil load after 1 min of contact time. In the carrier assay, NaOCl and PAA were tested at 200, 400, 600, and 1,000 ppm for 1 min and 200 and 1,000 ppm for 5 and 10 min. Stainless steel (SS) and high-density polyethylene (HDPE) disks were used as carriers. The virus was suspended in soil load and the disinfectants were prepared in 300 ppm of hard water. Virus quantification was done by TCID50 assay using Vero-E6 cell line. NaOCl and PAA were effective (> 3 log reduction in infectious virus) at 50 ppm in the absence of soil load. However, in the presence of soil load, 200 ppm was required for > 3 log reduction in virus infectivity. In contrast, NaOCl and PAA at 200 ppm and with a 1-min contact time were not effective against SARS-CoV-2 on either SS or HDPE surfaces. PAA at 200 ppm for 10 min was effective against SARS-CoV-2 on SS and HDPE surfaces, whereas NaOCl required 1,000 ppm for 10 min to be effective against SARS-CoV-2 on both surfaces. IMPORTANCE In the context of the COVID-19 pandemic, the World Health Organization (WHO) recommended the use of chlorine-based products such as sodium hypochlorite (NaOCl) at 1,000 ppm for a minimum of 1 min to disinfect environmental surfaces. However, this recommendation was not based on validated studies on the actual SARS-CoV-2 itself. In fact, over half of the chemical disinfectants, including many peracetic acid products, listed in EPA List N were approved based on "kills a harder-to-kill pathogen" without further validation on SARS-CoV-2. Research on SARS-CoV-2 is restricted to BSL3 laboratories and the urgency of tackling the pandemic might explain the lack of studies on the actual virus. Our results show that the WHO recommendation of 1 min contact time with 1,000 ppm NaOCl is not effective against SARS-CoV-2 on surfaces. Also, our results indicate that PAA is effective against SARS-CoV-2 on surfaces and can be used as safer and more environmentally friendly alternative to NaOCl at a lower concentration.

GLUT4 dynamic subcellular localization is controlled by AMP kinase activation as revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport into muscle and adipocytes, central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter in the plasma membrane ( PM ). Physiologic signals (activated insulin receptor or AMP kinase [ AMPK ]), acutely increase PM GLUT4 to enhance glucose uptake. Here we show in kinetic studies that intracellular GLUT4 is in equilibrium with the PM in unstimulated cultured human skeletal muscle cells, and that AMPK promotes GLUT4 redistribution to the PM by regulating both exocytosis and endocytosis. AMPK-stimulation of exocytosis requires Rab10 and Rab GTPase activating protein TBC1D4, requirements shared with insulin control of GLUT4 in adipocytes. Using APEX2 proximity mapping, we identify, at high-density and high-resolution, the GLUT4 proximal proteome, revealing GLUT4 traverses both PM proximal and distal compartments in unstimulated muscle cells. These data support intracellular retention of GLUT4 in unstimulated muscle cells by a dynamic mechanism dependent on the rates of internalization and recycling. AMPK promoted GLUT4 translocation to the PM involves redistribution of GLUT4 among the same compartments traversed in unstimulated cells, with a significant redistribution of GLUT4 from the PM distal Trans Golgi Network Golgi compartments. The comprehensive proximal protein mapping provides an integrated, whole cell accounting of GLUT4's localization at a resolution of ∼20 nm, a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in physiologically relevant cell type and as such, sheds new light on novel key pathways and molecular components as potential therapeutic approaches to modulate muscle glucose uptake.

Tolerability, safety and pharmacodynamics of oral, small-molecule glucagon-like peptide-1 receptor agonist danuglipron for type 2 diabetes: A 12-week, randomized, placebo-controlled, Phase 2 study comparing different dose-escalation schemes.

AIM: To evaluate the tolerability, safety and pharmacodynamics of different dose-escalation schemes of the oral small-molecule glucagon-like peptide-1 receptor (GLP-1R) agonist danuglipron. MATERIALS AND METHODS: This Phase 2a, double-blind, placebo-controlled, parallel-group study randomly assigned adults with type 2 diabetes (T2D) treated with metformin to placebo or danuglipron (low [5-mg] or high [10-mg] starting dose, with 1- or 2-week dose-escalation steps, to target doses of 80, 120 or 200 mg twice daily [BID]) and adults with obesity without diabetes to placebo or danuglipron 200 mg BID. RESULTS: Participants with T2D (n = 123, mean glycated haemoglobin [HbA1c] 8.19%) or obesity without diabetes (n = 28, mean body mass index 37.3 kg/m2 ) were randomly assigned and treated. Discontinuation from study medication occurred in 27.3% to 72.7% of participants across danuglipron groups versus 16.7% to 18.8% for placebo, most often due to adverse events. Nausea (20.0%-47.6% of participants across danuglipron groups vs. 12.5% for placebo) and vomiting (18.2%-40.9% danuglipron vs. 12.5% placebo, respectively) were most commonly reported in participants with T2D. Gastrointestinal adverse events were generally related to danuglipron target dose and were not substantially affected by starting dose. In participants with T2D, least squares mean changes from baseline in HbA1c (-1.04% to -1.57% across danuglipron groups vs. -0.32% for placebo), fasting plasma glucose (-23.34 mg/dL to -53.94 mg/dL danuglipron vs. -13.09 mg/dL placebo) and body weight (-1.93 to -5.38 kg danuglipron vs. -0.42 kg placebo) at Week 12 were generally statistically significant for danuglipron compared with placebo (P < 0.05). CONCLUSIONS: Danuglipron resulted in statistically significant reductions in HbA1c, FPG and body weight over 12 weeks, in the setting of higher discontinuation rates and incidence of gastrointestinal adverse events with higher target doses. CLINICALTRIALS: gov identifier: NCT04617275.