High-throughput screening for small-molecule stabilizers of misfolded glucocerebrosidase in Gaucher disease and Parkinson's disease.

Glucocerebrosidase (GCase) is implicated in both a rare, monogenic disorder (Gaucher disease, GD) and a common, multifactorial condition (Parkinson's disease, PD); hence, it is an urgent therapeutic target. To identify correctors of severe protein misfolding and trafficking obstruction manifested by the pathogenic L444P-variant of GCase, we developed a suite of quantitative, high-throughput, cell-based assays. First, we labeled GCase with a small proluminescent HiBiT peptide reporter tag, enabling quantitation of protein stabilization in cells while faithfully maintaining target biology. TALEN-based gene editing allowed for stable integration of a single HiBiT-GBA1 transgene into an intragenic safe-harbor locus in GBA1-knockout H4 (neuroglioma) cells. This GD cell model was amenable to lead discovery via titration-based quantitative high-throughput screening and lead optimization via structure-activity relationships. A primary screen of 10,779 compounds from the NCATS bioactive collections identified 140 stabilizers of HiBiT-GCase-L444P, including both pharmacological chaperones (ambroxol and noninhibitory chaperone NCGC326) and proteostasis regulators (panobinostat, trans-ISRIB, and pladienolide B). Two complementary high-content imaging-based assays were deployed to triage hits: The fluorescence-quenched substrate LysoFix-GBA captured functional lysosomal GCase activity, while an immunofluorescence assay featuring antibody hGCase-1/23 directly visualized GCase lysosomal translocation. NCGC326 was active in both secondary assays and completely reversed pathological glucosylsphingosine accumulation. Finally, we tested the concept of combination therapy by demonstrating synergistic actions of NCGC326 with proteostasis regulators in enhancing GCase-L444P levels. Looking forward, these physiologically relevant assays can facilitate the identification, pharmacological validation, and medicinal chemistry optimization of small molecules targeting GCase, ultimately leading to a viable therapeutic for GD and PD.

Patagonian sheepdog: Genomic analyses trace the footprints of extinct UK herding dogs to South America.

Most modern dog breeds were developed within the last two hundred years, following strong and recent human selection based predominantly on aesthetics, with few modern breeds constructed solely to maximize their work potential. In many cases, these working breeds represent the last remnants of now lost populations. The Patagonian sheepdog (PGOD), a rare herding breed, is a remarkable example of such a population. Maintained as an isolated population for over 130 years, the PGOD offers a unique opportunity to understand the genetic relationship amongst modern herding breeds, determine key genomic structure of the founder PGOD populations, and investigate how canine genomic data can mirror human migration patterns. We thus analyzed the population structure of 159 PGOD, comparing them with 1514 dogs representing 175 established breeds. Using 150,069 SNPs from a high-density SNP genotyping array, we establish the genomic composition, ancestry, and genetic diversity of the population, complementing genomic data with the PGOD's migratory history to South America. Our phylogenetic analysis reveals that PGODs are most closely related to modern herding breeds hailing from the United Kingdom. Admixture models illustrate a greater degree of diversity and genetic heterogeneity within the very small PGOD population than in Western European herding breeds, suggesting the PGOD predates the 200-year-old construction of most pure breeds known today. We thus propose that PGODs originated from the foundational herding dogs of the UK, prior to the Victorian explosion of breeds, and that they are the closest link to a now-extinct population of herding dogs from which modern herding breeds descended.

DNA methylation clocks for dogs and humans.

DNA methylation profiles have been used to develop biomarkers of aging known as epigenetic clocks, which predict chronological age with remarkable accuracy and show promise for inferring health status as an indicator of biological age. Epigenetic clocks were first built to monitor human aging, but their underlying principles appear to be evolutionarily conserved, as they have now been successfully developed for many mammalian species. Here, we describe reliable and highly accurate epigenetic clocks shown to apply to 93 domestic dog breeds. The methylation profiles were generated using the mammalian methylation array, which utilizes DNA sequences that are conserved across all mammalian species. Canine epigenetic clocks were constructed to estimate age and also average time to death. We also present two highly accurate human­dog dual species epigenetic clocks (R = 0.97), which may facilitate the ready translation from canine to human use (or vice versa) of antiaging treatments being developed for longevity and preventive medicine. Finally, epigenome-wide association studies here reveal individual methylation sites that may underlie the inverse relationship between breed weight and lifespan. Overall, we describe robust biomarkers to measure aging and, potentially, health status in canines.

Non-transport after Prehospital Naloxone Administration Is Associated with Higher Risk of Subsequent Non-fatal Overdose.

Objective: U.S. opioid overdoses increased nearly sixfold from 1999 to 2018, and greater than 1% of all emergency medical services (EMS) encounters now involve naloxone administration. While "treat and release" protocols may have low short-term mortality, the risk of subsequent non-fatal overdoses is not known. This study compares the risk of repeat overdose encounters between patients transported to an emergency department (ED) and those who refused transport after prehospital naloxone administration. Methods: All EMS charts within a large single-tier fire-based urban EMS system between January 1 and August 31, 2018 were reviewed if either naloxone administration or a clinical impression related to opioid overdose was documented. Charts were excluded if there was no documented evidence of an opioid toxidrome (respiratory depression or altered mental status), if there was another clear explanation for the symptoms (e.g., hypoglycemia), or if naloxone was not administered. Ten percent of charts were reviewed by a second author to assess reliability. Cox regression (survival analysis) was used to estimate the risk of a subsequent EMS encounter with naloxone administration following an index encounter with naloxone administration. Results: Of the 2143 charts reviewed, 1311 unique patients with 1600 overdose encounters involving naloxone administration were identified. Inter-rater reliability for chart inclusion was strong [κ = 0.83 (95% CI: 0.72-0.90)]. Police/bystanders administered naloxone in 208/1600 (13.0%) encounters. A substantial proportion of encounters resulted in transport refusal (674/1600, 42.1%). The final Cox model included only refusal vs. acceptance of transport to an ED during the index EMS encounter. Patient age, gender, and naloxone administration prior to EMS arrival were not statistically significant in univariate or multivariable analyses, nor were they significant confounders. Refusal of transport was associated with a hazard ratio of 1.66 (95% CI: 1.23-2.23) for subsequent EMS encounters with naloxone administration. Conclusions: Non-transport after prehospital naloxone administration is associated with an increased risk of subsequent non-fatal overdose requiring EMS intervention. Limitations include the use of a single EMS agency as patients may have had uncaptured overdose encounters in neighboring municipalities.

Appropriate Air Medical Services Utilization and Recommendations for Integration of Air Medical Services Resources into the EMS System of Care: A Joint Position Statement and Resource Document of NAEMSP, ACEP, and AMPA.

This update to the 2013 joint position statement, Appropriate and Safe Utilization of Helicopter Emergency Medical Services, provides guidance for air medical services utilization based on currently available evidence. Air medical services utilization considerations fall into three major categories: clinical considerations, safety considerations, and system integration and quality assurance.Clinically, air medical services should accomplish one or more of three primary patient-centered goals: initiation or continuation of locally unavailable advanced or specialty care; expedited delivery to definitive care for time-sensitive interventions; and/or extraction from physically remote or otherwise inaccessible locations that limit timely access to necessary care. Ground-EMS (GEMS) transport is preferred when it is able to provide the necessary level of care and timely transport to definitive care.Risk identification and safety of both the patient and crew must be uniformly balanced against the anticipated degree of patient medical benefit. While auto-ready and auto-launch practices may increase access to air medical services, they also risk over-use, and so must be rigorously reviewed. Safety is enhanced during multi-agency emergency responses by coordinated interagency communication, ideally through centralized communication centers. Helicopter shopping and reverse helicopter shopping both create significant safety risks and their use is discouraged.Regional EMS systems must integrate air medical services to facilitate appropriate utilization in alignment with the primary patient goals while being cognizant of local indications, resources, and needs. To maximize consistent, informed air medical services utilization decisions, specific indications for and limitations to air medical services utilization that align with local and regional system and patient needs should be identified, and requests routed through centralized coordinating centers supported by EMS physicians.To limit risk and promote appropriate utilization of air medical services, GEMS clinicians should be encouraged to cancel an air medical services response if it is not aligned with at least one of the three primary patient-centered goals. Similarly, air medical services clinicians should be empowered to redirect patient transport to GEMS. Air medical services should not routinely be used solely to allow GEMS to remain in their primary service area.

Emergent Dialysis and its Impact on Quality of Life in Undocumented Patients with End-Stage Renal Disease.

OBJECTIVE: This study aimed to define the ethnographic composition and assess the health-related quality of life (HRQoL) of a large population of undocumented patients with end-stage renal disease (ESRD) seeking emergent dialysis in the emergency department (ED) of a large public hospital in the United States. DESIGN: All ESRD patients presenting to the hospital's main ED were identified during a 4-week consecutive enrollment period. Consenting patients completed two surveys-an ethnographic questionnaire and the validated kidney disease quality of life-36 (KDQOL-36) instrument. SETTING: The study was conducted at a large county hospital in Dallas, Texas. In 2013, the hospital recorded >50,000 ED visits and administered approximately 6,000 dialysis treatments to ED patients. PARTICIPANTS: 88 of 101 unfunded patients presenting to the ED during the study period consented to participate, resulting in an 87.1% response rate. 65 of these patients were undocumented immigrants. MAIN OUTCOME MEASURES: Quantitative scores for the 5 subscales of the KDQOL-36 were calculated for the study population. RESULTS: Measures of physical and mental health in our study population were lower than those published for scheduled dialysis patients. 79.5% of our patients lost employment due to their dialysis requirements. At least 71.4% of the study patients were unaware that they required dialysis before immigrating to the United States. CONCLUSIONS: Quality of life scores were found to be low among our population of undocumented emergent dialysis patients. Our data also provide some evidence that availability of dialysis at no cost is not a primary driver of illegal immigration of ESRD patients to the United States.

Intra-arrest blood-based biomarkers for out-of-hospital cardiac arrest: A scoping review.

Objective: Blood-based biomarkers play a central role in the diagnosis and treatment of critically ill patients, yet none are routinely measured during the intra-arrest phase of out-of-hospital cardiac arrest (OHCA). Our objective was to describe methodological aspects, sources of evidence, and gaps in research surrounding intra-arrest blood-based biomarkers for OHCA. Methods: We used scoping review methodology to summarize existing literature. The protocol was designed a priori following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Extension for Scoping Reviews. Inclusion criteria were peer-reviewed scientific studies on OHCA patients with at least one blood draw intra-arrest. We excluded in-hospital cardiac arrest and animal studies. There were no language, date, or study design exclusions. We conducted an electronic literature search using PubMed and Embase and hand-searched secondary literature. Data charting/synthesis were performed in duplicate using standardized data extraction templates. Results: The search strategy identified 11,834 records, with 118 studies evaluating 105 blood-based biomarkers included. Only eight studies (7%) had complete reporting. The median number of studies per biomarker was 2 (interquartile range 1-4). Most studies were conducted in Asia (63 studies, 53%).  Only 22 studies (19%) had blood samples collected in the prehospital setting, and only six studies (5%) had samples collected by paramedics. Pediatric patients were included in only three studies (3%). Out of eight predefined biomarker categories of use, only two were routinely assessed: prognostic (97/105, 92%) and diagnostic (61/105, 58%). Conclusions: Despite a large body of literature on intra-arrest blood-based biomarkers for OHCA, gaps in methodology and knowledge are widespread.

Emergency department management of adults with infectious meningitis and encephalitis.

Infectious meningitis and encephalitis are often life-threatening illnesses, though prompt workup and targeted treatment can greatly reduce morbidity and mortality. Although presentation of central nervous system infection can sometimes be subtle, this issue focuses on evidence-based strategies for identifying combinations of signs and symptoms to narrow the diagnosis. Identifying meningitis versus encephalitis; bacterial versus viral, fungal, or iatrogenic causes; and providing prompt empiric antimicrobials and appropriate diagnostic testing are key to management. Cerebrospinal fluid testing findings are outlined to help determine a potential cause for symptoms, along with blood and serum testing options. International society guidelines and evidence regarding the need for computed tomography prior to lumbar puncture are presented, which can help reduce unnecessary imaging. Disposition criteria are expanded to help determine whether a patient can go home, or the level of hospital care that will be required for those admitted.

Natural and human-driven selection of a single non-coding body size variant in ancient and modern canids.

Domestic dogs (Canis lupus familiaris) are the most variable-sized mammalian species on Earth, displaying a 40-fold size difference between breeds.1 Although dogs of variable size are found in the archeological record,2-4 the most dramatic shifts in body size are the result of selection over the last two centuries, as dog breeders selected and propagated phenotypic extremes within closed breeding populations.5 Analyses of over 200 domestic breeds have identified approximately 20 body size genes regulating insulin processing, fatty acid metabolism, TGFß signaling, and skeletal formation.6-10 Of these, insulin-like growth factor 1 (IGF1) predominates, controlling approximately 15% of body size variation between breeds.8 The identification of a functional mutation associated with IGF1 has thus far proven elusive.6,10,11 Here, to identify and elucidate the role of an ancestral IGF1 allele in the propagation of modern canids, we analyzed 1,431 genome sequences from 13 species, including both ancient and modern canids, thus allowing us to define the evolutionary history of both ancestral and derived alleles at this locus. We identified a single variant in an antisense long non-coding RNA (IGF1-AS) that interacts with the IGF1 gene, creating a duplex. While the derived mutation predominates in both modern gray wolves and large domestic breeds, the ancestral allele, which predisposes to small size, was common in small-sized breeds and smaller wild canids. Our analyses demonstrate that this major regulator of canid body size nearly vanished in Pleistocene wolves, before its recent resurgence resulting from human-imposed selection for small-sized breed dogs.

Quantitative Translation of Dog-to-Human Aging by Conserved Remodeling of the DNA Methylome.

All mammals progress through similar physiological stages throughout life, from early development to puberty, aging, and death. Yet, the extent to which this conserved physiology reflects underlying genomic events is unclear. Here, we map the common methylation changes experienced by mammalian genomes as they age, focusing on comparison of humans with dogs, an emerging model of aging. Using oligo-capture sequencing, we characterize methylomes of 104 Labrador retrievers spanning a 16-year age range, achieving >150× coverage within mammalian syntenic blocks. Comparison with human methylomes reveals a nonlinear relationship that translates dog-to-human years and aligns the timing of major physiological milestones between the two species, with extension to mice. Conserved changes center on developmental gene networks, which are sufficient to translate age and the effects of anti-aging interventions across multiple mammals. These results establish methylation not only as a diagnostic age readout but also as a cross-species translator of physiological aging milestones.

Whole genome sequencing of canids reveals genomic regions under selection and variants influencing morphology.

Domestic dog breeds are characterized by an unrivaled diversity of morphologic traits and breed-associated behaviors resulting from human selective pressures. To identify the genetic underpinnings of such traits, we analyze 722 canine whole genome sequences (WGS), documenting over 91 million single nucleotide and small indels, creating a large catalog of genomic variation for a companion animal species. We undertake both selective sweep analyses and genome wide association studies (GWAS) inclusive of over 144 modern breeds, 54 wild canids and a hundred village dogs. Our results identify variants of strong impact associated with 16 phenotypes, including body weight variation which, when combined with existing data, explain greater than 90% of body size variation in dogs. We thus demonstrate that GWAS and selection scans performed with WGS are powerful complementary methods for expanding the utility of companion animal systems for the study of mammalian growth and biology.

Studies of modern Italian dog populations reveal multiple patterns for domestic breed evolution.

Through thousands of years of breeding and strong human selection, the dog (Canis lupus familiaris) exists today within hundreds of closed populations throughout the world, each with defined phenotypes. A singular geographic region with broad diversity in dog breeds presents an interesting opportunity to observe potential mechanisms of breed formation. Italy claims 14 internationally recognized dog breeds, with numerous additional local varieties. To determine the relationship among Italian dog populations, we integrated genetic data from 263 dogs representing 23 closed dog populations from Italy, seven Apennine gray wolves, and an established dataset of 161 globally recognized dog breeds, applying multiple genetic methods to characterize the modes by which breeds are formed within a single geographic region. Our consideration of each of five genetic analyses reveals a series of development events that mirror historical modes of breed formation, but with variations unique to the codevelopment of early dog and human populations. Using 142,840 genome-wide SNPs and a dataset of 1,609 canines, representing 182 breeds and 16 wild canids, we identified breed development routes for the Italian breeds that included divergence from common populations for a specific purpose, admixture of regional stock with that from other regions, and isolated selection of local stock with specific attributes.