GTExome: Modeling commonly expressed missense mutations in the human genome.

A web application, GTExome, is described that quickly identifies, classifies, and models missense mutations in commonly expressed human proteins. GTExome can be used to categorize genomic mutation data with tissue specific expression data from the Genotype-Tissue Expression (GTEx) project. Commonly expressed missense mutations in proteins from a wide range of tissue types can be selected and assessed for modeling suitability. Information about the consequences of each mutation is provided to the user including if disulfide bonds, hydrogen bonds, or salt bridges are broken, buried prolines introduced, buried charges are created or lost, charge is swapped, a buried glycine is replaced, or if the residue that would be removed is a proline in the cis configuration. Also, if the mutation site is in a binding pocket the number of pockets and their volumes are reported. The user can assess this information and then select from available experimental or computationally predicted structures of native proteins to create, visualize, and download a model of the mutated protein using Fast and Accurate Side-chain Protein Repacking (FASPR). For AlphaFold modeled proteins, confidence scores for native proteins are provided. Using this tool, we explored a set of 9,666 common missense mutations from a variety of tissues from GTEx and show that most mutations can be modeled using this tool to facilitate studies of protein-protein and protein-drug interactions. The open-source tool is freely available at https://pharmacogenomics.clas.ucdenver.edu/gtexome/.

Histologic characterization of spontaneous catecholamine-induced cardiomyopathy in laboratory New Zealand White rabbits.

Catecholamine-induced cardiomyopathy (CCM) is an entity associated with increased levels of catecholamines causing subendocardial and papillary muscle cardiomyocyte degeneration and necrosis. In 2020, 49 autopsies from early rabbit deaths in a colony used for medical device biocompatibility studies were submitted for microscopic examination. Of the 49 rabbits, 26 had histologic changes consistent with CCM. No common stressor for CCM was determined in affected rabbits. Animals were generally male, were 12-16-wk-old, and were found dead or had bloating, lethargy, and/or diarrhea. Those observed with clinical signs were euthanized and autopsied per the organization's standard operating procedures. Heart lesions consisted of various degrees of apical subendocardial myocardial degeneration and necrosis. Common non-cardiac lesions included pulmonary congestion and edema, hepatic congestion and centrilobular hepatocellular degeneration, and/or variable intestinal submucosal edema.

GTExome: Modeling commonly expressed missense mutations in the human genome.

A web application, GTExome, is described that quickly identifies, classifies, and models missense mutations in commonly expressed human proteins. GTExome can be used to categorize genomic mutation data with tissue specific expression data from the Genotype-Tissue Expression (GTEx) project. Commonly expressed missense mutations in proteins from a wide range of tissue types can be selected and assessed for modeling suitability. Information about the consequences of each mutation is provided to the user including if disulfide bonds, hydrogen bonds, or salt bridges are broken, buried prolines introduced, buried charges are created or lost, charge is swapped, a buried glycine is replaced, or if the residue that would be removed is a proline in the cis configuration. Also, if the mutation site is in a binding pocket the number of pockets and their volumes are reported. The user can assess this information and then select from available experimental or computationally predicted structures of native proteins to create, visualize, and download a model of the mutated protein using Fast and Accurate Side-chain Protein Repacking (FASPR). For AlphaFold modeled proteins, confidence scores for native proteins are provided. Using this tool, we explored a set of 9,666 common missense mutations from a variety of tissues from GTEx and show that most mutations can be modeled using this tool to facilitate studies of protein-protein and protein-drug interactions. The open-source tool is freely available at https://pharmacogenomics.clas.ucdenver.edu/gtexome/.

Assessing protein homology models with docking reproducibility.

Results of the recent Critical Assessment of Protein Structure (CASP) competitions demonstrate that protein backbones can be predicted with very high accuracy. In particular, the artificial intelligence methods of AlphaFold 2 from DeepMind were able to produce structures that were similar enough to experimental structures that many described the problem of protein prediction solved. However, for such structures to be used for drug docking studies requires precision in the placement of side chain atoms as well. Here we built a library of 1334 small molecules and examined how reproducibly they bound to the same site on a protein using QuickVina-W, a branch of the program Autodock that is optimized for blind searches. We discovered that the higher the backbone quality of the homology model the greater the similarity between the small molecule docking to the experimental and modeled structures. Furthermore, we found that specific subsets of this library were particularly useful for identifying small differences between the best of the best modeled structures. Specifically, when the number of rotatable bonds in the small molecule increased, differences in binding sites became more apparent.

Adipocyte-Specific Laminin Alpha 4 Deletion Preserves Adipose Tissue Health despite Increasing Adiposity.

Laminins are heterotrimeric glycoproteins with structural and functional roles in basement membranes. The predominant laminin alpha chain found in adipocyte basement membranes is laminin α4 (LAMA4). Global LAMA4 deletion in mice leads to reduced adiposity and increased energy expenditure, but also results in vascular defects that complicate the interpretation of metabolic data. Here, we describe the generation and initial phenotypic analysis of an adipocyte-specific LAMA4 knockout mouse (Lama4AKO). We first performed an in-silico analysis to determine the degree to which laminin α4 was expressed in human and murine adipocytes. Next, male Lama4AKO and control mice were fed chow or high-fat diets and glucose tolerance was assessed along with serum insulin and leptin levels. Adipocyte area was measured in both epididymal and inguinal white adipose tissue (eWAT and iWAT, respectively), and eWAT was used for RNA-sequencing. We found that laminin α4 was highly expressed in human and murine adipocytes. Further, chow-fed Lama4AKO mice are like control mice in terms of body weight, body composition, and glucose tolerance, although they have larger eWAT adipocytes and lower insulin levels. High-fat-fed Lama4AKO mice are fatter and more glucose tolerant when compared to control mice. Transcriptionally, the eWAT of high-fat fed Lama4AKO mice resembles that of chow-fed control mice. We conclude from these findings that adipocyte-specific LAMA4 deletion is protective in an obesogenic environment, even though overall adiposity is increased.

Metabolovigilance: Associating Drug Metabolites with Adverse Drug Reactions.

The Metabolovigilance database (https://pharmacogenomics.clas.ucdenver.edu/pharmacogenomics/side-effect/) is a single repository of information on over 15,920 pharmaceuticals and the compounds expected to result from metabolism of these drugs. Metabolovigilance functions as both a web server, providing data directly to users and as a web application, applying user inputs to create logic statements that curate the data presented or downloaded. Using this tool, it is easy to collect information on drugs, their side effects, and the metabolites associated with specific side effects. Information on these compounds can be sorted based on physical properties of the drugs and their metabolites. All of this information can be viewed, sorted, and downloaded for use in other applications. This open-access tool will facilitate molecular studies on the causes of adverse drug reactions and is well suited to integrate with genomic data furthering the goals of personalized medicine.

Biochemically prepared C-reactive protein conformational states differentially affect C1q binding.

C-reactive protein (CRP) is commonly measured as an inflammatory marker in patient studies for coronary heart disease, autoimmune disease and recent acute infections. Due to a correlation of CRP to a vast number of disease states, CRP is a well-studied protein in medical literature with over 16000 references in PubMed [1]. However, the biochemical and structural variations of CRP are not well understood in regards to their binding of complement immune response proteins. Conformations of CRP are thought to affect disease states differently, with a modified form showing neoepitopes and activating the complement immune response through C1q binding. In this work, we compare the unfolding of CRP using chemical denaturants and identify which states of CRP bind a downstream complement immune response binding partner (C1q). We used guanidine HCl (GndHCl), urea/EDTA, and 0.01% SDS with heat to perturb the pentameric state. All treatments give rise to a monomeric state in non-denaturing polyacrylamide gel electrophoresis experiments, but only treatment with certain concentrations of denaturant or dilute SDS with heat maintains CRP function with a key downstream binding partner, C1q, as measured by enzyme-linked immunosorbent assays. The results suggest that the final form of modified CRP and its ability to mimic biological binding is dependent on the preparation method.

Right Atrioventricular Valvular Dysplasia in a New Zealand White Rabbit.

A sixteen-week-old, male New Zealand White rabbit was euthanized following an acute onset of respiratory distress and cyanosis. On necropsy, the rabbit had marked right atrioventricular eccentric hypertrophy, absence or rudimentary presence of the septal leaflet of the right atrioventricular valve, focally extensive left ventricular infarction, diffuse hepatic chronic passive congestion, and diffuse pulmonary edema. To our knowledge, right atrioventricular valvular hypoplasia, dysplasia, or aplasia has not been previously described in rabbits.

Dynamics of Serine-8 Side-Chain in Amyloid-ß Fibrils and Fluorenylmethyloxycarbonyl Serine Amino Acid, Investigated by Solid-State Deuteron NMR.

Serine side-chains are strategic sites of post-translational modifications, and it is important to establish benchmarks of their internal dynamics. In this work, we compare the dynamics of serine side-chains in several biologically important systems: serine-8 in the disordered domain of Aß1-40 fibrils in the hydrated and dry states and fluorenylmethyloxycarbonyl (Fmoc) serine with the bulky group that mimics the hydrophobicity of the fibril contacts yet lacks the complexity of the protein system. Using deuterium solid-state NMR static line shape and longitudinal relaxation techniques in the 310 to 180 K temperature range, we compare the main features of the dynamics in these systems. The main motional modes in the fibrils are large-scale fluctuations in the hydrated state of the fibrils as well as local motions such as 3-site jumps of the Cß deuterons at high temperatures and small-angle fluctuations of the Cα-Cß axis at low temperatures. In the hydrated fibrils, two distinct states are present with vastly different extents of large-scale diffusive motions and 3-site-jump rate constants. The hydrated state at the physiological conditions is dominated by the "free" state undergoing large-scale diffusive motions and very fast local 3-site jumps, while in the "bound" state, these large-scale motions are quenched due to transient inter- and intramolecular interactions. Additionally, in the bound state, the 3-site-jump motions are orders of magnitude slower. Details of the dynamics in the serine side-chain are dependent on fine structural features and hydration levels of the systems.

Lipid-Coated Gold Nanoparticles and FRET Allow Sensitive Monitoring of Liposome Clustering Mediated by the Synaptotagmin-7 C2A Domain.

Synaptotagmin (Syt) family proteins contain tandem C2 domains, C2A and C2B, which insert into anionic membranes in response to increased cytosolic Ca2+ concentration and facilitate exocytosis in neuronal and endocrine cells. The C2A domain from Syt7 binds lipid membranes much more tightly than the corresponding domain from Syt1, but the implications of this difference for protein function are not yet clear. In particular, the ability of the isolated Syt7 C2A domain to initiate membrane apposition and/or aggregation has been previously unexplored. Here, we demonstrate that Syt7 C2A induces apposition and aggregation of liposomes using Förster resonance energy transfer (FRET) assays, dynamic light scattering, and spectroscopic techniques involving lipid-coated gold nanoparticles (LCAuNPs). Protein-membrane binding, membrane apposition, and macroscopic aggregation are three separate phenomena with distinct Ca2+ requirements: the threshold Ca2+ concentration for membrane binding is lowest, followed by apposition and aggregation. However, aggregation is highly sensitive to protein concentration and can occur even at submicromolar Syt7 C2A; thus, highly sensitive assays are needed for measuring apposition without complications arising from aggregation. Notably, the localized surface plasmon resonance of the LCAuNP is sensitive to ≤10 nM Syt7 C2A concentrations. Furthermore, when the LCAuNPs were added into a FRET-based liposome apposition assay, the resultant energy transfer increased; possible explanations are discussed. Overall, LCAuNP-based methods allow for highly sensitive detection of protein-induced membrane apposition under conditions that miminize large-scale aggregation.

Conformational Changes in C-Reactive Protein Affect Binding to Curved Membranes in a Lipid Bilayer Model of the Apoptotic Cell Surface.

C-reactive protein (CRP) is a serum protein that binds to damaged membranes through a phosphatidylcholine binding site. The membrane binding process can initiate the complement immune response and facilitates the clearance of apoptotic cells, likely aiding in the protection of autoimmunity. The initiation of an immune response relies on a conformation change from a native, pentameric form to a modified form, where the modified form binds complement proteins (i.e., C1q) and regulatory proteins substantially better than the native form. In vitro, this reactivity is observed when CRP is monomeric, and a modified form has also been observed at sites of inflammation. Despite evidence that the monomeric form has much higher affinities for almost all proteinaceous binding partners, the role of CRP conformation on lipid binding is yet unknown. In this work, we mimic the outer leaflet of apoptotic cell membranes using a nanopatterned substrate to create curved, supported lipid bilayers and then characterize how CRP conformation affects the interactions between CRP and target membranes. In this assay, the chemical composition and shape are separately tunable parameters. The lipids consisted primarily of palmitoyloleoylphosphatidylcholine, with and without lysophosphatidylcholine, and the curvature had a radius of 27-55 nm. Using this model system combined with quantitative fluorescence microscopy methods, CRP binding to lipid membranes was measured as a function of different conformations of CRP. The modified form of CRP bound curved membranes, but the pentameric form did not for the range of curvatures measured. Unlike most other curvature-sensing proteins, modified CRP accumulated more at a moderate curvature, rather than highly curved or flat regions, suggesting that the membrane bound form does not solely depend on a defect binding mechanism. The presence of lysophosphatidylcholine, a component of apoptotic membranes, increased CRP binding to all types of membranes. Overall, our results show that CRP interactions vary with protein form, lipid composition, and membrane shape. The mechanism by which CRP recognizes damaged membranes depends on the combination of all three.

Polymer mediated layer-by-layer assembly of different shaped gold nanoparticles.

Gold nanoparticles (GNPs) have a wide range of properties with potential applications in electronics, optics, catalysis, and sensing. In order to demonstrate that dense, stable, and portable samples could be created for these applications, multiple layers of GNPs were assembled via drop casting on glass substrates by layer-by-layer (LBL) techniques. Two cationic polyelectrolytes, poly(diallyldimethylammonium chloride) and polyethyleneimine, one anionic polyelectrolyte, poly(sodium 4-styrene sulfonate), and one neutral polymer, polyvinylpyrrolidone, were combined with four different shapes of GNPs (spherical, rod, triangular prismatic, and octahedral) to prepare thin films. A subset of these polymer nanoparticle combinations were assembled into thin films. Synthesized GNPs were characterized via dynamic light scattering, UV-vis spectroscopy, and transmission electron microscopy and the LBL thin films were characterized using UV-vis spectroscopy and atomic force microscopy. Sensing applications of the nanoparticles in solution and thin films were tested by monitoring the localized surface plasmon resonance of the GNPs. LBL thin films were prepared ranging from 25 to 100 layers with optical densities at plasmon from 0.5 to 3.0. Sensitivity in solutions ranged from 14 to 1002nm/refractive index units (RIU) and films ranged from 18.8 to 135.1nm/RIU suggesting reduced access to the GNPs within the films.

Oxyspirura petrowi infection leads to pathological consequences in Northern bobwhite (Colinus virginianus).

Debilitating ocular diseases are often reported in avian species. By and large, helminth parasites have been overlooked in avian diseases and regarded as inconsequential. The decline of Northern bobwhite quail (Colinus virginianus) in the Rolling Plains ecoregion of Texas has prompted an investigation of the factors influencing their disappearance. Infection by the eyeworm (Oxyspirura petrowi) has been documented in many avian species; however, the effect it has on its host is not well understood. Heavy eyeworm infection has been documented in Northern bobwhites throughout this ecoregion, leading to eye pathology in this host species. The present study further documents and supports the pathological changes associated with O. petrowi in bobwhites.

Improved methods for evaluating the environmental impact of nanoparticle synthesis†.

With the market for products containing nanoparticles growing, improvements in the efficiency of nanoparticle synthesis are poised to have significant positive economic and environmental impacts. While many metrics have been designed for measuring the efficiency of small molecule synthesis, the use of these metrics for evaluating nanoparticle preparation has not been optimized. Here a critical evaluation of various green chemistry metrics is provided as they are applied to a common set of nanoparticle synthetic methods. The effect of the nanoparticle polydispersity on the relative greenness of different synthetic methods is also examined. Using metrics modified to account for polydispersity, a case study of gold nanoparticle syntheses is provided and three different methods of preparing monodisperse gold nanoparticles are compared. Interestingly, not all of the metrics provide the same rankings for the synthetic methods. And when polydispersity is ignored, the metrics provide a different rank order of the methods, highlighting the importance of clearly defining the desired nanoparticle size range to avoid underestimating the environmental impact.

The Two-Wrongs model explains perception-action dissociations for illusions driven by distortions of the egocentric reference frame.

Several studies have demonstrated a dissociation of the effects of illusion on perception and action, with perception generally reported to be susceptible to illusions, while actions are seemingly immune. These findings have been interpreted to support Milner and Goodale's Two Visual Systems model, which proposes the existence of separate visual processing streams for perception and action. However, an alternative interpretation suggests that this type of behavioral dissociation will occur for any illusion that is caused by a distortion of the observer's egocentric reference frame, without requiring the existence of separate perception and action systems that are differently affected by the illusion. In this scenario, movements aimed at illusory targets will be accurate if they are guided within the same distorted reference frame used for target encoding, since the error of motor guidance will cancel with the error of encoding (hence, for actions, two wrongs do make a right). We further test this Two-Wrongs model by examining two illusions for which the hypothesis makes very different predictions: the rod-and-frame illusion (which affects perception but not actions) and the simultaneous-tilt illusion (which affects perception and actions equally). We demonstrate that the rod-and-frame illusion is caused by a distortion of the observer's egocentric reference frame suitable for the cancellation of errors predicted by the Two-Wrongs model. In contrast, the simultaneous-tilt illusion is caused by local interactions between stimulus elements within an undistorted reference frame, precluding the cancellation of errors associated with the Two-Wrongs model such that the illusion is reflected in both perception and actions. These results provide evidence for a class of illusions that lead to dissociations of perception and action through distortions of the observer's spatial reference frame, rather than through the actions of functionally separate visual processing streams.

Can routine trauma bay chest x-ray be bypassed with an extended focused assessment with sonography for trauma examination?

The objective of this study was to investigate the feasibility of using ultrasound (US) in place of portable chest x-ray (CXR) for the rapid detection of a traumatic pneumothorax (PTX) requiring urgent decompression in the trauma bay. All patients who presented as a trauma alert to a single institution from August 2011 to May 2012 underwent an extended focused assessment with sonography for trauma (FAST). The thoracic cavity was examined using four-view US imaging and were interpreted by a chief resident (Postgraduate Year 4) or attending staff. US results were compared with CXR and chest computed tomography (CT) scans, when obtained. The average age was 37.8 years and 68 per cent of the patients were male. Blunt injury occurred in 87 per cent and penetrating injury in 12 per cent of activations. US was able to predict the absence of PTX on CXR with a sensitivity of 93.8 per cent, specificity of 98 per cent, and a negative predictive value of 99.9 per cent compared with CXR. The only missed PTX seen on CXR was a small, low anterior, loculated PTX that was stable for transport to CT. The use of thoracic US during the FAST can rapidly and safely detect the absence of a clinically significant PTX. US can replace routine CXR obtained in the trauma bay and allow more rapid initiation of definitive imaging studies.

Leucine acts in the brain to suppress food intake but does not function as a physiological signal of low dietary protein.

Intracerebroventricular injections of leucine are sufficient to suppress food intake, but it remains unclear whether brain leucine signaling represents a physiological signal of protein balance. We tested whether variations in dietary and circulating levels of leucine, or all three branched-chain amino acids (BCAAs), contribute to the detection of reduced dietary protein. Of the essential amino acids (EAAs) tested, only intracerebroventricular injection of leucine (10 µg) was sufficient to suppress food intake. Isocaloric low- (9% protein energy; LP) or normal- (18% protein energy) protein diets induced a divergence in food intake, with an increased consumption of LP beginning on day 2 and persisting throughout the study (P < 0.05). Circulating BCAA levels were reduced the day after LP diet exposure, but levels subsequently increased and normalized by day 4, despite persistent hyperphagia. Brain BCAA levels as measured by microdialysis on day 2 of diet exposure were reduced in LP rats, but this effect was most prominent postprandially. Despite these diet-induced changes in BCAA levels, reducing dietary leucine or total BCAAs independently from total protein was neither necessary nor sufficient to induce hyperphagia, while chronic infusion of EAAs into the brain of LP rats failed to consistently block LP-induced hyperphagia. Collectively, these data suggest that circulating BCAAs are transiently reduced by dietary protein restriction, but variations in dietary or brain BCAAs alone do not explain the hyperphagia induced by a low-protein diet.

Adaptation to leftward-shifting prisms enhances local processing in healthy individuals.

In healthy individuals, adaptation to left-shifting prisms has been shown to simulate the symptoms of hemispatial neglect, including a reduction in global processing that approximates the local bias observed in neglect patients. The current study tested whether leftward prism adaptation can more specifically enhance local processing abilities. In three experiments, the impact of local and global processing was assessed through tasks that measure susceptibility to illusions that are known to be driven by local or global contextual effects. Susceptibility to the rod-and-frame illusion - an illusion disproportionately driven by both local and global effects depending on frame size - was measured before and after adaptation to left- and right-shifting prisms. A significant increase in rod-and-frame susceptibility was found for the left-shifting prism group, suggesting that adaptation caused an increase in local processing effects. The results of a second experiment confirmed that leftward prism adaptation enhances local processing, as assessed with susceptibility to the simultaneous-tilt illusion. A final experiment employed a more specific measure of the global effect typically associated with the rod-and-frame illusion, and found that although the global effect was somewhat diminished after leftward prism adaptation, the trend failed to reach significance (p=.078). Rightward prism adaptation had no significant effects on performance in any of the experiments. Combined, these findings indicate that leftward prism adaptation in healthy individuals can simulate the local processing bias of neglect patients primarily through an increased sensitivity to local visual cues, and confirm that prism adaptation not only modulates lateral shifts of attention, but also prompts shifts from one level of processing to another.