The hierarchical radiation of phyllostomid bats as revealed by adaptive molar morphology.

Adaptive radiations are bursts in biodiversity that generate new evolutionary lineages and phenotypes. However, because they typically occur over millions of years, it is unclear how their macroevolutionary dynamics vary through time and among groups of organisms. Phyllostomid bats radiated extensively for diverse diets-from insects to vertebrates, fruit, nectar, and blood-and we use their molars as a model system to examine the dynamics of adaptive radiations. Three-dimensional shape analyses of lower molars of Noctilionoidea (Phyllostomidae and close relatives) indicate that different diet groups exhibit distinct morphotypes. Comparative analyses further reveal that phyllostomids are a striking example of a hierarchical radiation; phyllostomids' initial, higher-level diversification involved an "early burst" in molar morphological disparity as lineages invaded new diet-affiliated adaptive zones, followed by subsequent lower-level diversifications within adaptive zones involving less dramatic morphological changes. We posit that strong selective pressures related to initial shifts to derived diets may have freed molars from morpho-functional constraints associated with the ancestral molar morphotype. Then, lineages with derived diets (frugivores and nectarivores) diversified within broad adaptive zones, likely reflecting finer-scale niche partitioning. Importantly, the observed early burst pattern is only evident when examining molar traits that are strongly linked to diet, highlighting the value of ecomorphological traits in comparative studies. Our results support the hypothesis that adaptive radiations are commonly hierarchical and involve different tempos and modes at different phylogenetic levels, with early bursts being more common at higher levels.

Remdesivir-ivermectin combination displays synergistic interaction with improved in vitro activity against SARS-CoV-2.

A key element for the prevention and management of coronavirus disease 2019 is the development of effective therapeutics. Drug combination strategies offer several advantages over monotherapies. They have the potential to achieve greater efficacy, to increase the therapeutic index of drugs and to reduce the emergence of drug resistance. We assessed the in vitro synergistic interaction between remdesivir and ivermectin, both approved by the US Food and Drug Administration, and demonstrated enhanced antiviral activity against severe acute respiratory syndrome coronavirus-2. Whilst the in vitro synergistic activity reported here does not support the clinical application of this combination treatment strategy due to insufficient exposure of ivermectin in vivo, the data do warrant further investigation. Efforts to define the mechanisms underpinning the observed synergistic action could lead to the development of novel treatment strategies.

SARS-CoV-2 infects an upper airway model derived from induced pluripotent stem cells.

As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal-like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal-like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air-liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS-CoV-2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs.

Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles.

Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3D56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3D56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance.

Evaluation of Dogs with Border Collie Collapse, Including Response to Two Standardized Strenuous Exercise Protocols.

Clinical and metabolic variables were evaluated in 13 dogs with border collie collapse (BCC) before, during, and following completion of standardized strenuous exercise protocols. Six dogs participated in a ball-retrieving protocol, and seven dogs participated in a sheep-herding protocol. Findings were compared with 16 normal border collies participating in the same exercise protocols (11 retrieving, five herding). Twelve dogs with BCC developed abnormal mentation and/or an abnormal gait during evaluation. All dogs had post-exercise elevations in rectal temperature, pulse rate, arterial blood pH, PaO2, and lactate, and decreased PaCO2 and bicarbonate, as expected with strenuous exercise, but there were no significant differences between BCC dogs and normal dogs. Electrocardiography demonstrated sinus tachycardia in all dogs following exercise. Needle electromyography was normal, and evaluation of muscle biopsy cryosections using a standard panel of histochemical stains and reactions did not reveal a reason for collapse in 10 dogs with BCC in which these tests were performed. Genetic testing excluded the dynamin-1 related exercise-induced collapse mutation and the V547A malignant hyperthermia mutation as the cause of BCC. Common reasons for exercise intolerance were eliminated. Although a genetic basis is suspected, the cause of collapse in BCC was not determined.

Evaluation of an enzyme immunoassay for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen as an aid in the diagnosis of blastomycosis in dogs.

OBJECTIVE: To evaluate the sensitivity and specificity of an enzyme immunoassay (EIA) for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen (rBAD-1) to aid in the diagnosis of blastomycosis in dogs and compare the findings with results from other tests used for this purpose. DESIGN: Prospective analytic study. SAMPLE: Serum and urine from 70 dogs with and without blastomycosis. PROCEDURES: Serum and urine samples were collected from dogs with blastomycosis (n = 21), histoplasmosis (8), or nonfungal pulmonary disease (21) and from healthy control dogs living in a blastomycosis-endemic area (20). Serum was tested for antibodies against Blastomyces dermatitidis with the rBAD-1 antibody EIA and an A-antigen antibody agar gel immunodiffusion (AGID) assay. Serum and urine were tested for B dermatitidis antigen with a quantitative EIA. RESULTS: Sensitivity of the quantitative antigen EIA was 100% in serum and urine samples from dogs with blastomycosis, with specificity of 95% in urine samples from dogs with nonfungal pulmonary disease and 100% in urine samples from healthy dogs. Sensitivity of the rBAD-1 antibody EIA (95%) was significantly greater than that of the A-antigen antibody AGID assay (65%). Specificity of the antibody EIA was 88% in dogs with histoplasmosis, 95% in healthy dogs, and 100% in dogs with nonfungal pulmonary disease. CONCLUSIONS AND CLINICAL RELEVANCE: The rBAD-1 antibody EIA had greater sensitivity than the A-antigen antibody AGID assay in dogs with blastomycosis. This antibody EIA may assist in distinguishing histoplasmosis from blastomycosis. Further evaluation in a larger prospective study is needed to verify these results.

Candidate genes and functional noncoding variants identified in a canine model of obsessive-compulsive disorder.

BACKGROUND: Obsessive-compulsive disorder (OCD), a severe mental disease manifested in time-consuming repetition of behaviors, affects 1 to 3% of the human population. While highly heritable, complex genetics has hampered attempts to elucidate OCD etiology. Dogs suffer from naturally occurring compulsive disorders that closely model human OCD, manifested as an excessive repetition of normal canine behaviors that only partially responds to drug therapy. The limited diversity within dog breeds makes identifying underlying genetic factors easier. RESULTS: We use genome-wide association of 87 Doberman Pinscher cases and 63 controls to identify genomic loci associated with OCD and sequence these regions in 8 affected dogs from high-risk breeds and 8 breed-matched controls. We find 119 variants in evolutionarily conserved sites that are specific to dogs with OCD. These case-only variants are significantly more common in high OCD risk breeds compared to breeds with no known psychiatric problems. Four genes, all with synaptic function, have the most case-only variation: neuronal cadherin (CDH2), catenin alpha2 (CTNNA2), ataxin-1 (ATXN1), and plasma glutamate carboxypeptidase (PGCP). In the 2 Mb gene desert between the cadherin genes CDH2 and DSC3, we find two different variants found only in dogs with OCD that disrupt the same highly conserved regulatory element. These variants cause significant changes in gene expression in a human neuroblastoma cell line, likely due to disrupted transcription factor binding. CONCLUSIONS: The limited genetic diversity of dog breeds facilitates identification of genes, functional variants and regulatory pathways underlying complex psychiatric disorders that are mechanistically similar in dogs and humans.

Evaluations of labrador retrievers with exercise-induced collapse, including response to a standardized strenuous exercise protocol.

Clinical and metabolic variables were evaluated in 14 Labrador retrievers with exercise-induced collapse (EIC) before, during, and following completion of a standardized strenuous exercise protocol. Findings were compared with previously reported variables from 14 normal Labrador retrievers that participated in the same protocol. Ten of 14 dogs with EIC developed an abnormal gait during evaluation, and these dogs were significantly more tachycardic and had a more severe respiratory alkalosis after exercise compared to the normal dogs. Muscle biopsy characteristics and sequential lactate and pyruvate concentrations were normal. Genetic testing and linkage analysis excluded malignant hyperthermia as the cause of EIC. Common causes of exercise intolerance were eliminated, but the cause of collapse in EIC was not determined.

A neonatal encephalopathy with seizures in standard poodle dogs with a missense mutation in the canine ortholog of ATF2.

Neonatal encephalopathy with seizures (NEWS) is a previously undescribed autosomal recessive disease of standard poodle puppies. Affected puppies are small and weak at birth. Many die in their first week of life. Those surviving past 1 week develop ataxia, a whole-body tremor, and, by 4 to 6 weeks of age, severe generalized clonic-tonic seizures. None have survived to 7 weeks of age. Cerebella from affected puppies were reduced in size and often contained dysplastic foci consisting of clusters of intermixed granule and Purkinje neurons. We used deoxyribonucleic acid samples from related standard poodles to map the NEWS locus to a 2.87-Mb segment of CFA36, which contains the canine ortholog of ATF2. This gene encodes activating transcription factor 2 (ATF-2), which participates in the cellular responses to a wide variety of stimuli. We amplified and sequenced all coding regions of canine ATF2 from a NEWS-affected puppy and identified a T > G transversion that predicts a methionine-to-arginine missense mutation at amino acid position 51. Methionine-51 lies within a hydrophobic docking site for mitogen-activated protein kinases that activate ATF-2 so the arginine substitution is likely to interfere with ATF-2 activation. All 20 NEWS-affected puppies in the standard poodle family were homozygous for the mutant G allele. The 58 clinically normal family members were either G/T heterozygotes or homozygous for the ancestral T allele. There are no previous reports of spontaneous ATF2 mutations in people or animals; however, atf2-knockout mice have cerebellar lesions that are similar to those in puppies with NEWS.

Inherited polyneuropathy in Leonberger dogs: a mixed or intermediate form of Charcot-Marie-Tooth disease?

A spontaneous distal, symmetrical polyneuropathy in related Leonberger dogs with onset between 1 to 9 years of age was characterized clinically, electrophysiologically, histologically, and morphometrically. Exercise intolerance and weakness was associated with a high-steppage pelvic-limb gait, a loss or change in the pitch of the bark, and dyspnea. Neurological examination revealed marked atrophy of the distal limb muscles, depressed spinal and cranial nerve reflexes, and weak or absent movement of the laryngeal and pharyngeal muscles. Electrophysiological evaluation was consistent with denervation and was characterized by loss or marked attenuation of compound muscle action potentials and slowed motor nerve conduction velocity. Muscle biopsy specimens showed neurogenic atrophy. Chronic nerve fiber loss associated with decreased myelinated fiber density and a shift of the axonal size-frequency distribution toward smaller fibers was the predominant finding in peripheral nerve specimens. Pedigree analysis of a large multigenerational family, including nine sibships with at least one affected individual, suggested X-linked inheritance. Mutational and linkage analysis of this family may aid in identification of the chromosomal loci and gene responsible for this inherited axonal neuropathy. Further characterization of this inherited axonal neuropathy may establish the Leonberger dog as a spontaneous animal model of inherited axonal neuropathy and possibly lead to the discovery of a new gene or genes associated with axonal variants.