Advanced variant classification framework reduces the false positive rate of predicted loss-of-function variants in population sequencing data.

Predicted loss of function (pLoF) variants are often highly deleterious and play an important role in disease biology, but many pLoF variants may not result in loss of function (LoF). Here we present a framework that advances interpretation of pLoF variants in research and clinical settings by considering three categories of LoF evasion: (1) predicted rescue by secondary sequence properties, (2) uncertain biological relevance, and (3) potential technical artifacts. We also provide recommendations on adjustments to ACMG/AMP guidelines' PVS1 criterion. Applying this framework to all high-confidence pLoF variants in 22 genes associated with autosomal-recessive disease from the Genome Aggregation Database (gnomAD v.2.1.1) revealed predicted LoF evasion or potential artifacts in 27.3% (304/1,113) of variants. The major reasons were location in the last exon, in a homopolymer repeat, in a low proportion expressed across transcripts (pext) scored region, or the presence of cryptic in-frame splice rescues. Variants predicted to evade LoF or to be potential artifacts were enriched for ClinVar benign variants. PVS1 was downgraded in 99.4% (162/163) of pLoF variants predicted as likely not LoF/not LoF, with 17.2% (28/163) downgraded as a result of our framework, adding to previous guidelines. Variant pathogenicity was affected (mostly from likely pathogenic to VUS) in 20 (71.4%) of these 28 variants. This framework guides assessment of pLoF variants beyond standard annotation pipelines and substantially reduces false positive rates, which is key to ensure accurate LoF variant prediction in both a research and clinical setting.

Natural history of TANGO2 deficiency disorder: Baseline assessment of 73 patients.

PURPOSE: TANGO2 deficiency disorder (TDD), an autosomal recessive disease first reported in 2016, is characterized by neurodevelopmental delay, seizures, intermittent ataxia, hypothyroidism, and life-threatening metabolic and cardiac crises. The purpose of this study was to define the natural history of TDD. METHODS: Data were collected from an ongoing natural history study of patients with TDD enrolled between February 2019 and May 2022. Data were obtained through phone or video based parent interviews and medical record review. RESULTS: Data were collected from 73 patients (59% male) from 57 unrelated families living in 16 different countries. The median age of participants at the time of data collection was 9.0 years (interquartile range = 5.3-15.9 years, range = fetal to 31.8 years). A total of 24 different TANGO2 alleles were observed. Patients showed normal development in early infancy, with progressive delay in developmental milestones thereafter. Symptoms included ataxia, dystonia, and speech difficulties, typically starting between the ages of 1 to 3 years. A total of 46/71 (65%) patients suffered metabolic crises, and of those, 30 (65%) developed cardiac crises. Metabolic crises were significantly decreased after the initiation of B-complex or multivitamin supplementation. CONCLUSION: We provide the most comprehensive review of natural history of TDD and important observational data suggesting that B-complex or multivitamins may prevent metabolic crises.

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities.

COVID-19 Exposure and Family Impact Scales for Adolescents and Young Adults.

OBJECTIVE: To understand the impact of the coronavirus disease 2019 (COVID-19) pandemic on adolescents and young adults (AYAs), we adapted the COVID-19 Exposure and Family Impact Scales (CEFIS; Kazak et al., 2021) for AYAs. Here, we report on the development, structure, and psychometric properties of the CEFIS-AYA. METHODS: The CEFIS-AYA was developed by a multidisciplinary, multi-institutional team using a rapid iterative process. Data from 3,912 AYAs from 21 programs at 16 institutions across the United States were collected from May 2020 to April 2021. We examined the underlying structure of the CEFIS-AYA using principal component analysis (PCA), calculated internal consistencies, and explored differences in scores by gender and age. RESULTS: Participants reported exposure to a range of COVID-19-related events (M = 9.08 events, of 28). On the bidirectional 4-point Impact scale, mean item scores were mostly above the midpoint, indicating a slightly negative impact. Kuder-Richardson 20/Cronbach's Alpha was good for Exposure (α = .76) and excellent for Impact (α = .93). PCA identified seven factors for Exposure (Severe COVID-19, Loss of Income, Limited Access to Essentials, COVID-19 Exposure, Disruptions to Activities, Disruptions to Living Conditions, and Designation as an Essential Worker) and five for Impact (Self and Family Relationships, Physical Well-Being, Emotional Well-Being, Social Well-Being, and Distress). Gender and age differences in CEFIS-AYA scores were identified. DISCUSSION: Initial reliability data are strong and support use of the CEFIS-AYA for measuring the effect of the COVID-19 pandemic on AYAs in research and clinical care.

Digestive Manifestations in Patients Hospitalized With Coronavirus Disease 2019.

BACKGROUND & AIMS: The prevalence and significance of digestive manifestations in coronavirus disease 2019 (COVID-19) remain uncertain. We aimed to assess the prevalence, spectrum, severity, and significance of digestive manifestations in patients hospitalized with COVID-19. METHODS: Consecutive patients hospitalized with COVID-19 were identified across a geographically diverse alliance of medical centers in North America. Data pertaining to baseline characteristics, symptomatology, laboratory assessment, imaging, and endoscopic findings from the time of symptom onset until discharge or death were abstracted manually from electronic health records to characterize the prevalence, spectrum, and severity of digestive manifestations. Regression analyses were performed to evaluate the association between digestive manifestations and severe outcomes related to COVID-19. RESULTS: A total of 1992 patients across 36 centers met eligibility criteria and were included. Overall, 53% of patients experienced at least 1 gastrointestinal symptom at any time during their illness, most commonly diarrhea (34%), nausea (27%), vomiting (16%), and abdominal pain (11%). In 74% of cases, gastrointestinal symptoms were judged to be mild. In total, 35% of patients developed an abnormal alanine aminotransferase or total bilirubin level; these were increased to less than 5 times the upper limit of normal in 77% of cases. After adjusting for potential confounders, the presence of gastrointestinal symptoms at any time (odds ratio, 0.93; 95% CI, 0.76-1.15) or liver test abnormalities on admission (odds ratio, 1.31; 95% CI, 0.80-2.12) were not associated independently with mechanical ventilation or death. CONCLUSIONS: Among patients hospitalized with COVID-19, gastrointestinal symptoms and liver test abnormalities were common, but the majority were mild and their presence was not associated with a more severe clinical course.

Taxonomic similarity does not predict necessary sample size for ex situ conservation: a comparison among five genera.

Effectively conserving biodiversity with limited resources requires scientifically informed and efficient strategies. Guidance is particularly needed on how many living plants are necessary to conserve a threshold level of genetic diversity in ex situ collections. We investigated this question for 11 taxa across five genera. In this first study analysing and optimizing ex situ genetic diversity across multiple genera, we found that the percentage of extant genetic diversity currently conserved varies among taxa from 40% to 95%. Most taxa are well below genetic conservation targets. Resampling datasets showed that ideal collection sizes vary widely even within a genus: one taxon typically required at least 50% more individuals than another (though Quercus was an exception). Still, across taxa, the minimum collection size to achieve genetic conservation goals is within one order of magnitude. Current collections are also suboptimal: they could remain the same size yet capture twice the genetic diversity with an improved sampling design. We term this deficiency the 'genetic conservation gap'. Lastly, we show that minimum collection sizes are influenced by collection priorities regarding the genetic diversity target. In summary, current collections are insufficient (not reaching targets) and suboptimal (not efficiently designed), and we show how improvements can be made.

Applying the zoo model to conservation of threatened exceptional plant species.

Maintaining a living plant collection is the most common method of ex situ conservation for plant species that cannot be seed banked (i.e., exceptional species). Viability of living collections, and their value for future conservation efforts, can be limited without coordinated efforts to track and manage individuals across institutions. Using a pedigree-focused approach, the zoological community has established an inter-institutional infrastructure to support long-term viability of captive animal populations. We assessed the ability of this coordinated metacollection infrastructure to support the conservation of 4 plant species curated in living collections at multiple botanic gardens around the world. Limitations in current practices include the inability to compile, share, and analyze plant collections data at the individual level, as well as difficulty in tracking original provenance of ex situ material. The coordinated metacollection framework used by zoos can be adopted by the botanical community to improve conservation outcomes by minimizing the loss of genetic diversity in collections. We suggest actions to improve ex situ conservation of exceptional plant species, including developing a central database to aggregate data and track unique individuals of priority threatened species among institutions and adapting a pedigree-based population management tool that incorporates life-history aspects unique to plants. If approached collaboratively across regional, national, and global scales, these actions could transform ex situ conservation of threatened plant species.

Aplicación del Modelo Zoológico a la Conservación de Especies Excepcionales de Plantas Amenazadas Resumen El mantenimiento de una colección de plantas vivas es el método más común para de conservación ex situ para especies de plantas que no pueden almacenarse en bancos de semillas (i. e., especies excepcionales). La viabilidad de las colecciones vivientes, junto con el valor que representan para los futuros esfuerzo de conservación, puede estar limitada si no existen esfuerzos coordinados para rastrear y manejar a los individuos entre las instituciones. Mediante una estrategia enfocada en el linaje, la comunidad de zoológicos ha establecido una infraestructura interinstitucional que respalda la viabilidad a largo plazo de las poblaciones de animales en cautiverio. Evaluamos la habilidad de esta infraestructura coordinada de metacolecciones para apoyar en la conservación de cuatro especies de plantas curadas en colecciones vivientes en varios jardines botánicos de todo el mundo. Las limitaciones de las prácticas contemporáneas incluyen la incapacidad de recopilar, compartir y analizar los datos de las colecciones de plantas a nivel individual, así como la dificultad de rastrear la procedencia original del material ex situ. El marco de trabajo de metacolecciones coordinadas que utilizan los zoológicos puede ser adoptado por la comunidad botánica para mejorar los resultados de conservación al minimizar la pérdida de la diversidad genética que ocurre en las colecciones. Sugerimos acciones que aumenten la conservación ex situ de las especies excepcionales de plantas. Estas acciones incluyen el desarrollo de una base de datos central para acumular datos y rastrear entre las instituciones a los individuos únicos de las especies amenazadas prioritarias y la adaptación de una herramienta de manejo poblacional basada en el linaje que incorpore los aspectos únicos de la historia de vida de las plantas. Si estas acciones se plantean colaborativamente a escala regional, nacional y global, podrían transformar la conservación ex situ de las especies amenazadas de plantas.

Circulating nutrients and hematological parameters in managed African elephants (Loxodonta Africana) over a 1-year period.

African elephants (Loxodonta Africana) are currently considered a vulnerable species. One key to improving methods of species management is to better monitor and understand elephant nutrition. Analyzing circulating nutrients is one of the best and least invasive methods of monitoring managed elephant nutrition, but limited reference values are available. This study examined the circulating basic hematology concentrations, minerals, vitamins A, D, and E, and fatty acids of six African elephants (two males and four females) at the North Carolina Zoo collected monthly from March 2016 to April 2017 and compared levels among seasons. Creatinine (CRE) and albumin had seasonal differences (p ≤ .05). Calcium, magnesium, phosphorus, potassium, selenium, zinc, cobalt, manganese, and molybdenum displayed seasonal differences (p ≤ .05). Retinol and 25-hydroxyvitamin D2 had seasonal differences (p ≤ .05). Linoleic acid, α-linolenic acid, arachidonic acid, total omega-3 fatty acids, total omega-6 fatty acids, and the omega-6 to omega-3 ratio showed seasonal differences (p ≤ .05). Findings suggest that exogenous vitamin E supplements may not be necessary with a mixed feedstuff diet (hay, fortified concentrate pellet, browse, and produce) based on circulating values. This data offer updated information on circulating reference values and novel circulating concentrations of nutrients for Southeastern US managed African elephants that can be used to inform nutritional and health management in all similar habitats.

Analyses of African elephant (Loxodonta africana) diet with various browse and pellet inclusion levels.

To more closely simulate the diet of free-ranging elephants, the diet of six (2.4) African elephants (Loxodonta africana) was altered to include more browse and less pelleted complete feed (5% total diet). Dietary proximate compounds, minerals, vitamins A (and carotenoids), D and E, and fatty acids were analyzed on pelleted diet items and forages including hay, grass, and browse. A total of 42 browse species were offered over 1 year with an average total diet inclusion of 5.2% (dry matter basis) per day. Dietary Na and Se were low while Fe and Mn were high compared to published intake levels for elephants. Analyzed nutrients within browse varied widely among seasons and species. Ingredient analyses were used to create predicted elephant nutrient intake for (a) the current diet, (b) a diet excluding pellets, and (c) a diet excluding pellets and providing browse at doubled levels. Formulated diets excluding pellets had lower mineral levels than the current diet and doubled browse did not alter mineral inclusions of concern. This study provides seasonal data on the nutrient levels of Southeastern browse species important for various pachyderm and herbivorous species. Predicted nutrient intake with new diet scenarios does not support the exclusion of pellets in the diets of African elephants without greater browse quantity availability, strict diet management, or additional supplements.

Aversive Behavior in the Nematode C. elegans Is Modulated by cGMP and a Neuronal Gap Junction Network.

All animals rely on their ability to sense and respond to their environment to survive. However, the suitability of a behavioral response is context-dependent, and must reflect both an animal's life history and its present internal state. Based on the integration of these variables, an animal's needs can be prioritized to optimize survival strategies. Nociceptive sensory systems detect harmful stimuli and allow for the initiation of protective behavioral responses. The polymodal ASH sensory neurons are the primary nociceptors in C. elegans. We show here that the guanylyl cyclase ODR-1 functions non-cell-autonomously to downregulate ASH-mediated aversive behaviors and that ectopic cGMP generation in ASH is sufficient to dampen ASH sensitivity. We define a gap junction neural network that regulates nociception and propose that decentralized regulation of ASH signaling can allow for rapid correlation between an animal's internal state and its behavioral output, lending modulatory flexibility to this hard-wired nociceptive neural circuit.

Hypoxia Upregulates Estrogen Receptor ß in Pulmonary Artery Endothelial Cells in a HIF-1α-Dependent Manner.

17ß-Estradiol (E2) attenuates hypoxia-induced pulmonary hypertension (HPH) through estrogen receptor (ER)-dependent effects, including inhibition of hypoxia-induced endothelial cell proliferation; however, the mechanisms responsible for this remain unknown. We hypothesized that the protective effects of E2 in HPH are mediated through hypoxia-inducible factor 1α (HIF-1α)-dependent increases in ERß expression. Sprague-Dawley rats and ERα or ERß knockout mice were exposed to hypobaric hypoxia for 2-3 weeks. The effects of hypoxia were also studied in primary rat or human pulmonary artery endothelial cells (PAECs). Hypoxia increased expression of ERß, but not ERα, in lungs from HPH rats as well as in rat and human PAECs. ERß mRNA time dependently increased in PAECs exposed to hypoxia. Normoxic HIF-1α/HIF-2α stabilization increased PAEC ERß, whereas HIF-1α knockdown decreased ERß abundance in hypoxic PAECs. In turn, ERß knockdown in hypoxic PAECs increased HIF-2α expression, suggesting a hypoxia-sensitive feedback mechanism. ERß knockdown in hypoxic PAECs also decreased expression of the HIF inhibitor prolyl hydroxylase 2 (PHD2), whereas ERß activation increased PHD2 and decreased both HIF-1α and HIF-2α, suggesting that ERß regulates the PHD2/HIF-1α/HIF-2α axis during hypoxia. Whereas hypoxic wild-type or ERα knockout mice treated with E2 demonstrated less pulmonary vascular remodeling and decreased HIF-1α after hypoxia compared with untreated hypoxic mice, ERß knockout mice exhibited increased HIF-2α and an attenuated response to E2 during hypoxia. Taken together, our results demonstrate a novel and potentially therapeutically targetable mechanism whereby hypoxia, via HIF-1α, increases ERß expression and the E2-ERß axis targets PHD2, HIF-1α, and HIF-2α to attenuate HPH development.

The C. elegans cGMP-dependent protein kinase EGL-4 regulates nociceptive behavioral sensitivity.

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.

Structural domains required for Caenorhabditis elegans G protein-coupled receptor kinase 2 (GRK-2) function in vivo.

G protein-coupled receptor kinases (GRKs) are key regulators of signal transduction that specifically phosphorylate activated G protein-coupled receptors (GPCRs) to terminate signaling. Biochemical and crystallographic studies have provided great insight into mammalian GRK2/3 interactions and structure. However, despite extensive in vitro characterization, little is known about the in vivo contribution of these described GRK structural domains and interactions to proper GRK function in signal regulation. We took advantage of the disrupted chemosensory behavior characteristic of Caenorhabditis elegans grk-2 mutants to discern the interactions required for proper in vivo Ce-GRK-2 function. Informed by mammalian crystallographic and biochemical data, we introduced amino acid substitutions into the Ce-grk-2 coding sequence that are predicted to selectively disrupt GPCR phosphorylation, Gα(q/11) binding, Gßγ binding, or phospholipid binding. Changing the most amino-terminal residues, which have been shown in mammalian systems to be required specifically for GPCR phosphorylation but not phosphorylation of alternative substrates or recruitment to activated GPCRs, eliminated the ability of Ce-GRK-2 to restore chemosensory signaling. Disrupting interaction between the predicted Ce-GRK-2 amino-terminal α-helix and kinase domain, posited to stabilize GRKs in their active ATP- and GPCR-bound conformation, also eliminated Ce-GRK-2 chemosensory function. Finally, although changing residues within the RH domain, predicted to disrupt interaction with Gα(q/11), did not affect Ce-GRK-2 chemosensory function, disruption of the predicted PH domain-mediated interactions with Gßγ and phospholipids revealed that both contribute to Ce-GRK-2 function in vivo. Combined, we have demonstrated functional roles for broadly conserved GRK2/3 structural domains in the in vivo regulation of organismal behavior.

Advanced variant classification framework reduces the false positive rate of predicted loss of function (pLoF) variants in population sequencing data.

Predicted loss of function (pLoF) variants are highly deleterious and play an important role in disease biology, but many of these variants may not actually result in loss-of-function. Here we present a framework that advances interpretation of pLoF variants in research and clinical settings by considering three categories of LoF evasion: (1) predicted rescue by secondary sequence properties, (2) uncertain biological relevance, and (3) potential technical artifacts. We also provide recommendations on adjustments to ACMG/AMP guidelines's PVS1 criterion. Applying this framework to all high-confidence pLoF variants in 22 autosomal recessive disease-genes from the Genome Aggregation Database (gnomAD, v2.1.1) revealed predicted LoF evasion or potential artifacts in 27.3% (304/1,113) of variants. The major reasons were location in the last exon, in a homopolymer repeat, in low per-base expression (pext) score regions, or the presence of cryptic splice rescues. Variants predicted to be potential artifacts or to evade LoF were enriched for ClinVar benign variants. PVS1 was downgraded in 99.4% (162/163) of LoF evading variants assessed, with 17.2% (28/163) downgraded as a result of our framework, adding to previous guidelines. Variant pathogenicity was affected (mostly from likely pathogenic to VUS) in 20 (71.4%) of these 28 variants. This framework guides assessment of pLoF variants beyond standard annotation pipelines, and substantially reduces false positive rates, which is key to ensure accurate LoF variant prediction in both a research and clinical setting.

Electron acceptors based on functionalizable cyclopenta[hi]aceanthrylenes and dicyclopenta[de,mn]tetracenes.

We report the synthesis and selective functionalization of two externally fused cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) and demonstrate their electron accepting behavior. 2,7-Bis(trimethylsilyl)cyclopenta[hi]aceanthrylene (1) and 2,8-bis(trimethylsilyl)dicyclopenta[de,mn]tetracene (4) were prepared in a one-pot, palladium-catalyzed cross-coupling of (trimethylsilyl)acetylene and either 9,10-dibromoanthracene or 5,11-dibromotetracene, respectively. The trimethylsilyl groups were selectively converted into bromides via substitution with N-bromosuccinimide to create universal partners (2 and 6) for metal-catalyzed cross-coupling reactions. To demonstrate the utility of the halogenated CP-PAHs, we successfully employed a Sonogashira cross-coupling between the CP-PAHs and a phenylacetylene derivative. The resulting compounds (3 and 7) were found to be highly conjugated between the CP-PAH core and the substituents, as demonstrated by large bathochromic shifts in the absorption spectra as well as density functional theory calculations. Ethynylated CP-PAHs 3 and 7 were found to possess low optical bandgaps (1.52 and 1.51 eV, respectively) and displayed two reversible reductions. We further demonstrated the fullerene-like electron-accepting behavior of 3 through solution-phase fluorescence quenching of the prototypical electron donor, poly(3-hexylthiophene).

Assessment of the effects of storage temperature on fatty acid analysis using dried blood spot cards from managed southern white rhinoceroses (Ceratotherium simum simum): implications for field collection and nutritional care.

BACKGROUND: Southern white rhinoceroses (Ceratotherium simum simum) are an endangered species in decline due to poaching and negative habitat changes. Conservation of the species has become increasingly important and a focus on better human management has become prevalent. One area of management that impacts southern white rhinoceroses is nutritional health monitoring, which is often conducted through blood analysis. Blood analysis conducted during field research can be difficult due to temperature, distance, and limited technological resources, so new methods of fast, and relatively stable blood collection are being pursued. One method that has been used in humans for many years is beginning to make its way into wildlife studies: the use of dried blood spot (DBS) cards. These cards are used as a tool to store single drops of whole blood on specialized filter paper and, once dried, can be used for nutritional biomarker analysis. An area of interest for southern white rhinoceroses and nutrition is monitoring fatty acid percentages for cardiovascular, immune, and reproductive health. The time and temperature limitations for storing blood fractions or liquid whole blood when analyzing fatty acids have been investigated, but few studies have performed storage studies on DBS cards colder than -20 °C or in non-human species. METHODS: In order to better understand the limitations of DBS cards and the impact of temperature on fatty acid DBS samples in long-term storage, triplicate samples from seven adult southern white rhinoceroses at the North Carolina Zoo were collected and subjected to three storage treatments (immediate, room temperature (23 °C), or frozen (-80 °C) for 1 year). RESULTS: Stearidonic (18:4w3) (Δ 0.3%), arachdic (20:0) (Δ 0.1%), eicosatetraenoic (20:4w3) (Δ 0.2%), and erucic acid (22:1w9) (Δ 0.1%) were in higher concentration in frozen than initial. Fatty acids in higher concentrations in the initial samples than frozen were myristic (14:0) (Δ 0.2%), mead (20:3w9) (Δ 0.1%), docosatetraenoic (22:4w6) (Δ 0.2%), nervonic (24:1) (Δ 0.1%), and total highly unsaturated fatty acids (HUFAs) (Δ 0.7%). Stearic (18:0) (Δ 2.2%), stearidonic (18:4w3) (Δ 0.3%), arachdic (20:0) (Δ 0.2%), paullinic (20:1w7) (Δ 0.4%), eicosatetraenoic (20:4w3) (Δ 0.1%), eicosapentaenoic (20:5w3) (Δ 0.1%), docosatetraenoic (22:4w6) (Δ 0.2%), nervonic acid (24:1) (Δ 0.2%), monoenes (Δ 1.9%), and total saturates (Δ 3.6%) had higher concentrations in room temperature than initial. Linoleic (18:2w6) (Δ 4.9%), mead acid (20:3w9) (Δ 0.1%), total polyunsaturated fatty acids (5.3%), and total omega-6 fatty acids (Δ 4.8%) had higher concentrations in initial compared to room temperature. Arachidonic (20:4w6) (Δ 0.4%) and omega-3 docosapentaenoic acid (22:5w3) (Δ 0.1%), had higher concentrations in frozen than in room temperature. DISCUSSION: The frozen samples had the fewest statistical differences compared to room temperature samples and essential omega-3 and -6 fatty acids were stable with freezing up to 1 year. While more research is still warranted, current results suggest that DBS samples are best utilized when immediate analysis or -80 °C storage is available.

Observable Metabolites and Metabolomic Sampling Protocols for Managed African Savanna Elephant (Loxodonta africana) Whole Blood Using H-NMR Spectroscopy.

We used nuclear magnetic spectroscopy (NMR) to evaluate the metabolomics of heparinized whole blood drawn from six African savanna elephants (Loxodonta africana) maintained on a well characterized diet. Whole blood samples obtained under behavioral restraint, then quickly frozen in liquid nitrogen, were stored at -80 °C until analysis. Frozen samples were thawed under controlled conditions and extracted with methanol and chloroform to separate the polar and non-polar metabolites. We identified 18 polar metabolites and 14 non-polar lipids using one-dimensional (1D) and two-dimensional (2D) NMR spectra. Despite unexpected rouleaux formation in the thawed frozen samples, spectra were consistent among animals and did not vary dramatically with age or the sex of the animal.

Recommendations for clinical interpretation of variants found in non-coding regions of the genome.

BACKGROUND: The majority of clinical genetic testing focuses almost exclusively on regions of the genome that directly encode proteins. The important role of variants in non-coding regions in penetrant disease is, however, increasingly being demonstrated, and the use of whole genome sequencing in clinical diagnostic settings is rising across a large range of genetic disorders. Despite this, there is no existing guidance on how current guidelines designed primarily for variants in protein-coding regions should be adapted for variants identified in other genomic contexts. METHODS: We convened a panel of nine clinical and research scientists with wide-ranging expertise in clinical variant interpretation, with specific experience in variants within non-coding regions. This panel discussed and refined an initial draft of the guidelines which were then extensively tested and reviewed by external groups. RESULTS: We discuss considerations specifically for variants in non-coding regions of the genome. We outline how to define candidate regulatory elements, highlight examples of mechanisms through which non-coding region variants can lead to penetrant monogenic disease, and outline how existing guidelines can be adapted for the interpretation of these variants. CONCLUSIONS: These recommendations aim to increase the number and range of non-coding region variants that can be clinically interpreted, which, together with a compatible phenotype, can lead to new diagnoses and catalyse the discovery of novel disease mechanisms.

Implications of Nutritional Management on Fatty Acid Profiles of Southern White Rhinoceroses (Ceratotherium simum simum) Housed at Two Zoological Institutions.

Southern white rhinoceroses (Ceratotherium simum simum) are African megaherbivores that are considered near threatened by the International Union for the Conservation of Nature. The fatty acid circulating values of these animals have not been thoroughly investigated. Fatty acids are critical for immune, heart, skin, and reproductive health, and may have a significant impact on the management and conservation of this species. Published data on fatty acids in this species is limited to incomplete profiles with very few animals in managed environments. The objectives of this research were to provide novel fatty acid percentage profiles for managed healthy southern white rhinoceroses, as well as to provide comparisons between two zoological institutions with differences in diet and climate during two distinct pasture growth periods. Whole blood samples were collected as dried blood spots from six rhinoceroses at the North Carolina Zoo (NC Zoo) and five rhinoceroses at Busch Gardens Tampa (BGT) in the low growth period (February to April) of 2019 and during the high growth period (July to September) of 2020. Fatty acid results indicated numerous differences when comparing the institutions within the same growth period and when comparing the same institution between its two growth periods. Most noteworthy were the higher levels of α-linolenic acid (18:3w3) and total omega-3 fatty acids and the lower linoleic acid (18:2w6), total omega-6 fatty acids, and omega-6 to omega-3 ratio found in the BGT population in both growth periods. This study provides novel percentages of fatty acids in managed southern white rhinoceroses and data on how fatty acid profiles may be altered between two housing locations via dietary differences in hay type and quantity, pasture availability via season, and pellet inclusion levels.

Comparison of African savanna elephant (Loxodonta africana) fatty acid profiles in whole blood, whole blood dried on blood spot cards, serum, and plasma.

BACKGROUND: African elephants in managed care have presented differences in the balance between omega-3 and omega-6 fatty acids, a situation primarily thought to be due to dietary differences between the managed animals and their free-ranging counterparts. Because of this, circulating fatty acid status is included in routine monitoring of elephant health. A method of blood collection that requires only a few drops of whole blood, dried on filter paper (DBS) and can be used for analyzing full fatty acid profiles offers advantages in clinical application. METHODS: This study compared the use of whole blood, and whole blood DBS, serum or plasma for use in evaluating circulating fatty acid composition in African savannah elephants. Samples from six African elephants (two males and four females) were collected during the same week at the NC Zoo, Asheboro, NC. RESULTS: Results found only 2 of 36 individual fatty acids and none of the 10 fatty acid groupings were different when comparing the four blood fraction sample types to each other with Mann-Whitney U-Test pairwise comparisons. Myristic acid (14:0) was lower in the DBS samples than in whole blood, serum, and plasma and pentadecaenoic acid (15:1) was slightly more concentrated in DBS and whole blood. DISCUSSION: Results indicate that fatty acid profile of serum, plasma, whole blood, and DBS are comparable in African elephants. The DBS method offers advantages in acquisition and handling and may be preferable to other methods in both routine health assessment of captive animals and field research on free ranging animals.