An autosomal recessive variant in PYGM causes myophosphorylase deficiency in Red Angus composite cattle.

BACKGROUND: Between 2020 and 2022, eight calves in a Nebraska herd (composite Simmental, Red Angus, Gelbvieh) displayed exercise intolerance during forced activity. In some cases, the calves collapsed and did not recover. Available sire pedigrees contained a paternal ancestor within 2-4 generations in all affected calves. Pedigrees of the calves' dams were unavailable, however, the cows were ranch-raised and retained from prior breeding seasons, where bulls used for breeding occasionally had a common ancestor. Therefore, it was hypothesized that a de novo autosomal recessive variant was causative of exercise intolerance in these calves. RESULTS: A genome-wide association analysis utilizing SNP data from 6 affected calves and 715 herd mates, followed by whole-genome sequencing of 2 affected calves led to the identification of a variant in the gene PYGM (BTA29:g.42989581G > A). The variant, confirmed to be present in the skeletal muscle transcriptome, was predicted to produce a premature stop codon (p.Arg650*). The protein product of PYGM, myophosphorylase, breaks down glycogen in skeletal muscle. Glycogen concentrations were fluorometrically assayed as glucose residues demonstrating significantly elevated glycogen concentrations in affected calves compared to cattle carrying the variant and to wild-type controls. The absence of the PYGM protein product in skeletal muscle was confirmed by immunohistochemistry and label-free quantitative proteomics analysis; muscle degeneration was confirmed in biopsy and necropsy samples. Elevated skeletal muscle glycogen persisted after harvest, resulting in a high pH and dark-cutting beef, which is negatively perceived by consumers and results in an economic loss to the industry. Carriers of the variant did not exhibit differences in meat quality or any measures of animal well-being. CONCLUSIONS: Myophosphorylase deficiency poses welfare concerns for affected animals and negatively impacts the final product. The association of the recessive genotype with dark-cutting beef further demonstrates the importance of genetics to not only animal health but to the quality of their product. Although cattle heterozygous for the variant may not immediately affect the beef industry, identifying carriers will enable selection and breeding strategies to prevent the production of affected calves.

Pharmacotherapy for Alzheimer's disease: what's new on the horizon?

INTRODUCTION: Alzheimer's disease (AD) is a debilitating disease, with no cure. Recently, a monoclonal antibody (aducanumab) directed toward amyloid aggregates was approved as a disease-modifying treatment (DMT) for the disease. Other compounds (symptomatic or DMTs) are at different stages of clinical trial development. AREAS COVERED: The authors conducted a search on PUBMED, MEDLINE, and clinicaltrials.gov for compounds in phase III clinical trials for cognitive impairment due to AD. Mechanisms of action and clinical trial data related to these compounds are discussed in this paper. EXPERT OPINION: There is an unmet need for both treatment approaches (symptomatic and DMTs) to improve outcomes in individuals at different stages of the AD continuum. Future trials with symptomatic therapies should rely on biomarkers to improve enrollment of participants with pure AD. More sensitive, innovative, and composite assessment tools should be used. Given the complexity and heterogeneity of AD, combining several DMTs with synergistic mechanisms of action is a promising approach to achieve a significant impact on reversing cognitive decline. We recommend testing DMTs early on in the disease continuum, even in asymptomatic individuals at risk for AD. Longer duration of follow-up in clinical trials with DMTs is recommended.

Increased connectivity and depth improve the effectiveness of marine reserves.

Marine reserves are a key tool for the conservation of marine biodiversity, yet only ~2.5% of the world's oceans are protected. The integration of marine reserves into connected networks representing all habitats has been encouraged by international agreements, yet the benefits of this design has not been tested empirically. Australia has one of the largest systems of marine reserves, providing a rare opportunity to assess how connectivity influences conservation success. An Australia-wide dataset was collected using baited remote underwater video systems deployed across a depth range from 0 to 100 m to assess the effectiveness of marine reserves for protecting teleosts subject to commercial and recreational fishing. A meta-analytical comparison of 73 fished species within 91 marine reserves found that, on average, marine reserves had 28% greater abundance and 53% greater biomass of fished species compared to adjacent areas open to fishing. However, benefits of protection were not observed across all reserves (heterogeneity), so full subsets generalized additive modelling was used to consider factors that influence marine reserve effectiveness, including distance-based and ecological metrics of connectivity among reserves. Our results suggest that increased connectivity and depth improve the aforementioned marine reserve benefits and that these factors should be considered to optimize such benefits over time. We provide important guidance on factors to consider when implementing marine reserves for the purpose of increasing the abundance and size of fished species, given the expected increase in coverage globally. We show that marine reserves that are highly protected (no-take) and designed to optimize connectivity, size and depth range can provide an effective conservation strategy for fished species in temperate and tropical waters within an overarching marine biodiversity conservation framework.

Assessing the current state of ecological connectivity in a large marine protected area system.

The establishment of marine protected areas (MPAs) is a critical step in ensuring the continued persistence of marine biodiversity. Although the area protected in MPAs is growing, the movement of individuals (or larvae) among MPAs, termed connectivity, has only recently been included as an objective of many MPAs. As such, assessing connectivity is often neglected or oversimplified in the planning process. For promoting population persistence, it is important to ensure that protected areas in a system are functionally connected through dispersal or adult movement. We devised a multi-species model of larval dispersal for the Australian marine environment to evaluate how much local scale connectivity is protected in MPAs and determine whether the extensive system of MPAs truly functions as a network. We focused on non-migratory species with simplified larval behaviors (i.e., passive larval dispersal) (e.g., no explicit vertical migration) as an illustration. Of all the MPAs analyzed (approximately 2.7 million km2 ), outside the Great Barrier Reef and Ningaloo Reef, <50% of MPAs (46-80% of total MPA area depending on the species considered) were functionally connected. Our results suggest that Australia's MPA system cannot be referred to as a single network, but rather a collection of numerous smaller networks delineated by natural breaks in the connectivity of reef habitat. Depending on the dispersal capacity of the taxa of interest, there may be between 25 and 47 individual ecological networks distributed across the Australian marine environment. The need to first assess the underlying natural connectivity of a study system prior to implementing new MPAs represents a key research priority for strategically enlarging MPA networks. Our findings highlight the benefits of integrating multi-species connectivity into conservation planning to identify opportunities to better incorporate connectivity into the design of MPA systems and thus to increase their capacity to support long-term, sustainable biodiversity outcomes.

Valoración del Estado Actual de la Conectividad Ecológica en un Sistema Extenso de Áreas Marinas Protegidas Resumen La creación de áreas marinas protegidas (AMP) es un paso muy importante para asegurar la persistencia de la biodiversidad marina. Aunque el área protegida dentro de las AMP está creciendo, el movimiento de individuos (o larvas) entre las AMP, denominado conectividad, sólo ha sido incluido recientemente como un objetivo para muchas AMP. Por lo anterior es normal que con frecuencia se ignora la evaluación de la conectividad o se sobresimplifica durante el proceso de planeación. Para promover la persistencia poblacional es importante asegurar que las áreas protegidas en un sistema estén conectadas funcionalmente por medio de la dispersión o el movimiento de individuos adultos. Diseñamos un modelo multiespecie de la dispersión larval para el ambiente marino australiano y así evaluar cuán protegida está la conectividad a escala local en las AMP y determinar si el sistema extensivo de AMP realmente funciona como una red. Nuestro diseño se enfocó en especies no migratorias con comportamientos larvales simplificados (es decir, dispersión larval pasiva) (p. ej.: sin migración vertical explícita) como un ejemplo. De todas las AMP analizadas (aproximadamente 2.7 millones de km2 ), fuera de la Gran Barrera de Arrecifes y el Arrecife Ningaloo, <50% de las AMP (46-80% del área total de la MPA dependiendo de la especie considerada) estaba conectado funcionalmente. Nuestros resultados sugieren que el sistema de AMP australiano no puede ser considerado como una sola red sino más bien como una colección de numerosas redes más pequeñas delineadas por interrupciones naturales en la conectividad del hábitat arrecifal. De acuerdo con la capacidad de dispersión del taxón de interés, puede haber entre 25 y 47 redes ecológicas individuales distribuidas a lo largo del ambiente marino australiano. La necesidad de primero evaluar la conectividad natural subyacente de un sistema de estudio previo a la implementación de nuevas AMP representa una prioridad de investigación clave para aumentar estratégicamente las redes de AMP. Nuestros resultados resaltan los beneficios de la integración de la conectividad multiespecie dentro de la planeación de la conservación para identificar las oportunidades que mejor incorporen la conectividad en el diseño de los sistemas de AMP y así incrementar su capacidad para soportar resultados sustentables de biodiversidad a largo plazo.

Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit.

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.

Granular Activated Carbon Adsorption of Carcinogenic Volatile Organic Compounds at Low Influent Concentrations.

The effectiveness of granular activated carbon (GAC) for carcinogenic volatile organic compounds (cVOCs) has not been evaluated in the low- to sub- microgram per liter range. Rapid small scale column tests (RSSCTs) were employed to determine the GAC performance at empty bed contact times (EBCTs) of 7.5 and 15 minutes for 13 cVOCs at a target influent concentration of 5 µg/L in a typical groundwater matrix. Breakthrough was assessed for vinyl chloride, dichloromethane, 1,1-dichloroethane, 1,2-dichloroethane, 1,2-dichloropropane, carbon tetrachloride, 1,3-butadiene, 1,1,1,2-tetrachloroethane, 1,2,3-trichloropropane, trichloroethylene and tetrachloroethylene. The throughput to breakthrough was found to be linearly correlated to capacities calculated with single-solute equilibrium isotherm parameters. Modest decreases, 9 to 13% on average, in throughput to 50% and 75% breakthrough were found when the EBCT was increased from 7.5 to 15 minutes. The carbon use rate (CUR), when scaled to simulate full-scale adsorption, indicated that GAC would be a viable technology for seven of the VOCs evaluated, with a CUR threshold less than 0.2 lbs/1000 gal. It may be possible to use 1,1 DCA and 1,2 DCA as surrogates for assessing chemicals near the feasibility limit.

Health Impact of Hurricanes Irma and Maria on St Thomas and St John, US Virgin Islands, 2017-2018.

Objectives. To assess the health impact of Hurricane Irma and Hurricane Maria on St Thomas, US Virgin Islands.Methods. We collected data from interviews conducted 6 and 9 months after the hurricanes, a review of 597 randomly selected emergency department (ED) encounters, and administrative records from 10 716 ED visits 3 months before, between, and 3 months after the hurricanes.Results. Informants described damaged hospital infrastructure, including flooding, structural damage, and lost staff. The greatest public health impact was on the elderly and persons with chronic diseases. In the setting of loss of the electronic medical record system, ED chart reviews were limited by problems with missing data. ED administrative data demonstrated that posthurricane patients, compared with prehurricane patients, were older and had less severe complaints. There was a significant increase in patients being seen for diabetes-related and respiratory complaints, especially asthma. Suboptimal recordkeeping for medical evacuees limited the ability to assess outcomes for patients with severe illnesses.Conclusions. Hurricanes Irma and Maria caused major disruptions to health care on St Thomas. Emphasis should be given to building a resilient health care system that will optimally respond to future hurricanes.

Smoothened stimulation by membrane sterols drives Hedgehog pathway activity.

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3-5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results-combined with signalling studies and molecular dynamics simulations-delineate the structural basis for PTCH1-SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.

Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

Repeated Dietary Exposure to Low Levels of Domoic Acid and Problems with Everyday Memory: Research to Public Health Outreach.

Domoic Acid (DA) is a marine-based neurotoxin. Dietary exposure to high levels of DA via shellfish consumption has been associated with Amnesic Shellfish Poisoning, with milder memory decrements found in Native Americans (NAs) with repetitive, lower level exposures. Despite its importance for protective action, the clinical relevance of these milder memory problems remains unknown. The purpose of this study was to determine whether repeated, lower-level exposures to DA impact everyday memory (EM), i.e., the frequency of memory failures in everyday life. A cross-sectional sample of 60 NA men and women from the Pacific NW was studied with measures of dietary exposure to DA via razor clam (RC) consumption and EM. Findings indicated an association between problems with EM and elevated consumption of RCs with low levels of DA throughout the previous week and past year after controlling for age, sex, and education. NAs who eat a lot of RCs with presumably safe levels of DA are at risk for clinically significant memory problems. Public health outreach to minimize repetitive exposures are now in place and were facilitated by the use of community-based participatory research methods, with active involvement of state regulatory agencies, tribe leaders, and local physicians.

Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium.

Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1-Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1-Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na+ to regulate Smoothened in our assays, raising the possibility that a Na+ gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na+ gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation.

The Stromal Niche for Epithelial Stem Cells: A Template for Regeneration and a Brake on Malignancy.

Stromal restraint of cancer growth and progression-emerging as a widespread phenomenon in epithelial cancers such as bladder, pancreas, colon, and prostate-appears rooted in stromal cell niche activity. During normal tissue repair, stromal niche signals, often Hedgehog-induced, promote epithelial stem cell differentiation as well as self-renewal, thus specifying a regenerating epithelial pattern. In the case of cancerous tissue, stromal cell-derived differentiation signals in particular may provide a brake on malignant growth. Understanding and therapeutic harnessing of the role of stroma in cancer restraint may hinge on our knowledge of the signaling programs elaborated by the stromal niche.

A General, Simple Catalyst for Enantiospecific Cross Couplings of Benzylic Ammonium Triflates and Boronic Acids: No Phosphine Ligand Required.

Highly improved conditions for the enantiospecific cross coupling of benzylic ammonium triflates with boronic acids are reported. This method relies on the use of Ni(cod)2 without ancillary phosphine or N-heterocyclic carbene ligands as catalyst. These conditions enable the coupling of new classes of boronic acids and benzylic ammonium triflates. In particular, both heteroaromatic and vinyl boronic acids are well tolerated as coupling partners. In addition, these conditions enable the use of ammonium triflates with a variety of substituents at the benzylic stereocenter. Further, naphthyl-substitution is not required on the benzylic ammonium triflate; ammonium triflates with simple aromatic substituents also undergo this coupling. Good to high yields and levels of stereochemical fidelity are observed. This new catalyst system greatly expands the utility of enantiospecific cross couplings of these amine-derived substrates for the preparation of highly enantioenriched products.

Production of recombineering substrates with standard-size PCR primers.

Recombineering is a powerful method for DNA manipulation. It has advantages over restriction endonuclease-based methods and is usually rapid. Typically, recombineering uses long PCR primers (c. 65 bases), each of which contains a small region of target homology (c. 45 bases). We have developed a simple, albeit somewhat less rapid, strategy to create recombineering substrates that can use primers of ≤ 35 bases for all steps. The regions of homology can be several hundred base pairs in length to (1) increase the chance of obtaining the desired clone and/or (2) allow coliphage-based recombineering in some non-Escherichia coli bacteria. The method uses cloning techniques to construct a template for the generation of the recombineering substrate. Because the template is made from cloned DNA segments, the segments (including those for the homology regions) can be readily changed. During construction of the template plasmid, potential background transformants arising from the vector without insert are significantly reduced by cloning each segment with two restriction endonucleases that produce noncompatible ends. We have used this method to change the bla gene of pACYC177 to aadA, to add the MCS-lacZα region from pBBR1MCS to IncQ plasmid vectors, and to make an oriT(IncP) -aacC1 cassette and add it to a plasmid.

Consistency of the counting talk test for exercise prescription.

The purpose of this study was to assess the consistency of the counting talk test (CTT) method for estimating exercise intensity across various modes of exercise in healthy young adults. Thirty-six individuals completed the study, which required participation in 3 separate sessions within a 2-week time period. During the first session, the individuals completed a maximal effort treadmill test from which each individual's heart rate reserve (HRR) was calculated. During the second and third sessions, the subjects participated in 2 modes of exercise in each session for a total of 4 different modes of exercise. The individuals exercised at 40% HRR, 50% HRR, 60% HRR, 75% HRR, and 85% HRR. The heart rate (HR), CTT, and rating of perceived exertion (RPE) were recorded at each workload. Based on the individual's resting CTT (CTT(rest)), the %CTT for each exercise stage was then calculated. Pearson correlations demonstrated moderate to good correlations between the CTT and HRR methods and the CTT and RPE methods for estimating exercise intensity. This study found that for the individuals with CTT(rest) <25, moderate to vigorous intensity exercise as recommended by the American College of Sports Medicine HRR guidelines could be achieved by exercising at a level of 40-50% CTT(rest). Individuals with a CTT(rest) ≥25, exercising at a level of 30-40% CTT(rest) would place them in the moderate to vigorous exercise intensity range. A high degree of reliability was demonstrated using the CTT method across the various modes of aerobic exercise. As such, independent of the exercise mode, the CTT was found to be an easy and consistent method for prescribing moderate to vigorous aerobic exercise intensity.