Integrative analysis of pathogenic variants in glucose-6-phosphatase based on an AlphaFold2 model.

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein glucose-6-phosphate catalytic subunit 1 (G6PC1) regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 causes glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. In this study, we determine the atomic interactions governing G6P binding as well as explore the perturbations imposed by disease-linked missense variants by subjecting an AlphaFold2 G6PC1 structural model to molecular dynamics simulations and in silico predictions of thermodynamic stability validated with robust in vitro and in situ biochemical assays. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. The introduction of GSD type 1a mutations modified the thermodynamic landscape, altered side chain packing and substrate-binding interactions, and induced trapping of catalytic intermediates. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm the active-site structural organization but also identify previously unobserved mechanistic contributions of catalytic and noncatalytic side chains.

A pragmatic trial of a family-centered approach to childhood obesity treatment: Rationale and study design.

BACKGROUND: Family-based behavioral treatment (FBT) is an effective intensive health behavior and lifestyle treatment for obesity reduction in children and adolescents, but families have limited access. The purpose of this randomized, pragmatic, comparative effectiveness trial was to examine changes in child relative weight in a 12-month, enhanced standard of care (eSOC) intervention combined with FBT (eSOC+FBT) vs. eSOC alone. METHODS: Children aged 6 to 15 years with obesity, and their primary caregiver, were recruited from primary care clinics. Families were randomized 1:1 to eSOC, a staged approach led by the primary care provider that gradually intensified dependent on a child's response to care and aligns with the American Medical Association guidelines, or the eSOC+FBT arm, which included regular meetings with a health coach for healthy eating, physical activity, positive parenting strategies, and managing social and environmental cues. Both treatments align with the 2023 American Academy of Pediatrics clinical practice guidelines. Assessments occurred at baseline, midpoint (month 6), end-of-intervention (month 12), and follow-up (month 18). Primary outcome was change from baseline to 12 months in child percent overweight (percentage above the median body mass index in the general US population normalized for age and sex). Secondary outcomes were parent weight, child psychosocial factors, heterogeneity of treatment effects, and cardiometabolic risk factors. Exploratory outcomes assessed reach, effectiveness, adoption, implementation, and maintenance. CONCLUSION: This pragmatic trial will generate evidence for the comparative effectiveness of implementing two guidelines-based approaches in primary care for obesity reduction in children and adolescents. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03843424.

Approximating Projections of Conformational Boltzmann Distributions with AlphaFold2 Predictions: Opportunities and Limitations.

Protein thermodynamics is intimately tied to biological function and can enable processes such as signal transduction, enzyme catalysis, and molecular recognition. The relative free energies of conformations that contribute to these functional equilibria evolved for the physiology of the organism. Despite the importance of these equilibria for understanding biological function and developing treatments for disease, computational and experimental methods capable of quantifying the energetic determinants of these equilibria are limited to systems of modest size. Recently, it has been demonstrated that the artificial intelligence system AlphaFold2 can be manipulated to produce structurally valid protein conformational ensembles. Here, we extend these studies and explore the extent to which AlphaFold2 contact distance distributions can approximate projections of the conformational Boltzmann distributions. For this purpose, we examine the joint probability distributions of inter-residue contact distances along functionally relevant collective variables of several protein systems. Our studies suggest that AlphaFold2 normalized contact distance distributions can correlate with conformation probabilities obtained with other methods but that they suffer from peak broadening. We also find that the AlphaFold2 contact distance distributions can be sensitive to point mutations. Overall, we anticipate that our findings will be valuable as the community seeks to model the thermodynamics of conformational changes in large biomolecular systems.

Rosetta Energy Analysis of AlphaFold2 models: Point Mutations and Conformational Ensembles.

There has been an explosive growth in the applications of AlphaFold2, and other structure prediction platforms, to accurately predict protein structures from a multiple sequence alignment (MSA) for downstream structural analysis. However, two outstanding questions persist in the field regarding the robustness of AlphaFold2 predictions of the consequences of point mutations and the completeness of its prediction of protein conformational ensembles. We combined our previously developed method SPEACH_AF with model relaxation and energetic analysis with Rosetta to address these questions. SPEACH_AF introduces residue substitutions across the MSA and not just within the input sequence. With respect to conformational ensembles, we combined SPEACH_AF and a new MSA subsampling method, AF_cluster, and for a benchmarked set of proteins, we found that the energetics of the conformational ensembles generated by AlphaFold2 correspond to those of experimental structures and explored by standard molecular dynamic methods. With respect to point mutations, we compared the structural and energetic consequences of having the mutation(s) in the input sequence versus in the whole MSA (SPEACH_AF). Both methods yielded models different from the wild-type sequence, with more robust changes when the mutation(s) were in the whole MSA. While our findings demonstrate the robustness of AlphaFold2 in analyzing point mutations and exploring conformational ensembles, they highlight the need for multi parameter structural and energetic analyses of these models to generate experimentally testable hypotheses.

Validation of remote child weight and height measurements within a weight management trial.

OBJECTIVE: The aim of this substudy within the Treatment Efforts Addressing Child Weight Management by Unifying Patients, Parents, and Providers (TEAM UP) pragmatic clinical trial was to compare the validity of anthropometric measurements collected remotely versus in person (≤7 days apart) among youth with obesity who were 6 to 15 years of age. METHODS: Child (n = 37) weight and height were measured in person by a trained data assessor. These were compared with measurements taken remotely by the child's parent with live videoconferencing observation by a study data assessor. In-person and remote measurements were compared using Bland-Altman plots, Pearson correlations, and two one-sided paired t tests. A priori bounds of acceptability were set at ±0.68 kg to allow for typical weight fluctuations within the 7-day comparison period. RESULTS: Measurements were highly correlated (height: r = 0.991, p < 0.0001; weight: r = 0.999; p = 0.03). For height, two one-sided t tests for upper, t(36) = 3.95, and lower, t(36) = -2.63, bounds (-1, 1) revealed an overall p = 0.006; absolute error was 3.5 cm. For weight, two one-sided t tests for upper, t(36) = 1.93, and lower, t(36) = -7.91, bounds (-0.68, 0.68) revealed an overall p = 0.03; absolute error was 1.7 kg. CONCLUSIONS: The present findings support the utility and interpretation of remotely assessed weight management outcomes for both research and clinical purposes. These procedures may offer greater accessibility to evidence-based measurement.

Epigenetic changes induced by pathogenic Chlamydia spp.

Chlamydia trachomatis, C. pneumoniae, and C. psittaci, the three Chlamydia species known to cause human disease, have been collectively linked to several pathologies, including conjunctivitis, trachoma, respiratory disease, acute and chronic urogenital infections and their complications, and psittacosis. In vitro, animal, and human studies also established additional correlations, such as between C. pneumoniae and atherosclerosis and between C. trachomatis and ovarian cancer. As part of their survival and pathogenesis strategies as obligate intracellular bacteria, Chlamydia spp. modulate all three major types of epigenetic changes, which include deoxyribonucleic acid (DNA) methylation, histone post-translational modifications, and microRNA-mediated gene silencing. Some of these epigenetic changes may be implicated in key aspects of pathogenesis, such as the ability of the Chlamydia spp. to induce epithelial-to-mesenchymal transition, interfere with DNA damage repair, suppress cholesterol efflux from infected macrophages, act as a co-factor in human papillomavirus (HPV)-mediated cervical cancer, prevent apoptosis, and preserve the integrity of mitochondrial networks in infected host cells. A better understanding of the individual and collective contribution of epigenetic changes to pathogenesis will enhance our knowledge about the biology of Chlamydia spp. and facilitate the development of novel therapies and biomarkers. Pathogenic Chlamydia spp. contribute to epigenetically-mediated gene expression changes in host cells by multiple mechanisms.

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least 1 order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase in the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of interspin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4-9, compared to the common spin label such as MTSL, which is not affected by CB-7. Interspin distances of 3 nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating an undisturbed structure and conformation of the protein.

Asymmetric conformations and lipid interactions shape the ATP-coupled cycle of a heterodimeric ABC transporter.

Here we used cryo-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy (DEER), and molecular dynamics (MD) simulations, to capture and characterize ATP- and substrate-bound inward-facing (IF) and occluded (OC) conformational states of the heterodimeric ATP binding cassette (ABC) multidrug exporter BmrCD in lipid nanodiscs. Supported by DEER analysis, the structures reveal that ATP-powered isomerization entails changes in the relative symmetry of the BmrC and BmrD subunits that propagates from the transmembrane domain to the nucleotide binding domain. The structures uncover asymmetric substrate and Mg2+ binding which we hypothesize are required for triggering ATP hydrolysis preferentially in one of the nucleotide-binding sites. MD simulations demonstrate that multiple lipid molecules differentially bind the IF versus the OC conformation thus establishing that lipid interactions modulate BmrCD energy landscape. Our findings are framed in a model that highlights the role of asymmetric conformations in the ATP-coupled transport with general implications to the mechanism of ABC transporters.

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins.

We report complex formation between the chloroacetamide 2,6-diazaadamantane nitroxide radical (ClA-DZD) and cucurbit[7]uril (CB-7), for which the association constant in water, Ka = 1.9 × 106 M-1, is at least one order of magnitude higher than the previously studied organic radicals. The radical is highly immobilized by CB-7, as indicated by the increase of the rotational correlation time, τrot, by a factor of 36, relative to that in the buffer solution. The X-ray structure of ClA-DZD@CB-7 shows the encapsulated DZD guest inside the undistorted CB-7 host, with the pendant group protruding outside. Upon addition of CB-7 to T4 Lysozyme (T4L) doubly spin-labeled with the iodoacetamide derivative of DZD, we observe the increase in τrot and electron spin coherence time, Tm, along with the narrowing of inter-spin distance distributions. Sensitivity of the DEER measurements at 83 K increases by a factor 4 - 9, compared to the common spin label such as MTSL, which is not affected by CB-7. Inter-spin distances of 3-nm could be reliably measured in water/glycerol up to temperatures near the glass transition/melting temperature of the matrix at 200 K, thus bringing us closer to the goal of supramolecular recognition-enabled long-distance DEER measurements at near physiological temperatures. The X-ray structure of DZD-T4L 65 at 1.12 Å resolution allows for unambiguous modeling of the DZD label (0.88 occupancy), indicating undisturbed structure and conformation of the protein.

Determining how MAFA and MAFB transcription factors activity is influenced by structural differences predicted by AlphaFold2.

MAFA and MAFB are related basic-leucine-zipper domain containing transcription factors which have important regulatory roles in a variety of cellular contexts, including pancreatic islet hormone producing α and ß cells. These proteins have similar as well as distinct functional properties, and here we first used AlphaFold2, an artificial intelligence-based structural prediction program, to obtain insight into the three-dimensional organization of their non-DNA binding/dimerization sequences. This analysis was conducted on the wildtype (WT) proteins as well the pathogenic MAFA Ser64Phe (MAFA S64F ) and MAFB Ser70Ala (MAFB S70A ) mutants, with structural differences revealed between MAFA WT and MAFB WT in addition to MAFA S64F and MAFA WT , but not MAFB S70A and MAFB WT . Functional analysis disclosed that the inability to properly phosphorylate at S70 in MAFB S70A , like S65 in MAFA S64F , greatly increased protein stability and enabled MAFB S70A to accelerate cellular senescence in cultured cells. Significant differences were also observed in the ability of MAFA, MAFA S64F , MAFB, and MAFB S70A to cooperatively stimulate Insulin enhancer-driven activity in the presence of other islet-enriched transcription factors. Experiments performed on protein chimeras disclosed that these properties were greatly influenced by structural differences found between the WT and mutant proteins. In general, these results revealed that AlphaFold2 predicts features essential to protein activity.

Approximating conformational Boltzmann distributions with AlphaFold2 predictions.

Protein dynamics are intimately tied to biological function and can enable processes such as signal transduction, enzyme catalysis, and molecular recognition. The relative free energies of conformations that contribute to these functional equilibria are evolved for the physiology of the organism. Despite the importance of these equilibria for understanding biological function and developing treatments for disease, the computational and experimental methods capable of quantifying them are limited to systems of modest size. Here, we demonstrate that AlphaFold2 contact distance distributions can approximate conformational Boltzmann distributions, which we evaluate through examination of the joint probability distributions of inter-residue contact distances along functionally relevant collective variables of several protein systems. Further, we show that contact distance probability distributions generated by AlphaFold2 are sensitive to points mutations thus AF2 can predict the structural effects of mutations in some systems. We anticipate that our approach will be a valuable tool to model the thermodynamics of conformational changes in large biomolecular systems.

Asymmetric conformations and lipid interactions shape the ATP-coupled cycle of a heterodimeric ABC transporter.

To illuminate the structural origin of catalytic asymmetry of heterodimeric ABC transporters and how it shapes the energetics of their conformational cycles, we used cryo-electron microscopy (cryo-EM), double electron-electron resonance spectroscopy (DEER), and molecular dynamics (MD) simulations, to capture and characterize conformational states of the heterodimeric ABC multidrug exporter BmrCD in lipid nanodiscs. In addition to multiple ATP- and substrate-bound inward-facing (IF) conformations, we obtained the structure of an occluded (OC) conformation wherein the unique extracellular domain (ECD) twists to partially open the extracellular gate. In conjunction with DEER analysis of the populations of these conformations, the structures reveal that ATP-powered isomerization entails changes in the relative symmetry of the BmrC and BmrD subunits that propagates from the transmembrane domain (TMD) to the nucleotide binding domain (NBD). The structures uncover asymmetric substrate and Mg 2+ binding which we hypothesize are required for triggering ATP hydrolysis preferentially in one of the nucleotide-binding sites. MD simulations demonstrated that multiple lipid molecules, identified from the cryo-EM density maps, differentially bind the IF versus the OC conformation thus modulating their relative stability. In addition to establishing how lipid interactions with BmrCD modulate the energy landscape, our findings are framed in a distinct transport model that highlights the role of asymmetric conformations in the ATP-coupled cycle with implications to the mechanism of ABC transporters in general.

Epigenetic effects of short-chain fatty acids from the large intestine on host cells.

Adult humans harbor at least as many microbial cells as eukaryotic ones. The largest compartment of this diverse microbial population, the gut microbiota, encompasses the collection of bacteria, archaea, viruses, and eukaryotic organisms that populate the gastrointestinal tract, and represents a complex and dynamic ecosystem that has been increasingly implicated in health and disease. The gut microbiota carries ∼100-to-150-times more genes than the human genome and is intimately involved in development, homeostasis, and disease. Of the several microbial metabolites that have been studied, short-chain fatty acids emerge as a group of molecules that shape gene expression in several types of eukaryotic cells by multiple mechanisms, which include DNA methylation changes, histone post-translational modifications, and microRNA-mediated gene silencing. Butyric acid, one of the most extensively studied short-chain fatty acids, reaches higher concentrations in the colonic lumen, where it provides a source of energy for healthy colonocytes, and its concentrations decrease towards the bottom of the colonic crypts, where stem cells reside. The lower butyric acid concentration in the colonic crypts allows undifferentiated cells, such as stem cells, to progress through the cell cycle, pointing towards the importance of the crypts in providing them with a protective niche. In cancerous colonocytes, which metabolize relatively little butyric acid and mostly rely on glycolysis, butyric acid preferentially acts as a histone deacetylase inhibitor, leading to decreased cell proliferation and increased apoptosis. A better understanding of the interface between the gut microbiota metabolites and epigenetic changes in eukaryotic cells promises to unravel in more detail processes that occur physiologically and as part of disease, help develop novel biomarkers, and identify new therapeutic modalities.

A model-informed method to retrieve intrinsic from apparent cooperativity and project cellular target occupancy for ternary complex-forming compounds.

There is an increasing interest to develop therapeutics that modulate challenging or undruggable target proteins via a mechanism that involves ternary complexes. In general, such compounds can be characterized by their direct affinities to a chaperone and a target protein and by their degree of cooperativity in the formation of the ternary complex. As a trend, smaller compounds have a greater dependency on intrinsic cooperativity to their thermodynamic stability relative to direct target (or chaperone) binding. This highlights the need to consider intrinsic cooperativity of ternary complex-forming compounds early in lead optimization, especially as they provide more control over target selectivity (especially for isoforms) and more insight into the relationship between target occupancy and target response via estimation of ternary complex concentrations. This motivates the need to quantify the natural constant of intrinsic cooperativity (α) which is generally defined as the gain (or loss) in affinity of a compound to its target in pre-bound vs. unbound state. Intrinsic cooperativities can be retrieved via a mathematical binding model from EC50 shifts of binary binding curves of the ternary complex-forming compound with either a target or chaperone relative to the same experiment but in the presence of the counter protein. In this manuscript, we present a mathematical modeling methodology that estimates the intrinsic cooperativity value from experimentally observed apparent cooperativities. This method requires only the two binary binding affinities and the protein concentrations of target and chaperone and is therefore suitable for use in early discovery therapeutic programs. This approach is then extended from biochemical assays to cellular assays (i.e., from a closed system to an open system) by accounting for differences in total ligand vs. free ligand concentrations in the calculations of ternary complex concentrations. Finally, this model is used to translate biochemical potency of ternary complex-forming compounds into expected cellular target occupancy, which could ultimately serve as a way for validation or de-validation of hypothesized biological mechanisms of action.

Insulin Sensitivity and ß-Cell Function During Early and Late Pregnancy in Women With and Without Gestational Diabetes Mellitus.

OBJECTIVE: To evaluate the metabolic alterations associated with gestational diabetes mellitus (GDM) in women with overweight or obesity. RESEARCH DESIGN AND METHODS: We compared fasting and postprandial plasma glucose and free fatty acid (FFA) concentrations, insulin sensitivity (IS; Matsuda index), and ß-cell function (i.e., ß-cell responsiveness to glucose) by using a frequently sampled oral glucose tolerance test (OGTT) at 15 and 35 weeks' gestation in women with overweight or obesity who had GDM (n = 29) or did not have GDM (No-GDM; n = 164) at 35 weeks. RESULTS: At 15 weeks, IS and ß-cell function were lower, and fasting, 1-h, and total area-under-the-curve plasma glucose concentrations during the OGTT were higher (all P < 0.05) in the GDM than in the No-GDM group. At 35 weeks compared with 15 weeks, IS decreased, ß-cell function increased, and postprandial suppression of plasma FFA was blunted in both the GDM and No-GDM groups, but the decrease in IS and the increase in postprandial FFA concentration were greater and the increase in ß-cell function was less (all P ≤ 0.05) in the GDM than in the No-GDM group. A receiver operating characteristic curve analysis showed that both fasting plasma glucose and 1-h OGTT glucose concentration at 15 weeks are predictors of GDM, but the predictive power was <30%. CONCLUSIONS: Women with overweight or obesity and GDM, compared with those without GDM, have worse IS and ß-cell function early during pregnancy and a greater subsequent decline in IS and blunted increase in ß-cell function. Increased fasting and 1-h OGTT plasma glucose concentration early during pregnancy are markers of increased GDM risk, albeit with weak predictive power.

Efficacy and safety of MAS825 (anti-IL-1ß/IL-18) in COVID-19 patients with pneumonia and impaired respiratory function.

MAS825, a bispecific IL-1ß/IL-18 monoclonal antibody, could improve clinical outcomes in COVID-19 pneumonia by reducing inflammasome-mediated inflammation. Hospitalized non-ventilated patients with COVID-19 pneumonia (n = 138) were randomized (1:1) to receive MAS825 (10 mg/kg single i.v.) or placebo in addition to standard of care (SoC). The primary endpoint was the composite Acute Physiology and Chronic Health Evaluation II (APACHE II) score on Day 15 or on the day of discharge (whichever was earlier) with worst-case imputation for death. Other study endpoints included safety, C-reactive protein (CRP), SARS-CoV-2 presence, and inflammatory markers. On Day 15, the APACHE II score was 14.5 ± 1.87 and 13.5 ± 1.8 in the MAS825 and placebo groups, respectively (P = 0.33). MAS825 + SoC led to 33% relative reduction in intensive care unit (ICU) admissions, ~1 day reduction in ICU stay, reduction in mean duration of oxygen support (13.5 versus 14.3 days), and earlier clearance of virus on Day 15 versus placebo + SoC group. On Day 15, compared with placebo group, patients treated with MAS825 + SoC showed a 51% decrease in CRP levels, 42% lower IL-6 levels, 19% decrease in neutrophil levels, and 16% lower interferon-γ levels, indicative of IL-1ß and IL-18 pathway engagement. MAS825 + SoC did not improve APACHE II score in hospitalized patients with severe COVID-19 pneumonia; however, it inhibited relevant clinical and inflammatory pathway biomarkers and resulted in faster virus clearance versus placebo + SoC. MAS825 used in conjunction with SoC was well tolerated. None of the adverse events (AEs) or serious AEs were treatment-related.

Human endogenous retroviruses: our genomic fossils and companions.

Approximately 8% of the human genome, over four times more than its protein-coding regions, comprises sequences of viral origin that are known as human endogenous retroviral elements (HERVs). Present in the genome of all human cells, HERVs resulted from the integration of now-extinct exogenous retroviruses into mammalian ancestor germ cells or their precursors on several occasions, sometimes as long as tens of millions of years ago. Most HERVs have become silenced because of mutations such as substitutions, insertions, or deletions, and as a result of epigenetic changes, and are vertically transmitted in the population. Considered for a long time to be part of the "junk DNA," HERVs were shown, in more recent years, to perform critical functions in the host. Two of the very few HERVs known to encode functional proteins, syncytin-1 and syncytin-2, are critical during embryogenesis, when they contribute to the formation of the placenta and facilitate tolerance of the maternal immune system toward the developing fetus. Homologs of syncytin-encoding genes were described in several other species, and it appears that during evolution they were stably endogenized into the respective genomes on multiple occasions and became co-opted for critical physiological functions. The aberrant expression of HERVs has been linked to conditions that include infectious, autoimmune, malignant, and neurological diseases. HERVs, our genomic fossils and storytellers, provide a fascinating and somewhat mysterious insight into our co-evolution with viruses, and will undoubtedly offer many teachings, surprises, and paradigm changes for years to come.

Molecular mechanisms of catalytic inhibition for active site mutations in glucose-6-phosphatase catalytic subunit 1 linked to glycogen storage disease.

Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein G6PC1 regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 cause glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. Exploiting a computational model of G6PC1 derived from the groundbreaking structure prediction algorithm AlphaFold2 (AF2), we combine molecular dynamics (MD) simulations and computational predictions of thermodynamic stability with a robust in vitro screening platform to define the atomic interactions governing G6P binding as well as explore the energetic perturbations imposed by disease-linked variants. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. Introduction of GSD type 1a mutations into the G6PC1 sequence elicits changes in G6P binding energy, thermostability and structural properties, suggesting multiple pathways of catalytic impairment. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm active site structural organization but also suggest novel mechanistic contributions of catalytic and non-catalytic side chains.

The far-reaching impact of abortion bans: reproductive care and beyond.

On 24 June 2022, the US Supreme Court overturned Roe v. Wade, a 49-year-old precedent that provided federal constitutional protection for abortions up to the point of foetal viability, returning jurisdiction to the individual states. Restrictions that came into effect automatically in several states, and are anticipated in others, will severely limit access to abortions in approximately half of the US. Even though every state allows for exceptions to the abortion bans, in some instances these exceptions can be used to preserve the health of a pregnant patient, while in other instances, only to preserve their life. The vague and confusing nature of the abortion ban exceptions threatens to compromise the standard of care for patients with pregnancy complications that are distinct from abortions, such as nonviable pregnancies, miscarriages, and ectopic pregnancies. Additionally, we envision challenges for the treatment of women with certain autoimmune conditions, pregnant cancer patients, and patients contemplating preimplantation genetic diagnosis as part of assisted reproductive technologies. The abortion ban exceptions will impact and interfere with the medical care of pregnant and non-pregnant patient populations alike and are poised to create a medical and public health crisis unlike any other one from the recent past.

Change in parent and child psychopathology following obesity treatment and maintenance: A secondary data analysis.

BACKGROUND: General and eating disorder (ED) psychopathology are common among children and adults with overweight/obesity; few studies have examined their course of change throughout family-based behavioural obesity treatment (FBT) and maintenance. OBJECTIVES: Examine: (1) the changes in the parent and child general and ED psychopathology during FBT and maintenance interventions; (2) the associations between change in psychopathology and change in weight among children or parents; (3) the associations between change in psychopathology within parent-child dyads. METHODS: 172 parent-child dyads participated in 4-month FBT and were subsequently randomized to one of three 8-month maintenance interventions. General psychopathology (child anxiety/depressive symptoms, parent severity of global psychological distress), ED psychopathology (shape/weight concern), and percent overweight were assessed at baseline, post-FBT, and post-maintenance. Separate linear mixed-effects models evaluated change in general and ED psychopathology, including an interaction between maintenance condition and time. Partial correlations examined associations between change in psychopathology and percent overweight among children or parents, and associations between change in psychopathology within parent-child dyads. RESULTS: Among children, significant reductions were observed from baseline to post-FBT in all forms of psychopathology and from post-FBT to post-maintenance in general psychopathology. Among parents, significant reductions were observed from baseline to post-FBT in all forms of psychopathology; reductions were maintained from post-FBT to post-maintenance. There was no significant interaction between maintenance condition and time. Correlations between change in most forms of parent or child psychopathology and percent overweight were observed. CONCLUSIONS: Participation in FBT and maintenance was associated with improvements in general and ED psychopathology in both parents and children.