Dental sculpting and compensatory shearing crests demonstrated in a Wear series of Presbytis rubicunda (Cercopithecoidea, Colobidae) with dental topography analysis.

OBJECTIVES: Maintaining effective and efficient occlusal morphology presents adaptive challenges for mammals, particularly because mastication produces interactions with foods and other materials that alters the geometry of occlusal surfaces through macrowear and/or catastrophic failure (i.e. "chipping"). Altered occlusal morphologies are often less effective for masticating materials of given diet-but not always-some species exhibit dental sculpting, meaning their dentitions are set up to harness macrowear to hone their occlusal surfaces into more effective morphologies (i.e. secondary morphologies). Here we show that dental sculpting is present in the folivorous Presbytis rubicunda of Borneo. METHODS: Thirty-one undamaged lower second molars of P. rubicunda exhibiting various stages of macroscopic wear were micro-CT scanned and processed into digital surfaces. The surfaces were measured for convex Dirichlet normal energy (vDNE, a measure of surface sharpness), and degree of surface wear. Regression analyses compared surface sharpness with several measures of wear to test for the presence and magnitude of dental sculpting. RESULTS: Positive correlations between the wear proxies and vDNE reveal that P. rubicunda wear in such a way as to become sharper, and therefore more effective chewing surfaces by exposing enamel-dentine junctions on their occlusal surfaces and then honing these junctions into sharpened edges. Compared to another primate folivore in which increasing surface sharpness with macrowear has been demonstrated (i.e., Alouatta palliata), the worn surfaces are similarly sharp, but the dental sculpting process appears to be different. DISCUSSION: The results presented here suggest that not only do some primates exhibit dental sculpting and the attendant secondary morphology, but that there appear to be multiple different morphological configurations that can achieve this result. P. rubicunda has thicker enamel and a more stereotyped wear pattern than A. palliata, although both show positive correlations of occlusal surface sharpness (vDNE) with various wear proxies. These findings shed light on the varied approaches for the maintenance of effective and efficient occlusal surfaces in primates.

Motilin fluctuations in healthy volunteers determined by liquid chromatography mass spectrometry.

Introduction: Motilin is a hormone secreted by specialised enteroendocrine cells in the small intestine, and is known to modulate gastrointestinal motility in humans, regulating the migratory motor complex. It is understudied at least in part due to the lack of commercially available immunoassays. Method: A multiplexed liquid chromatography mass spectrometry (LC-MS/MS) method was optimised to measure motilin, insulin, C-peptide, GIP (1-42) and GIP (3-42). Corresponding active ghrelin concentrations were determined by immunoassay. Ten healthy volunteers with no prior history of gastroenterological or endocrine condition attended after overnight fast and had blood samples taken every 15 minutes for 4 hours whilst continuing to fast, and then further sampling for 2 hours following a liquid mixed meal. Hunger scores were taken at each time point using a visual analogue scale. Normal bowel habit was confirmed by 1 week stool diary. Results: Motilin levels fluctuated in the fasting state with an average period between peaks of 109.5 mins (SD:30.0), but with no evidence of a relationship with either ghrelin levels or hunger scores. The mixed meal interrupted cyclical motilin fluctuations, increased concentrations of motilin, insulin, C-peptide, GIP(1-42) and GIP(3-42), and suppressed ghrelin levels. Discussion: This study highlights the utility of LC-MS/MS for parallel measurement of motilin alongside other peptide hormones, and supports previous reports of the cyclical nature of motilin levels in the fasting state and interruption with feeding. This analytical method has utility for further clinical studies into motilin and gut hormone physiology in human volunteers.

Efficacy of oral 20-hydroxyecdysone (BIO101), a MAS receptor activator, in adults with severe COVID-19 (COVA): a randomized, placebo-controlled, phase 2/3 trial.

Background: SARS-CoV-2 binding to ACE2 is potentially associated with severe pneumonia due to COVID-19. The aim of the study was to test whether Mas-receptor activation by 20-hydroxyecdysone (BIO101) could restore the Renin-Angiotensin System equilibrium and limit the frequency of respiratory failure and mortality in adults hospitalized with severe COVID-19. Methods: Double-blind, randomized, placebo-controlled phase 2/3 trial. Randomization: 1:1 oral BIO101 (350 mg BID) or placebo, up to 28 days or until an endpoint was reached. Primary endpoint: mortality or respiratory failure requiring high-flow oxygen, mechanical ventilation, or extra-corporeal membrane oxygenation. Key secondary endpoint: hospital discharge following recovery (ClinicalTrials.gov Number, NCT04472728). Findings: Due to low recruitment the planned sample size of 310 was not reached and 238 patients were randomized between August 26, 2020 and March 8, 2022. In the modified ITT population (233 patients; 126 BIO101 and 107 placebo), respiratory failure or early death by day 28 was 11.4% lower in the BIO101 (13.5%) than in the placebo (24.3%) group, (p = 0.0426). At day 28, proportions of patients discharged following recovery were 80.1%, and 70.9% in the BIO101 and placebo group respectively, (adjusted difference 11.0%, 95% CI [-0.4%, 22.4%], p = 0.0586). Hazard Ratio for time to death over 90 days: 0.554 (95% CI [0.285, 1.077]), a 44.6% mortality reduction in the BIO101 group (not statistically significant). Treatment emergent adverse events of respiratory failure were more frequent in the placebo group. Interpretation: BIO101 significantly reduced the risk of death or respiratory failure supporting its use in adults hospitalized with severe respiratory symptoms due to COVID-19. Funding: Biophytis.

Trial of N-Acetyl-l-Leucine in Niemann-Pick Disease Type C.

BACKGROUND: Niemann-Pick disease type C is a rare lysosomal storage disorder. We evaluated the safety and efficacy of N-acetyl-l-leucine (NALL), an agent that potentially ameliorates lysosomal and metabolic dysfunction, for the treatment of Niemann-Pick disease type C. METHODS: In this double-blind, placebo-controlled, crossover trial, we randomly assigned patients 4 years of age or older with genetically confirmed Niemann-Pick disease type C in a 1:1 ratio to receive NALL for 12 weeks, followed by placebo for 12 weeks, or to receive placebo for 12 weeks, followed by NALL for 12 weeks. NALL or matching placebo was administered orally two to three times per day, with patients 4 to 12 years of age receiving weight-based doses (2 to 4 g per day) and those 13 years of age or older receiving a dose of 4 g per day. The primary end point was the total score on the Scale for the Assessment and Rating of Ataxia (SARA; range, 0 to 40, with lower scores indicating better neurologic status). Secondary end points included scores on the Clinical Global Impression of Improvement, the Spinocerebellar Ataxia Functional Index, and the Modified Disability Rating Scale. Crossover data from the two 12-week periods in each group were included in the comparisons of NALL with placebo. RESULTS: A total of 60 patients 5 to 67 years of age were enrolled. The mean baseline SARA total scores used in the primary analysis were 15.88 before receipt of the first dose of NALL (60 patients) and 15.68 before receipt of the first dose of placebo (59 patients; 1 patient never received placebo). The mean (±SD) change from baseline in the SARA total score was -1.97±2.43 points after 12 weeks of receiving NALL and -0.60±2.39 points after 12 weeks of receiving placebo (least-squares mean difference, -1.28 points; 95% confidence interval, -1.91 to -0.65; P<0.001). The results for the secondary end points were generally supportive of the findings in the primary analysis, but these were not adjusted for multiple comparisons. The incidence of adverse events was similar with NALL and placebo, and no treatment-related serious adverse events occurred. CONCLUSIONS: Among patients with Niemann-Pick disease type C, treatment with NALL for 12 weeks led to better neurologic status than placebo. A longer period is needed to determine the long-term effects of this agent in patients with Niemann-Pick disease type C. (Funded by IntraBio; ClinicalTrials.gov number, NCT05163288; EudraCT number, 2021-005356-10.).

Optimized LC-MS/MS Method for the Detection of ppCCK(21-44): A Surrogate to Monitor Human Cholecystokinin Secretion.

The hormone cholecystokinin (CCK) is secreted postprandially from duodenal enteroendocrine cells and circulates in the low picomolar range. Detection of this digestion and appetite-regulating hormone currently relies on the use of immunoassays, many of which suffer from insufficient sensitivity in the physiological range and cross-reactivity problems with gastrin, which circulates at higher plasma concentrations. As an alternative to existing techniques, a liquid chromatography and mass spectrometry-based method was developed to measure CCK-derived peptides in cell culture supernatants. The method was initially applied to organoid studies and was capable of detecting both CCK8 and an N-terminal peptide fragment (prepro) ppCCK(21-44) in supernatants following stimulation. Extraction optimization was performed using statistical modeling software, enabling a quantitative LC-MS/MS method for ppCCK(21-44) capable of detecting this peptide in the low pM range in human plasma and secretion buffer solutions. Plasma samples from healthy individuals receiving a standardized meal (Ensure) after an overnight fast were analyzed; however, the method only had sensitivity to detect ppCCK(21-44). Secretion studies employing human intestinal organoids and meal studies in healthy volunteers confirmed that ppCCK(21-44) is a suitable surrogate analyte for measuring the release of CCK in vitro and in vivo.

Assessing the Interchangeability of AVT02 and Humira® in Participants with Moderate­to­Severe Chronic Plaque Psoriasis: Pharmacokinetics, Efficacy, Safety, and Immunogenicity Results from a Multicenter, Double-Blind, Randomized, Parallel-Group Study.

BACKGROUND: The US Food and Drug Administration (FDA) interchangeability guidelines state that the primary endpoint in a switching study should assess the impact of switching between the proposed interchangeable product and the reference product on clinical pharmacokinetics (PK) and pharmacodynamics (if available), as these assessments are generally sensitive to changes in immunogenicity and/or exposure that may arise due to switching. In addition, interchangeability designation requires no clinically meaningful difference in safety and efficacy of switching between the biosimilar and reference, compared with when using the reference product alone. OBJECTIVES:  The aim of this study was to investigate the PK, immunogenicity, efficacy, and safety in participants undergoing repeated switches between Humira® and AVT02 as part of a global interchangeable development program. METHODS: This multicenter, randomized, double-blind, parallel-group study in patients with moderate-to-severe plaque psoriasis comprises three parts: lead-in period (weeks 1-12), switching module (weeks 12-28), and the optional extension phase (weeks 28-52). Following the lead-in period during which all participants received the reference product (80 mg in week 1, followed by 40 mg every other week), participants with a clinical response of ≥ 75% improvement in the Psoriasis Area and Severity Index (PASI75) were randomized 1:1 to receive AVT02 alternating with the reference product (switching arm) or reference product only (non-switching arm). At week 28, participants who were PASI50 responders could opt to take part in an open-label extension phase receiving AVT02 up to week 50, with an end of study visit at week 52. PK, safety, immunogenicity, and efficacy were evaluated at various timepoints throughout the study for both switching and non-switching arms. RESULTS: In total, 550 participants were randomized to switching (277) and non-switching arms (273). The switching versus non-switching arithmetic least square means ratio [90% confidence intervals (CIs)] was 101.7% (91.4-112.0%) for the area under the concentration-time curve over the dosing interval from weeks 26-28 (AUCtau, W26-28) and 108.1% (98.3-117.9%) for maximum concentration over the dosing interval from weeks 26-28 (Cmax, W26-28). The 90% CIs for the switching versus non-switching arithmetic means ratio for primary endpoints AUCtau, W26-28 and Cmax, W26-28 were within the prespecified limits of 80-125%, demonstrating comparable PK profiles between groups. In addition, the PASI, Dermatology Life Quality Index, and static Physician's Global Assessment efficacy scores were highly similar for both treatment groups. There were no clinically meaningful differences between the immunogenicity and safety assessments of repeated switching between AVT02 and the reference product, versus the reference product alone. CONCLUSIONS: This study demonstrated that the risk, in terms of safety or diminished efficacy of switching between the biosimilar and the reference product, is not greater than the risk of using the reference product alone, as required by the FDA for interchangeability designation. Beyond the scope of interchangeability, a consistent long-term safety and immunogenicity profile, with no impact on the trough levels up to 52 weeks, was established. CLINICAL TRIAL REGISTRATION: NCT04453137; date of registration: 1 July 2020.

Dental topography of the Oligocene anthropoids Aegyptopithecus zeuxis and Apidium phiomense: Paleodietary insights from analysis of wear series.

Fossil primate dietary inference is enhanced when ascertained through multiple, distinct proxies. Dental topography can be used to assess changes in occlusal morphology with macrowear, providing insight on tooth use and function across the lifespans of individuals. We measured convex Dirichlet normal energy-a dental topography metric reflecting occlusal sharpness of features such as cusps and crests-in macrowear series of the second mandibular molars of two African anthropoid taxa from ∼30 Ma (Aegyptopithecus zeuxis and Apidium phiomense). Wear was quantified via three proxies: occlusal dentine exposure, inverse relief index, and inverse occlusal relief. The same measurements were calculated on macrowear series of four extant platyrrhine taxa (Alouatta, Ateles, Plecturocebus, and Sapajus apella) to provide an analogical framework for dietary inference in the fossil taxa. We predicted that Ae. zeuxis and Ap. phiomense would show similar patterns in topographic change with wear to one another and to extant platyrrhine frugivores like Ateles and Plecturocebus. The fossil taxa have similar distributions of convex Dirichlet normal energy to one another, and high amounts of concave Dirichlet normal energy 'noise' in unworn molars-a pattern shared with extant hominids that may distort dietary interpretations. Inverse relief index was the most useful wear proxy for comparison among the taxa in this study which possess disparate enamel thicknesses. Contrary to expectations, Ae. zeuxis and Ap. phiomense both resemble S. apella in exhibiting an initial decline in convex Dirichlet normal energy followed by an increase at the latest stages of wear as measured by inverse relief index, lending support to previous suggestions that hard-object feeding played a role in their dietary ecology. Based on these results and previous analyses of molar shearing quotients, microwear, and enamel microstructure, we suggest that Ae. zeuxis had a pitheciine-like strategy of seed predation, whereas Ap. phiomense potentially consumed berry-like compound fruits with hard seeds.

Rapid and Quantitative Enrichment of Peptides from Plasma for Mass Spectrometric Analysis.

Mass spectrometric analysis of peptides enables the assignment of their exact mass and confirmation of all or a significant portion of the peptide's amino acid sequence. LC-MS/MS analysis has proven invaluable in peptidomics research and can identify new biomarkers and assign their circulatory concentrations to aid research into disease processes. However, due to the high background plasma protein content, which masks the presence of the naturally low abundance circulatory peptidome, extraction of peptides from plasma prior to mass spectrometric analysis is therefore crucial. Organic solvents efficiently precipitate these high molecular weight plasma proteins while leaving small molecular weight peptides in solution, providing a rapid and effective technique for separating peptides from the contaminating plasma proteins. A secondary cleanup step involving solid phase extraction is required to remove lipids and highly hydrophobic contaminants before LC-MS/MS analysis. The method described within this chapter is effective at enriching circulatory plasma peptides prior to LC-MS/MS analysis and has been used in multiple peptidomic studies to improve peptide detection and quantification. Peptides studied using this methodology include insulin, C-peptide, glucagon, PYY, GIP, and a number of other challenging gut peptide hormones. Quantitative analyses of peptides using the described method showed good correlation with existing immunoassays.

Efficacy and Safety of N-Acetyl-l-Leucine in Children and Adults With GM2 Gangliosidoses.

BACKGROUND AND OBJECTIVES: GM2 gangliosidoses (Tay-Sachs and Sandhoff diseases) are rare, autosomal recessive, neurodegenerative diseases with no available symptomatic or disease-modifying treatments. This clinical trial investigated N-acetyl-l-leucine (NALL), an orally administered, modified amino acid in pediatric (≥6 years) and adult patients with GM2 gangliosidoses. METHODS: In this phase IIb, multinational, open-label, rater-blinded study (IB1001-202), male and female patients aged ≥6 years with a genetically confirmed diagnosis of GM2 gangliosidoses received orally administered NALL for a 6-week treatment period (4 g/d in patients ≥13 years, weight-tiered doses for patients 6-12 years), followed by a 6-week posttreatment washout period. For the primary Clinical Impression of Change in Severity analysis, patient performance on a predetermined primary anchor test (the 8-Meter Walk Test or the 9-Hole Peg Test) at baseline, after 6 weeks on NALL, and again after a 6-week washout period was videoed and evaluated centrally by blinded raters. Secondary outcomes included assessments of ataxia, clinical global impression, and quality of life. RESULTS: Thirty patients between the age of 6 and 55 years were enrolled. Twenty-nine had an on-treatment assessment and were included in the primary modified intention-to-treat analysis. The study met its CI-CS primary end point (mean difference 0.71, SD = 2.09, 90% CI 0.00, 1.50, p = 0.039), as well as secondary measures of ataxia and global impression. NALL was safe and well tolerated, with no serious adverse reactions. DISCUSSION: Treatment with NALL was associated with statistically significant and clinically relevant changes in functioning and quality of life in patients with GM2 gangliosidosis. NALL was safe and well tolerated, contributing to an overall favorable risk:benefit profile. NALL is a promising, easily administered (oral) therapeutic option for these rare, debilitating diseases with immense unmet medical needs. TRIAL REGISTRATION INFORMATION: The trial is registered with ClinicalTrials.gov (NCT03759665; registered on November 30, 2018), EudraCT (2018-004406-25), and DRKS (DRKS00017539). The first patient was enrolled on June 7, 2019. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that NALL improves outcomes for patients with GM2 gangliosidoses.

LC-MS/MS based detection of circulating proinsulin derived peptides in patients with altered pancreatic beta cell function.

Routine immunoassays for insulin and C-peptide have the potential to cross-react with partially processed proinsulin products, although in healthy patients these are present at such low levels that the interference is insignificant. Elevated concentrations of proinsulin and des-31,32 proinsulin arising from pathological conditions, or injected insulin analogues, however can cause significant assay interferences, complicating interpretation. Clinical diagnosis and management therefore sometimes require methods that can distinguish true insulin and C-peptide from partially processed proinsulin or injected insulin analogues. In this scenario, the high specificity of mass spectrometric analysis offers potential benefit for patient care. A high throughput targeted LC-MS/MS method was developed as a fit for purpose investigation of insulin, insulin analogues, C-peptide and proinsulin processing intermediates in plasma samples from different patient groups. Using calibration standards and bovine insulin as an internal standard, absolute concentrations of insulin and C-peptide were quantified across a nominal human plasma postprandial range and correlated strongly with immunoassay-based measurements. The ability to distinguish between insulin, insulin analogues and proinsulin intermediates in a single extraction is an improvement over existing immunological based techniques, offering the advantage of exact identification of the species being measured. The method promises to aid in the detection of circulating peptides which have previously been overlooked but may interfere with standard insulin and C-peptide immunoassays.

Identification of Structural and Molecular Signatures Mediating Adaptive Changes in the Mouse Kidney in Response to Pregnancy.

Pregnancy is characterized by adaptations in the function of several maternal body systems that ensure the development of the fetus whilst maintaining health of the mother. The renal system is responsible for water and electrolyte balance, as well as waste removal. Thus, it is imperative that structural and functional changes occur in the kidney during pregnancy. However, our knowledge of the precise morphological and molecular mechanisms occurring in the kidney during pregnancy is still very limited. Here, we investigated the changes occurring in the mouse kidney during pregnancy by performing an integrated analysis involving histology, gene and protein expression assays, mass spectrometry profiling and bioinformatics. Data from non-pregnant and pregnant mice were used to identify critical signalling pathways mediating changes in the maternal kidneys. We observed an expansion of renal medulla due to proliferation and infiltration of interstitial cellular constituents, as well as alterations in the activity of key cellular signalling pathways (e.g., AKT, AMPK and MAPKs) and genes involved in cell growth/metabolism (e.g., Cdc6, Foxm1 and Rb1) in the kidneys during pregnancy. We also generated plasma and urine proteomic profiles, identifying unique proteins in pregnancy. These proteins could be used to monitor and study potential mechanisms of renal adaptations during pregnancy and disease.

Lipidomic Approaches to Study HDL Metabolism in Patients with Central Obesity Diagnosed with Metabolic Syndrome.

The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated "omics" approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the "lysophosphatidylcholines to phosphatidylcholines" and "cholesteryl ester to free cholesterol" ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated "omics" approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.

Unique nasal turbinal morphology reveals Homunculus patagonicus functionally converged on modern platyrrhine olfactory sensitivity.

The phyletic position of early Miocene platyrrhine Homunculus patagonicus is currently a matter of debate. Some regard it to be an early member of the Pitheciidae, represented today by the sakis, uakaris, and titi monkeys. Others view Homunculus as a stem platyrrhine, part of a group that diversified in Patagonia and converged in some respects on modern pitheciine dental and gnathic morphology and perhaps seed-eating specialization. New details of its internal nasal anatomy are pertinent to resolving this debate. In addition, they provide a new perspective on how modern platyrrhine olfactory sensitivity evolved. Here we reconstruct the internal nasal anatomy of Homunculus from high-resolution computed tomography scans. This species has three ethmoturbinals, the scrolls of bone in the nasal fossa that were covered in sensory epithelium in vivo. This condition stands in stark contrast to extant platyrrhines, and indeed to all other haplorhines, which have only two ethmoturbinals or, in the case of all pitheciid platyrrhines, only one ethmoturbinal. Quantitatively, however, Homunculus has an olfactory turbinal surface area that falls within the modern platyrrhine distribution, suggesting that while turbinal numbers differ, olfactory sensitivity in this taxon was likely comparable to that of modern platyrrhines. These new data from the fossil record provide further support for the hypothesis that Homunculus is a stem platyrrhine that functionally converged on modern platyrrhines rather than being an early representative of any extant clade.

A randomized, adaptive design, double-blind, 3-arm, parallel study assessing the pharmacokinetics and safety of AVT02, a high-concentration (100 mg/mL) Adalimumab biosimilar, in healthy adult subjects (ALVOPAD FIRST).

BACKGROUND: This study (ALVOPAD FIRST) assessed bioequivalence, safety, and immunogenicity of AVT02, an adalimumab biosimilar, compared with reference product adalimumab (EU- and US-approved Humira®). METHODS: Healthy subjects (N = 392) were randomized 1:1:1 to receive one 40 mg dose of AVT02, EU-reference product, or US-reference product subcutaneously. An interim analysis was planned when ~30 subjects per arm had completed the study, to optimize final sample size. The primary PK parameters were Cmax, AUC0-t, and AUC0-inf. Bioequivalence was demonstrated if the 90% confidence intervals (CI) for the ratio of geometric means for the primary pharmacokinetic (PK) parameters were all contained within the prespecified margins of 80% and 125%. Safety and immunogenicity were assessed until Day 64. RESULTS: The 90% CI for the ratio of geometric means for the primary PK parameters, based on Fisher's Combination test analysis, were all contained within the prespecified bioequivalence margins of 80% and 125%, supporting the demonstration of bioequivalence between AVT02 and both EU- and US-reference product. The safety and immunogenicity profiles were comparable across all three treatment arms. CONCLUSION: PK bioequivalence was supported between AVT02, US-licensed- and EU-approved-reference product adalimumab. Similar safety and immunogenicity were also demonstrated. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov (CT.gov identifier: NCT03849313).

Stimulation of motilin secretion by bile, free fatty acids, and acidification in human duodenal organoids.

OBJECTIVE: Motilin is a proximal small intestinal hormone with roles in gastrointestinal motility, gallbladder emptying, and hunger initiation. In vivo motilin release is stimulated by fats, bile, and duodenal acidification but the underlying molecular mechanisms of motilin secretion remain poorly understood. This study aimed to establish the key signaling pathways involved in the regulation of secretion from human motilin-expressing M-cells. METHODS: Human duodenal organoids were CRISPR-Cas9 modified to express the fluorescent protein Venus or the Ca2+ sensor GCaMP7s under control of the endogenous motilin promoter. This enabled the identification and purification of M-cells for bulk RNA sequencing, peptidomics, calcium imaging, and electrophysiology. Motilin secretion from 2D organoid-derived cultures was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), in parallel with other gut hormones. RESULTS: Human duodenal M-cells synthesize active forms of motilin and acyl-ghrelin in organoid culture, and also co-express cholecystokinin (CCK). Activation of the bile acid receptor GPBAR1 stimulated a 3.4-fold increase in motilin secretion and increased action potential firing. Agonists of the long-chain fatty acid receptor FFA1 and monoacylglycerol receptor GPR119 stimulated secretion by 2.4-fold and 1.5-fold, respectively. Acidification (pH 5.0) was a potent stimulus of M-cell calcium elevation and electrical activity, an effect attributable to acid-sensing ion channels, and a modest inducer of motilin release. CONCLUSIONS: This study presents the first in-depth transcriptomic and functional characterization of human duodenal motilin-expressing cells. We identify several receptors important for the postprandial and interdigestive regulation of motilin release.

Efficacy, Safety and Immunogenicity of AVT02 Versus Originator Adalimumab in Subjects with Moderate to Severe Chronic Plaque Psoriasis: A Multicentre, Double-Blind, Randomised, Parallel Group, Active Control, Phase III Study.

BACKGROUND: AVT02 (adalimumab) is a proposed biosimilar to Humira®. AVT02 is produced at a 100 mg/mL concentration with a citrate-free formulation. OBJECTIVES: The aim of this study was to compare the efficacy, safety and immunogenicity of AVT02 versus Humira® in subjects with moderate to severe chronic plaque psoriasis. METHODS: This double-blind, randomised, parallel group, active control study of adult subjects compared (at a 1:1 ratio) AVT02 with originator adalimumab 80 mg subcutaneously in Week 1, then 40 mg every other week. At Week 16, subjects who had received originator adalimumab were re-randomised at a 1:1 ratio to continue receiving originator adalimumab, or to switch to AVT02, every other week until Week 48, with final efficacy endpoint at Week 50. Subjects who initially received AVT02 continued to receive AVT02 from Week 16 to Week 48. The primary endpoint was percentage improvement in Psoriasis Area and Severity Index (PASI) score at Week 16. Secondary efficacy endpoints included percentage improvement in PASI score at additional timepoints, change from baseline in Dermatology Life Quality Index (DLQI) score and number and percentage of subjects achieving static Physician's Global Assessment (sPGA) responses of 'clear' or 'almost clear'. Additional secondary endpoints included comparison of adverse event profiles, anti-drug antibodies and neutralising antibodies, and serum trough levels of adalimumab at steady state. RESULTS: A total of 413 subjects were randomised (205 to AVT02 and 208 to originator). The percentage improvement in PASI score at Week 16 was 91.6% for AVT02-treated subjects and 89.6% for originator adalimumab. The 90% confidence intervals for the primary endpoint were within the pre-defined equivalence margin of ±10% (90% CI - 0.76 to 5.29; 95% CI - 1.34 to 5.88), and a comparable pattern for DLQI score (11.4-point and 10.6-point improvement in AVT02-treated and originator adalimumab-treated groups, respectively) and sPGA (90.5% in both groups achieving 'clear' or 'almost clear') at Week 16 supported the assessment. Efficacy persisted through Week 50 of the study in all treatment groups, including those who switched from originator adalimumab to AVT02, for percent improvement in PASI score, quality-of-life assessment and sPGA. The safety, tolerability and immunogenicity profiles between AVT02 and originator adalimumab were similar at Week 16, and this persisted in the switched and continued groups through Week 50. CONCLUSION: Objective and subjective measures of efficacy supported the evaluation of biosimilarity between AVT02 and originator adalimumab at Week 16 and until Week 50, in switched and continued treatment groups. AVT02 was safe and well tolerated, with a safety and immunogenicity profile similar to that observed in originator adalimumab with no clinically meaningful difference between the two. CLINICAL TRIAL REGISTRATION: EudraCT: 2017-003367-35; ClinicalTrials.gov: NCT03849404.

Murine neuronatin deficiency is associated with a hypervariable food intake and bimodal obesity.

Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat+/-p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat+/-p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat+/-p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat+/-p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat+/-p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat+/-p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.

The Human and Mouse Islet Peptidome: Effects of Obesity and Type 2 Diabetes, and Assessment of Intraislet Production of Glucagon-like Peptide-1.

To characterize the impact of metabolic disease on the peptidome of human and mouse pancreatic islets, LC-MS was used to analyze extracts of human and mouse islets, purified mouse alpha, beta, and delta cells, supernatants from mouse islet incubations, and plasma from patients with type 2 diabetes. Islets were obtained from healthy and type 2 diabetic human donors, and mice on chow or high fat diet. All major islet hormones were detected in lysed islets as well as numerous peptides from vesicular proteins including granins and processing enzymes. Glucose-dependent insulinotropic peptide (GIP) was not detectable. High fat diet modestly increased islet content of proinsulin-derived peptides in mice. Human diabetic islets contained increased content of proglucagon-derived peptides at the expense of insulin, but no evident prohormone processing defects. Diabetic plasma, however, contained increased ratios of proinsulin and des-31,32-proinsulin to insulin. Active GLP-1 was detectable in human and mouse islets but 100-1000-fold less abundant than glucagon. LC-MS offers advantages over antibody-based approaches for identifying exact peptide sequences, and revealed a shift toward islet insulin production in high fat fed mice, and toward proglucagon production in type 2 diabetes, with no evidence of systematic defective prohormone processing.

In vitro metabolism of synthetic Elabela/Toddler (ELA-32) peptide in human plasma and kidney homogenates analyzed with mass spectrometry and validation of endogenous peptide quantification in tissues by ELISA.

BACKGROUND: Elabela/Toddler (ELA) is a novel endogenous ligand of the apelin receptor, whose signalling has emerged as a therapeutic target, for example, in cardiovascular disease and cancer. Shorter forms of ELA-32 have been predicted, including ELA-21 and ELA-11, but metabolism and stability of ELA-32 in humans is poorly understood. We, therefore, developed an LC-MS/MS assay to identify ELA-32 metabolites in human plasma and tissues. METHOD: Human kidney homogenates or plasma were incubated at 37 °C with ELA-32 and aliquots withdrawn over 2-4 h into guanidine hydrochloride. Proteins were precipitated and supernatant solid-phase extracted. Peptides were extracted from coronary artery, brain and kidney by immunoprecipitation or solid-phase extraction following acidification. All samples were reduced and alkylated before analysis on an Orbitrap mass spectrometer in high and nano flow mode. RESULTS: The half-life of ELA-32 in plasma and kidney were 47.2 ±â€¯5.7 min and 44.2 ±â€¯3 s, respectively. Using PEAKS Studio and manual data analysis, the most important fragments of ELA-32 with potential biological activity identified were ELA-11, ELA-16, ELA-19 and ELA-20. The corresponding fragments resulting from the loss of C-terminal amino acids were also identified. Endogenous levels of these peptides could not be measured, as ELA peptides are prone to oxidation and poor chromatographic peaks. CONCLUSIONS: The relatively long ELA plasma half-life observed and identification of a potentially more stable fragment, ELA-16, may suggest that ELA could be a better tool compound and novel template for the development of new drugs acting at the apelin receptor.