Structural basis of nearest-neighbor cooperativity in the ring-shaped gene regulatory protein TRAP from protein engineering and cryo-EM.

Homotropic cooperativity is widespread in biological regulation. The homo-oligomeric ring-shaped trp RNA binding attenuation protein (TRAP) from bacillus binds multiple tryptophan ligands (Trp) and becomes activated to bind a specific sequence in the 5' leader region of the trp operon mRNA. Ligand-activated binding to this specific RNA sequence regulates downstream biosynthesis of Trp in a feedback loop. Characterized TRAP variants form 11- or 12-mer rings and bind Trp at the interface between adjacent subunits. Various studies have shown that a pair of loops that gate each Trp binding site is flexible in the absence of the ligand and become ordered upon ligand binding. Thermodynamic measurements of Trp binding have revealed a range of cooperative behavior for different TRAP variants, even if the averaged apparent affinities for Trp have been found to be similar. Proximity between the ligand binding sites, and the ligand-coupled disorder-to-order transition has implicated nearest-neighbor interactions in cooperativity. To establish a solid basis for describing nearest-neighbor cooperativity we engineered dodecameric (12-mer) TRAP variants constructed with two subunits connected by a flexible linker (dTRAP). We mutated one of the protomers such that only every other site was competent for Trp binding. Thermodynamic and structural studies using native mass spectrometry, NMR spectroscopy, and cryo-EM provided unprecedented detail into the thermodynamic and structural basis for the observed ligand binding cooperativity. Such insights can be useful for understanding allosteric control networks and for the development of new ones with defined ligand sensitivity and regulatory control.

Obesity disrupts the pituitary-hepatic UPR communication leading to NAFLD progression.

Obesity alters levels of pituitary hormones that govern hepatic immune-metabolic homeostasis, dysregulation of which leads to nonalcoholic fatty liver disease (NAFLD). However, the impact of obesity on intra-pituitary homeostasis is largely unknown. Here, we uncovered a blunted unfolded protein response (UPR) but elevated inflammatory signatures in pituitary glands of obese mice and humans. Furthermore, we found that obesity inflames the pituitary gland, leading to impaired pituitary inositol-requiring enzyme 1α (IRE1α)-X-box-binding protein 1 (XBP1) UPR branch, which is essential for protecting against pituitary endocrine defects and NAFLD progression. Intriguingly, pituitary IRE1-deletion resulted in hypothyroidism and suppressed the thyroid hormone receptor B (THRB)-mediated activation of Xbp1 in the liver. Conversely, activation of the hepatic THRB-XBP1 axis improved NAFLD in mice with pituitary UPR defect. Our study provides the first evidence and mechanism of obesity-induced intra-pituitary cellular defects and the pathophysiological role of pituitary-liver UPR communication in NAFLD progression.

Analytical solutions for single and multiple scattering from rib-stiffened plates in water.

The interaction of an acoustic plane wave with a pair of plates connected by periodically spaced stiffeners in water is considered. The rib-stiffened structure is called a "flex-layer" because its low frequency response is dominated by bending stiffness. The quasi-static behavior is equivalent to a homogeneous layer of compressible fluid, which we identify as air for the purposes of comparison. In this way, an air layer is acoustically the same as a pair of thin elastic plates connected by a periodic spacing of ribs. At discrete higher frequencies, the flex-layer exhibits perfect acoustic transmission, the cause of which is identified as fluid-loaded plate waves propagating back and forth between the ribs. Both the low and finite frequency behavior of the flex-layer are fully explained by closed-form solutions for reflection and transmission. The analytical model is extended to two flex-layers in series, introducing new low and high frequency phenomena that are explained in terms of simple lumped parameter models.

Using Exogenous Social Media Exposure Measures to Assess the Effects of Smokeless Tobacco-Related Social Media Content on Smokeless Tobacco Sales in the United States.

INTRODUCTION: Prior research on the effects of social media promotion of tobacco products has predominantly relied on survey-based self-report measures of marketing exposure, which potentially introduce endogeneity, recall, and selection biases. New approaches can enhance measurement and help better understand the effects of exposure to tobacco-related messages in a dynamic social media marketing environment. We used geolocation-specific tweet rate as an exogenous indicator of exposure to smokeless tobacco (ST)-related content and employed this measure to examine the influence of social media marketing on ST sales. AIMS AND METHODS: Autoregressive error models were used to analyze the association between the ST-relevant tweet rate (aggregated by 4-week period from February 12, 2017 to June 26, 2021 and scaled by population density) and logarithmic ST unit sales across time by product type (newer, snus, conventional) in the United States, accounting for autocorrelated errors. Interrupted time series approach was used to control for policy change effects. RESULTS: ST product category-related tweet rates were associated with ST unit sales of newer and conventional products, controlling for price, relevant policy events, and the coronavirus disease 2019 (COVID-19) pandemic. On average, 100-unit increase in the number of newer ST-related tweets was associated with 14% increase in unit sales (RR = 1.14; p = .01); 100-unit increase in conventional ST tweets was associated with ~1% increase in unit sales (p = .04). Average price was negatively associated with the unit sales. CONCLUSIONS: Study findings reveal that ST social media tweet rate was related to increased ST consumption and illustrate the utility of exogenous measures in conceptualizing and assessing effects in the complex media environment. IMPLICATIONS: Tobacco control initiatives should include efforts to monitor the role of social media in promoting tobacco use. Surveillance of social media platforms is critical to monitor emerging tobacco product-related marketing strategies and promotional content reach. Exogenous measures of potential exposure to social media messages can supplement survey data to study media effects on tobacco consumption.

Monovalent SARS-CoV-2 mRNA Vaccine Does not Boost Omicron-Specific Immune Response in Diabetic and Control Pediatric Patients.

While the immunogenicity of SARS-CoV-2 vaccines has been well described in adults, pediatric populations have been less studied. In particular, children with type 1 diabetes are generally at elevated risk for more severe disease after infections, but are understudied in terms of COVID-19 and SARS-CoV-2 vaccine responses. We investigated the immunogenicity of COVID-19 mRNA vaccinations in 35 children with type 1 diabetes (T1D) and 23 controls and found that these children develop levels of SARS-CoV-2 neutralizing antibody titers and spike protein-specific T cells comparable to nondiabetic children. However, in comparing the neutralizing antibody responses in children who received 2 doses of mRNA vaccines (24 T1D; 14 controls) with those who received a third, booster dose (11 T1D; 9 controls), we found that the booster dose increased neutralizing antibody titers against ancestral SARS-CoV-2 strains but, unexpectedly, not Omicron lineage variants. In contrast, boosting enhanced Omicron variant neutralizing antibody titers in adults.

Screening for Turner syndrome-associated hyperglycemia: Evaluating hemoglobin A1c and fasting blood glucose.

INTRODUCTION: Individuals with Turner syndrome (TS) are at increased risk of developing diabetes mellitus (DM). Currently, annual DM screening with hemoglobin A1c (HbA1c) with or without fasting blood glucose (FBG) is recommended starting at age 10. However, the optimal DM screening for individuals with TS is not known. The purpose of this study was to evaluate the correlation between HbA1c, FBG, and the 2-hour oral glucose tolerance test (OGTT). A second goal was to query whether optimal HbA1c and fasting (FBG) cut points for TS-associated DM and impaired glucose tolerance (IGT), as defined by the OGTT 2-hour blood glucose (BG), might differ from those for the general population. METHODS: Individuals with TS ≥ age 10 from the TS: Genotype Phenotype study in the National Institute of Child Health and Human Development's Data and Specimen Hub (DASH) who had 2-hour OGTT BG, HbA1c, and FBG were included. Correlations between HbA1c, FBG, and 2-hour OGTT BG were evaluated. Areas under the receiver operative characteristic (ROC-AUC) curves were generated. Optimal cut points for predicting TS-associated IGT (2-hour BG ≥ 7.77 mmol/L ) and DM 2-hour BG ≥11.10 mmol/L) were determined. RESULTS: 348 individuals had complete data (2-hour OGTT BG < 7.77 mmol/L, n = 239; TS-associated IGT, n = 79; DM, n = 30). ROC-AUC was poor for HbA1c to predict IGT (0.57, 0.49-0.65) but better for DM (0.81, 0.71-0.90). ROC-AUC was also poor for FBG to predict IGT (0.63, 0.56-0.70) but better for DM (0.85, 0.77-0.93). At a cut point of 38 mmol/mol (5.6%), HbA1c had 67% sensitivity (95% CI: 47-83%) and 86% specificity (95% CI: 82-90%) for identifying TS-associated DM defined by 2-hour OGTT BG. DISCUSSION/CONCLUSIONS: The correlation of HbA1c and 2-hour OGTT BG are lower in TS than other published studies regarding type 2 DM. HbA1c is fairly specific for DM in TS but lacks sensitivity especially at currently utilized levels. Future research should focus on characterizing individuals with TS whose glycemic status is discordant, as this may provide additional insights into the pathophysiology of glucose metabolism in TS. Longitudinal assessement of glycemia as it relates to micro- and macrovascular complications in individuals with TS will further inform DM screening in this population.

Deficiency of endothelial sirtuin1 in mice stimulates skeletal muscle insulin sensitivity by modifying the secretome.

Downregulation of endothelial Sirtuin1 (Sirt1) in insulin resistant states contributes to vascular dysfunction. Furthermore, Sirt1 deficiency in skeletal myocytes promotes insulin resistance. Here, we show that deletion of endothelial Sirt1, while impairing endothelial function, paradoxically improves skeletal muscle insulin sensitivity. Compared to wild-type mice, male mice lacking endothelial Sirt1 (E-Sirt1-KO) preferentially utilize glucose over fat, and have higher insulin sensitivity, glucose uptake, and Akt signaling in fast-twitch skeletal muscle. Enhanced insulin sensitivity of E-Sirt1-KO mice is transferrable to wild-type mice via the systemic circulation. Endothelial Sirt1 deficiency, by inhibiting autophagy and activating nuclear factor-kappa B signaling, augments expression and secretion of thymosin beta-4 (Tß4) that promotes insulin signaling in skeletal myotubes. Thus, unlike in skeletal myocytes, Sirt1 deficiency in the endothelium promotes glucose homeostasis by stimulating skeletal muscle insulin sensitivity through a blood-borne mechanism, and augmented secretion of Tß4 by Sirt1-deficient endothelial cells boosts insulin signaling in skeletal muscle cells.

Oxidative Imidation of Benzylic and Cycloalkane C(sp3)-H Bond Donors Using N-Aroyloxyquinuclidinium Salts and Nitriles under Photoredox Catalysis.

A series of N-aroyloxyquinuclidinium salts were prepared and used as reagents to perform efficient three-component Ritter-Mumm-type oxidative C-H imidation of donors of 1° and 2° benzylic C-H bonds used as limiting reagents with nitriles as a source of imide nitrogen under photocatalytic conditions; these reagents also exhibit somewhat lower reactivity toward cycloalkanes.

Cystic Fibrosis-Related Diabetes Workshop: Research Priorities Spanning Disease Pathophysiology, Diagnosis, and Outcomes.

Cystic fibrosis (CF) is a recessive disorder arising from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR is expressed in numerous tissues, with high expression in the airways, small and large intestine, pancreatic and hepatobiliary ducts, and male reproductive tract. CFTR loss in these tissues disrupts regulation of salt, bicarbonate, and water balance across their epithelia, resulting in a systemic disorder with progressive organ dysfunction and damage. Pancreatic exocrine damage ultimately manifests as pancreatic exocrine insufficiency that begins as early as infancy. Pancreatic remodeling accompanies this early damage, during which abnormal glucose tolerance can be observed in toddlers. With increasing age, however, insulin secretion defects progress such that CF-related diabetes (CFRD) occurs in 20% of teens and up to half of adults with CF. The relevance of CFRD is highlighted by its association with increased morbidity, mortality, and patient burden. While clinical research on CFRD has greatly assisted in the care of individuals with CFRD, key knowledge gaps on CFRD pathogenesis remain. Furthermore, the wide use of CFTR modulators to restore CFTR activity is changing the CFRD clinical landscape and the field's understanding of CFRD pathogenesis. For these reasons, the National Institute of Diabetes and Digestive and Kidney Diseases and the Cystic Fibrosis Foundation sponsored a CFRD Scientific Workshop, 23-25 June 2021, to define knowledge gaps and needed research areas. This article describes the findings from this workshop and plots a path for CFRD research that is needed over the next decade.

Cystic Fibrosis-Related Diabetes Workshop: Research Priorities Spanning Disease Pathophysiology, Diagnosis, and Outcomes.

Cystic fibrosis (CF) is a recessive disorder arising from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR is expressed in numerous tissues, with high expression in the airways, small and large intestine, pancreatic and hepatobiliary ducts, and male reproductive tract. CFTR loss in these tissues disrupts regulation of salt, bicarbonate, and water balance across their epithelia, resulting in a systemic disorder with progressive organ dysfunction and damage. Pancreatic exocrine damage ultimately manifests as pancreatic exocrine insufficiency that begins as early as infancy. Pancreatic remodeling accompanies this early damage, during which abnormal glucose tolerance can be observed in toddlers. With increasing age, however, insulin secretion defects progress such that CF-related diabetes (CFRD) occurs in 20% of teens and up to half of adults with CF. The relevance of CFRD is highlighted by its association with increased morbidity, mortality, and patient burden. While clinical research on CFRD has greatly assisted in the care of individuals with CFRD, key knowledge gaps on CFRD pathogenesis remain. Furthermore, the wide use of CFTR modulators to restore CFTR activity is changing the CFRD clinical landscape and the field's understanding of CFRD pathogenesis. For these reasons, the National Institute of Diabetes and Digestive and Kidney Diseases and the Cystic Fibrosis Foundation sponsored a CFRD Scientific Workshop, 23-25 June 2021, to define knowledge gaps and needed research areas. This article describes the findings from this workshop and plots a path for CFRD research that is needed over the next decade.

Hyperglycemia in Turner syndrome: Impact, mechanisms, and areas for future research.

Turner syndrome (TS) is a common chromosomal disorder resulting from complete or partial absence of the second sex chromosome. Hyperglycemia, ranging from impaired glucose tolerance (IGT) to diabetes mellitus (DM), is common in TS. DM in individuals with TS is associated with an 11-fold excess in mortality. The reasons for the high prevalence of hyperglycemia in TS are not well understood even though this aspect of TS was initially reported almost 60 years ago. Karyotype, as a proxy for X chromosome (Xchr) gene dosage, has been associated with DM risk in TS - however, no specific Xchr genes or loci have been implicated in the TS hyperglycemia phenotype. The molecular genetic study of TS-related phenotypes is hampered by inability to design analyses based on familial segregation, as TS is a non-heritable genetic disorder. Mechanistic studies are confounded by a lack of adequate TS animal models, small and heterogenous study populations, and the use of medications that alter carbohydrate metabolism in the management of TS. This review summarizes and assesses existing data related to the physiological and genetic mechanisms hypothesized to underlie hyperglycemia in TS, concluding that insulin deficiency is an early defect intrinsic to TS that results in hyperglycemia. Diagnostic criteria and therapeutic options for treatment of hyperglycemia in TS are presented, while emphasizing the pitfalls and complexities of studying glucose metabolism and diagnosing hyperglycemia in the TS population.

Diabetes Device Downloading: Benefits and Barriers Among Youth With Type 1 Diabetes.

BACKGROUND: The majority of youth with type 1 diabetes (T1D) fail to meet glycemic targets despite increasing continuous glucose monitoring (CGM) use. We therefore aimed to determine the proportion of caregivers who review recent glycemic trends ("retrospective review") and make ensuant insulin adjustments based on this data ("retroactive insulin adjustments"). We additionally considered that fear of hypoglycemia and frequency of severe hypoglycemia would be associated with performing retrospective review. METHODS: We conducted a cross-sectional survey of caregivers of youth with T1D, collecting demographics, diabetes technology usage, patterns of glucose data review/insulin dose self-adjustment, and Hypoglycemia Fear Survey (HFS). RESULTS: Nineteen percent of eligible caregivers (191/1003) responded. Performing retrospective review was associated with younger child age (12.2 versus 15.4, P = .0001) and CGM use (92% versus 73%, P = .004), but was not associated with a significant improvement in child's HbA1c (7.89 versus 8.04, P = .65). Retrospective reviewers had significantly higher HFS-behavior scores (31.9 versus 27.7, P = .0002), which remained significantly higher when adjusted for child's age and CGM use (P = .005). Linear regression identified a significant negative association between HbA1c (%) and number of retroactive insulin adjustments (0.24 percent lower mean HbA1c per additional adjustment made, P = .02). CONCLUSIONS: Retrospective glucose data review is associated with improved HbA1c when coupled with data-driven retroactive insulin adjustments. Barriers to data downloading existed even in this cohort of predominantly CGM-using T1D families.

Structure of a RecT/Redß family recombinase in complex with a duplex intermediate of DNA annealing.

Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA). These proteins are apparently related to RAD52, the primary human SSA protein. The best studied protein, Redß from bacteriophage λ, binds weakly to ssDNA, not at all to dsDNA, but tightly to a duplex intermediate of annealing formed when two complementary DNA strands are added to the protein sequentially. We used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a Redß homolog from a prophage of Listeria innocua in complex with two complementary 83mer oligonucleotides. The structure reveals a helical protein filament bound to a DNA duplex that is highly extended and unwound. Native mass spectrometry confirms that the complex seen by cryo-EM is the predominant species in solution. The protein shares a common core fold with RAD52 and a similar mode of ssDNA-binding. These data provide insights into the mechanism of protein-catalyzed SSA.

De novo design of obligate ABC-type heterotrimeric proteins.

The de novo design of three protein chains that associate to form a heterotrimer (but not any of the possible two-chain heterodimers) and that can drive the assembly of higher-order branching structures is an important challenge for protein design. We designed helical heterotrimers with specificity conferred by buried hydrogen bond networks and large aromatic residues to enhance shape complementary packing. We obtained ten designs for which all three chains cooperatively assembled into heterotrimers with few or no other species present. Crystal structures of a helical bundle heterotrimer and extended versions, with helical repeat proteins fused to individual subunits, showed all three chains assembling in the designed orientation. We used these heterotrimers as building blocks to construct larger cyclic oligomers, which were structurally validated by electron microscopy. Our three-way junction designs provide new routes to complex protein nanostructures and enable the scaffolding of three distinct ligands for modulation of cell signaling.

Constant intensity acoustic propagation in the presence of non-uniform properties and impedance discontinuities: Hermitian and non-Hermitian solutions.

Propagation of sound through a non-uniform medium without scattering is possible, in principle, if the density and acoustic compressibility assume complex values, requiring passive and active mechanisms, also known as Hermitian and non-Hermitian solutions, respectively. Two types of constant intensity wave conditions are identified: in the first, the propagating acoustic pressure has constant amplitude, while in the second, the energy flux remains constant. The fundamental problem of transmission across an impedance discontinuity without reflection or energy loss is solved using a combination of monopole and dipole resonators in parallel. The solution depends on an arbitrary phase angle that can be chosen to give a unique acoustic metamaterial with both resonators undamped and passive, requiring purely Hermitian acoustic elements. For other phase angles, one of the two elements must be active and the other passive, resulting in a gain/loss non-Hermitian system. These results prove that uni-directional and reciprocal transmission through a slab separating two half spaces is possible using passive Hermitian acoustic elements without the need to resort to active gain/loss energetic mechanisms.

The fatty acid imbalance of cystic fibrosis exists at birth independent of feeding in pig and ferret models.

Persons with cystic fibrosis (CF) exhibit a unique alteration of fatty acid composition, marked especially among polyunsaturates by relative deficiency of linoleic acid and excess of Mead acid. Relative deficiency of docosahexaenoic acid is variably found. However, the initial development of these abnormalities is not understood. We examined fatty acid composition in young CF ferrets and pigs, finding abnormalities from the day of birth onward including relative deficiency of linoleic acid in both species. Fatty acid composition abnormalities were present in both liver and serum phospholipids of newborn CF piglets even prior to feeding, including reduced linoleic acid and increased Mead acid. Serum fatty acid composition evolved over the first weeks of life in both non-CF and CF ferrets, though differences between CF and non-CF persisted. Although red blood cell phospholipid fatty acid composition was normal in newborn animals, it became perturbed in juvenile CF ferrets including relative deficiencies of linoleic and docosahexaenoic acids and excess of Mead acid. In summary, fatty acid composition abnormalities in CF pigs and ferrets exist from a young age including at birth independent of feeding and overlap extensively with the abnormalities found in humans with CF. That the abnormalities exist prior to feeding implies that dietary measures alone will not address the mechanisms of imbalance.

Broadband acoustic lens design by reciprocity and optimization.

A broadband acoustic lens is designed based on the principle of reciprocity and gradient-based optimization. Acoustic reciprocity is used to define the pressure at the focal point due to a source located in a far-field and to relate the response by a configuration of scatterers for an incident plane wave. The pressure at the focal point is maximized by rearranging the scatterers and supplying the gradients of absolute pressure at the focal point with respect to scatterer positions. Numerical examples are given for clusters of cylindrical voids and sets of elastic thin shells in water.

Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand-dependent population distributions.

Homo-oligomeric ligand-activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand-binding sites. Understanding the basis for this regulation requires both quantifying the free energy ΔG transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring-shaped homo-oligomeric trp RNA-binding attenuation protein (TRAP). First, we developed a nearest-neighbor statistical thermodynamic binding model comprising microscopic free energies for ligand binding to isolated sites ΔG0 , and for coupling between adjacent sites, ΔGα . Using the resulting partition function (PF) we explored the effects of these parameters on simulated population distributions for the 2N possible liganded states. We then experimentally monitored ligand-dependent population shifts using conventional spectroscopic and calorimetric methods and using native mass spectrometry (MS). By resolving species with differing numbers of bound ligands by their mass, native MS revealed striking differences in their ligand-dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. Uniquely, this approach predicts which of the possible 2N liganded states are populated at different ligand concentrations, providing necessary insights into regulation. The combination of statistical thermodynamic modeling with native MS may provide the thermodynamic foundation for a meaningful understanding of the structure-thermodynamic linkage that drives cooperativity.

Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein.

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5' leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae-RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein-protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.