Sequencing of multiple related species followed by comparative genomics analysis constitutes a powerful approach for the systematic understanding of any genome. Here, we use the genomes of 12 Drosophila species for the de novo discovery of functional elements in the fly. Each type of functional element shows characteristic patterns of change, or 'evolutionary signatures', dictated by its precise selective constraints. Such signatures enable recognition of new protein-coding genes and exons, spurious and incorrect gene annotations, and numerous unusual gene structures, including abundant stop-codon readthrough. Similarly, we predict non-protein-coding RNA genes and structures, and new microRNA (miRNA) genes. We provide evidence of miRNA processing and functionality from both hairpin arms and both DNA strands. We identify several classes of pre-and post-transcriptional regulatory motifs, and predict individual motif instances with high confidence. We also study how discovery power scales with the divergence and number of species compared, and we provide general guidelines for comparative studies.The sequencing of the human genome and the genomes of dozens of other metazoan species has intensified the need for systematic methods to extract biological information directly from DNA sequence. Comparative genomics has emerged as a powerful methodology for this endeavour 1,2 . Comparison of few (two-four) closely related genomes has proven successful for the discovery of protein-coding genes 3-5 , RNA genes 6,7 , miRNA genes 8-11 and catalogues of regulatory elements 3,4,12-14 . The resolution and discovery power of these studies should increase with the number of genomes [15][16][17][18][19][20] , in principle enabling the systematic discovery of all conserved functional elements.The fruitfly Drosophila melanogaster is an ideal system for developing and evaluating comparative genomics methodologies. Over the past century, Drosophila has been a pioneering model in which many of the basic principles governing animal development and population biology were established 21 . In the past decade, the genome sequence of D. melanogaster provided one of the first systematic views *These authors contributed equally to this work. {Lists of participants and affiliations appear at the end of the paper.

Background: The use of serological markers to diagnose inflammatory bowel disease (IBD) in humans is well-established. Because of the frequency of IBD in dogs and resources required for its diagnosis with current methods, new approaches are desired.Objective: The goal is to evaluate novel serologic markers to differentiate clinical cohorts in dogs with gastrointestinal (GI) disease and assess their potential to develop a serum-based IBD diagnostic test.Animals: Seventy dogs diagnosed with biopsy-confirmed IBD, 23 dogs with non-IBD predominantly acute GI diseases, and 58 normal dogs.Methods: Prospective control study. ELISA methods were developed to detect autoantibodies to polymorphonuclear leukocytes (APMNA) and calprotectin (ACNA), antibodies against gliadins (AGA), microbial outer membrane porin C (ACA), and flagellins (AFA) isolated from diseased dogs based on clinical and histopathological scoring.Results: IBD dogs displayed a 39%-76% prevalence of seropositivity against selected serologic markers that markedly decreased to 0%-13% in non-IBD and normal dogs.ROC analysis showed statistical significance in differentiating the cohorts, with seropositivity against OmpC being the highest single performance marker. The combination of markers such as OmpC and APMNA reached specificities of 93%-99% and 79%-98% and sensitivities of 76%-97% and 66%-86% when comparing IBD versus normal cohorts and non-IBD cohorts, respectively. Conclusion and Clinical Importance: Seropositivity of canine immunoglobulinsA against selected serologic markers in dogs appears promising in the detection and differentiation of IBD versus other acute GI conditions. Among them, antibody

During Drosophila development Suppressor of Hairless ½Su(H)-dependent Notch activation upregulates transcription of the Enhancer of split-Complex ½E(spl)-C genes. Drosophila melanogaster E(spl) genes share common transcription regulators including binding sites for Su(H), proneural, and E(spl) basic-helix-loophelix (bHLH) proteins. However, the expression patterns of E(spl) genes during development suggest that additional factors are involved. To better understand regulators responsible for these expression patterns, recently available sequence and annotation data for multiple Drosophila genomes were used to compare the E(spl) upstream regulatory regions from more than nine Drosophila species. The mg and mb regulatory regions are the most conserved of the bHLH genes. Fine analysis of Su(H) sites showed that high-affinity Su(H) paired sites and the Su(H) paired site plus proneural site (SPS 1 A) architecture are completely conserved in a subset of Drosophila E(spl) genes. The SPS 1 A module is also present in the upstream regulatory regions of the more ancient mosquito and honeybee E(spl) bHLH genes. Additional transcription factor binding sites were identified upstream of the E(spl) genes and compared between species of Drosophila. Conserved sites provide new understandings about E(spl) regulation during development. Conserved novel sequences found upstream of multiple E(spl) genes may play a role in the expression of these genes.

CASE DESCRIPTION A 6-year-old castrated male Australian Shepherd was evaluated because of a recent onset of persistent hypoglycemia. CLINICAL FINDINGS Physical examination results were generally unremarkable. No abnormalities were detected on thoracic radiographs, and abdominal ultrasonography revealed no obvious pancreatic lesion. Hematologic analysis revealed hypoglycemia with a high serum insulin-to-glucose concentration ratio. TREATMENT AND OUTCOME Insulinoma was suspected; medical treatment with prednisone was initiated, and exploratory laparotomy was performed. No pancreatic lesions or masses were observed. Partial left pancreatectomy and hepatic and local lymph node biopsies were performed. Histologic examination revealed islet cell hypertrophy and hyperplasia, with no evidence of neoplasia. Results of a PCR assay of the pancreatic tissue for Bartonella infection were negative. Clinical, biochemical, and histopathologic findings were compatible with nesidioblastosis. The clinical signs, including hypoglycemia, resolved after surgery. On follow-up examination 8 months later, the dog was apparently healthy and results of a CBC and serum biochemical analysis, including blood glucose concentration, were within respective reference ranges. CLINICAL RELEVANCE To our knowledge, this is the first report of nesidioblastosis in a dog for which clinical signs and clinicopathologic abnormalities resolved after partial pancreatectomy. Although extremely rare, nesidioblastosis should be considered a differential diagnosis in dogs with signs suggestive of insulinoma.

OBJECTIVE To describe dogs with detected Ancylostoma caninum anthelmintic treatment resistance markers in Canada. ANIMALS 11 client-owned dogs with fecal quantitative PCR (qPCR) assay detected A caninum with benzimidazole (BZ) resistance genotypic markers. METHODS Signalment, presenting concern, duration of clinical signs, fecal testing, treatment, and outcomes were obtained. Where available, follow-up data were collected via telephone or email with the primary veterinarian. RESULTS Ancylostoma spp was detected from 184/32,205 dog fecal samples by reference laboratory qPCR surveillance, between May 15, 2022, and April 26, 2023. 11 of these 184 samples had A caninum with genetic BZ F167Y resistance marker detection. 4 dogs had not traveled outside Canada, 6 had been imported from the US, and the travel history was unclear in 1 dog. 7 of the dogs had gastro-intestinal signs (diarrhea or soft stool) on initial presentation. Clinical improvement was reported in 6 of these dogs (resolution of diarrhea and soft stool), with 1 dog lost to follow-up. All 11 dogs received anthelmintic treatment (varied drugs and duration). CLINICAL RELEVANCE Identification of genetic markers of BZ resistance raises concerns about the potential animal and human impacts of resistant hookworms. 4 dogs lacked an origin from or travel history to the US, indicating true emergence and/or novel spread within Canada, not just importation from an area where resistance has been reported. Fecal surveillance was performed with a qPCR test incorporating treatment (BZ) resistance markers. There is a need to raise clinician awareness around treatment-resistant hookworm in dogs and the capability of fecal surveillance for genotypic and phenotypic resistance.