Ten years of the horse reference genome: insights into equine biology, domestication and population dynamics in the post-genome era.

The horse reference genome from the Thoroughbred mare Twilight has been available for a decade and, together with advances in genomics technologies, has led to unparalleled developments in equine genomics. At the core of this progress is the continuing improvement of the quality, contiguity and completeness of the reference genome, and its functional annotation. Recent achievements include the release of the next version of the reference genome (EquCab3.0) and generation of a reference sequence for the Y chromosome. Horse satellite-free centromeres provide unique models for mammalian centromere research. Despite extremely low genetic diversity of the Y chromosome, it has been possible to trace patrilines of breeds and pedigrees and show that Y variation was lost in the past approximately 2300 years owing to selective breeding. The high-quality reference genome has led to the development of three different SNP arrays and WGSs of almost 2000 modern individual horses. The collection of WGS of hundreds of ancient horses is unique and not available for any other domestic species. These tools and resources have led to global population studies dissecting the natural history of the species and genetic makeup and ancestry of modern breeds. Most importantly, the available tools and resources, together with the discovery of functional elements, are dissecting molecular causes of a growing number of Mendelian and complex traits. The improved understanding of molecular underpinnings of various traits continues to benefit the health and performance of the horse whereas also serving as a model for complex disease across species.

Genetic diversity and relationships among native Japanese horse breeds, the Japanese Thoroughbred and horses outside of Japan using genome-wide SNP data.

Eight horse breeds-Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara, Miyako and Yonaguni-are native to Japan. Although Japanese native breeds are believed to have originated from ancient Mongolian horses imported from the Korean Peninsula, the phylogenetic relationships among these breeds are not well elucidated. In the present study, we compared genetic diversity among 32 international horse breeds previously evaluated by the Equine Genetic Diversity Consortium, the eight Japanese native breeds and Japanese Thoroughbreds using genome-wide SNP genotype data. The proportion of polymorphic loci and expected heterozygosity showed that the native Japanese breeds, with the exception of the Hokkaido, have relatively low diversity compared to the other breeds sampled. Phylogenetic and cluster analyses demonstrated relationships among the breeds that largely reflect their geographic distribution in Japan. Based on these data, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The Japanese Thoroughbreds were distinct from the native breeds, and although they maintain similar overall diversity as Thoroughbreds from outside Japan, they also show evidence of uniqueness relative to the other Thoroughbred samples. This is the first study to place the eight native Japanese breeds and Japanese Thoroughbred in context with an international sample of diverse breeds.

Generation of an equine biobank to be used for Functional Annotation of Animal Genomes project.

The Functional Annotation of Animal Genomes (FAANG) project aims to identify genomic regulatory elements in both sexes across multiple stages of development in domesticated animals. This study represents the first stage of the FAANG project for the horse, Equus caballus. A biobank of 80 tissue samples, two cell lines and six body fluids was created from two adult Thoroughbred mares. Ante-mortem assessments included full physical examinations, lameness, ophthalmologic and neurologic evaluations. Complete blood counts and serum biochemistries were also performed. At necropsy, in addition to tissue samples, aliquots of serum, ethylenediaminetetraacetic acid (EDTA) plasma, heparinized plasma, cerebrospinal fluid, synovial fluid, urine and microbiome samples from all regions of the gastrointestinal and urogenital tracts were collected. Epidermal keratinocytes and dermal fibroblasts were cultured from skin samples. All tissues were grossly and histologically evaluated by a board-certified veterinary pathologist. The results of the clinical and pathological evaluations identified subclinical eosinophilic and lymphocytic infiltration throughout the length of the gastrointestinal tract as well as a mild clinical lameness in both animals. Each sample was cryo-preserved in multiple ways, and nuclei were extracted from selected tissues. These samples represent the first published systemically healthy equine-specific biobank with extensive clinical phenotyping ante- and post-mortem. The tissues in the biobank are intended for community-wide use in the functional annotation of the equine genome. The use of the biobank will improve the quality of the reference annotation and allow all equine researchers to elucidate unknown genomic and epigenomic causes of disease.

Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes.

BACKGROUND: The ability to perform comprehensive profiling of cancers at high resolution is essential for precision medicine. Liquid biopsies using shed exosomes provide high-quality nucleic acids to obtain molecular characterization, which may be especially useful for visceral cancers that are not amenable to routine biopsies. PATIENTS AND METHODS: We isolated shed exosomes in biofluids from three patients with pancreaticobiliary cancers (two pancreatic, one ampullary). We performed comprehensive profiling of exoDNA and exoRNA by whole genome, exome and transcriptome sequencing using the Illumina HiSeq 2500 sequencer. We assessed the feasibility of calling copy number events, detecting mutational signatures and identifying potentially actionable mutations in exoDNA sequencing data, as well as expressed point mutations and gene fusions in exoRNA sequencing data. RESULTS: Whole-exome sequencing resulted in 95%-99% of the target regions covered at a mean depth of 133-490×. Genome-wide copy number profiles, and high estimates of tumor fractions (ranging from 56% to 82%), suggest robust representation of the tumor DNA within the shed exosomal compartment. Multiple actionable mutations, including alterations in NOTCH1 and BRCA2, were found in patient exoDNA samples. Further, RNA sequencing of shed exosomes identified the presence of expressed fusion genes, representing an avenue for elucidation of tumor neoantigens. CONCLUSIONS: We have demonstrated high-resolution profiling of the genomic and transcriptomic landscapes of visceral cancers. A wide range of cancer-derived biomarkers could be detected within the nucleic acid cargo of shed exosomes, including copy number profiles, point mutations, insertions, deletions, gene fusions and mutational signatures. Liquid biopsies using shed exosomes has the potential to be used as a clinical tool for cancer diagnosis, therapeutic stratification and treatment monitoring, precluding the need for direct tumor sampling.

Long-term selection for litter size in swine results in shifts in allelic frequency in regions involved in reproductive processes.

High-density genotype data were analyzed in three lines of swine that express substantial variation in sow fertility to uncover regions of the genome potentially influenced during selection for litter size traits. The experimental lines examined include the Nebraska Index Line (NIL), which has been subjected to long-term selection for litter size; a control line derived from the same population that founded NIL; and a commercial Duroc × Hampshire (D × H) population, in which no selection for litter size was practiced. Regions of the genome potentially affected by selection for litter size traits in NIL were determined by multiple lines of evidence, including altered allelic frequency compared to the other lines, loss of heterozygosity and relative extended haplotype homozygosity. Additionally, a genome-wide association study for litter size traits was conducted in a population based on NIL and commercial maternal line genetics. Several genomic regions identified as putative signatures of selection overlapped with QTL for litter size traits. One of these regions, located on SSC2 (13-14 Mb), includes the candidate gene P2X3R, which plays a role in implantation and sustained release of hormones associated with reproductive processes. Sequencing identified synonymous SNPs in P2X3R that are fixed in NIL but polymorphic with nearly equal frequencies in the D × H line, indicating a potential role of P2X3R in sow fertility. These results suggest that data derived from these lines can help to uncover and understand a portion of the genetic variance associated with fertility traits in swine.

An application of MeSH enrichment analysis in livestock.

An integral part of functional genomics studies is to assess the enrichment of specific biological terms in lists of genes found to be playing an important role in biological phenomena. Contrasting the observed frequency of annotated terms with those of the background is at the core of overrepresentation analysis (ORA). Gene Ontology (GO) is a means to consistently classify and annotate gene products and has become a mainstay in ORA. Alternatively, Medical Subject Headings (MeSH) offers a comprehensive life science vocabulary including additional categories that are not covered by GO. Although MeSH is applied predominantly in human and model organism research, its full potential in livestock genetics is yet to be explored. In this study, MeSH ORA was evaluated to discern biological properties of identified genes and contrast them with the results obtained from GO enrichment analysis. Three published datasets were employed for this purpose, representing a gene expression study in dairy cattle, the use of SNPs for genome-wide prediction in swine and the identification of genomic regions targeted by selection in horses. We found that several overrepresented MeSH annotations linked to these gene sets share similar concepts with those of GO terms. Moreover, MeSH yielded unique annotations, which are not directly provided by GO terms, suggesting that MeSH has the potential to refine and enrich the representation of biological knowledge. We demonstrated that MeSH can be regarded as another choice of annotation to draw biological inferences from genes identified via experimental analyses. When used in combination with GO terms, our results indicate that MeSH can enhance our functional interpretations for specific biological conditions or the genetic basis of complex traits in livestock species.

A major effect quantitative trait locus for whirling disease resistance identified in rainbow trout (Oncorhynchus mykiss).

Whirling disease, caused by the pathogen Myxobolus cerebralis, leads to skeletal deformation, neurological impairment and under certain conditions, mortality of juvenile salmonid fishes. The disease has impacted the propagation and survival of many salmonid species over six continents, with particularly negative consequences for rainbow trout. To assess the genetic basis of whirling disease resistance in rainbow trout, genome-wide mapping was initiated using a large outbred F(2) rainbow trout family (n=480) and results were confirmed in three additional outbred F(2) families (n=96 per family). A single quantitative trait locus (QTL) region on chromosome Omy9 was identified in the large mapping family and confirmed in all additional families. This region explains 50-86% of the phenotypic variance across families. Therefore, these data establish that a single QTL region is capable of explaining a large percentage of the phenotypic variance contributing to whirling disease resistance. This is the first genetic region discovered that contributes directly to the whirling disease phenotype and the finding moves the field closer to a mechanistic understanding of resistance to this important disease of salmonid fish.

Reduction of serum-induced endothelial STAT3(Y705) activation is associated with preeclampsia.

OBJECTIVES: Preeclampsia is associated with maternal morbidity and mortality during pregnancy, and also an increased cardiovascular disease (CVD) risk later in life. During preeclampsia, alterations in secreted placental factors leading to systemic maternal endothelial dysfunction are evident. However, little is known about the associated endothelial intracellular signaling. STAT3 is a latent cytoplasmic transcription factor involved in endothelial cell differentiation, survival, and angiogenesis. We aimed to test if preeclampsia and preeclampsia-related placental factors could alter serum-induced STAT3(Y705) activation in endothelial cells. Furthermore, if altered serum-induced endothelial STAT3 (Y705) activation is related to post-preeclamptic CVD risk. STUDY DESIGN: HUVECs were used as a model of maternal endothelium. Experiments entailed addition of 20% human pregnancy serum as well as addition of recombinant PlGF, sFLT1 and VEGF-A165a to the cells. MAIN OUTCOME MEASURES: Levels of pSTAT3(Y705) related to STAT3 levels were evaluated by immunoblotting analysis. RESULTS: Our results show that preeclamptic serum induces significantly lower STAT3(Y705) phosphorylation compared with uncomplicated pregnancy serum (P = 0.0089) in endothelial cells. Furthermore, STAT3(Y705) phosphorylation was not changed upon addition of PlGF, sFLT1, or VEGF-A165a together with pregnancy sera compared with sera alone. Finally, sera from women with previous preeclampsia and current hypertension and carotid atherosclerotic plaques show significantly lower STAT3(Y705) phosphorylation capabilities compared with healthy women with previous uncomplicated pregnancies 8-18 years after deliveries (P = 0.029). CONCLUSIONS: Reduction in serum-induced endothelial STAT3(Y705) activation may play an important role in the preeclampsia-associated endothelial dysfunction. Additionally, reduced endothelial STAT3(Y705) phosphorylation may contribute to increased post-preeclamptic CVD risk 8-18 years after delivery.

Efficacy studies of Aquaprene (1.8% and 2.8% AI) sand granules and Altosid XR-G (1.5% AI) sand granules against first and second instars of Ochlerotatus taeniorhynchus as a preflood treatment in small field plots.

An efficacy study was conducted to evaluate sand granule formulations of Aquaprene (1.8% and 2.8% active ingredient [AI]) and Altosid XR-G (1.5% AI) as a preflood application against Ochlerotatus taeniorhynchus larvae in small field test plots. Aquaprene sand granules (2.8% Al) were applied at 2.5 and 5 lb/acre and the 1.8% AI formulation at 4.2 lb/acre. The 1.8% AI formulation was compared with Altosid XR-G sand granules (1.5% AI) applied at 5 lb/acre. Plots were flooded after 7 days, and 1st and 2nd instars were introduced, pupae were collected, and the plots were drained and dried for 9 days. Assessments were made 21 and 35 days posttreatment. Both Aquaprene sand granules formulations exhibited excellent control throughout the 35-day study.

A gene required for the novel activation of a class II DNA photolyase in Chlamydomonas.

DNA photolyases catalyze the blue light-dependent repair of UV light-induced damage in DNA. DNA photolyases are specific for either cyclobutane-type pyrimidine dimers or (6-4) photoproducts. PHR2 is a gene that in Chlamydomonas reinhardtii encodes a class II DNA photolyase which catalyzes the photorepair of cyclobutane-type pyrimidine dimers. Based on amino acid sequence analysis of PHR2, which indicates the presence of a chloroplast targeting sequence, PHR2 was predicted to encode the chloroplast photolyase of Chlamydomonas. Using a sensitive gene-specific in vivo repair assay, we found that overexpression of PHR2 in Chlamydomonas results in targeting of the protein to not only the chloroplast, but also to the nucleus. Overexpression of PHR2 photolyase in a photoreactivation-deficient mutant, phr1, results in a largely inactive product. The phr1 mutant was found to be deficient in both photorepair of a chloroplast gene, rbcL, and a nuclear gene, rDNA. These results suggest that PHR2 is the structural gene for the photolyase targeted to both the chloroplast and the nucleus, and that the PHR1 gene product is necessary for full activity of PHR2 protein. To our knowledge, the requirement for a second gene for full activity of a DNA photolyase is novel.

Efficacy studies of Aquaprene (emulsifiable concentrate and wettable powder) insect growth regulator formulations against third- and fourth-stage larvae of Ochlerotatus taeniorhynchus in small-plot field studies.

Efficacy studies were conducted with Aquaprene emulsifiable concentrate (EC) (33.6% active ingredient [AI]) and wettable powder (WP) (40% AI) (S)-methoprene insect growth regulator formulations against larval Ochlerotatus taeniorhynchus in small field test plots. Aquaprene EC was applied at 7.18 and 9.57 g/acre. Approximately one thousand 4th-stage larvae were added to each plot before treatment, and 24 h later pupae were collected to determine emergence inhibition. At both applications rates, Aquaprene EC was extremely effective (99%) at significantly reducing adult emergence in the studies. Two application rates ((S)-methoprene at 2.4 and 4.8 g/acre) of the Aquaprene WP also were evaluated against Oc. taeniorhynchus at 1, 3, 7, and 14 days after treatment. Emergence inhibition and statistical analysis showed that significant differences were found between the (S)-methoprene rates of 2.4 and 4.8 g/acre at each posttreatment assessment. The (S)-methoprene rate of 4.8 g/acre was effective in controlling adult emergence for up to 14 days after treatment.

New antiserum against Ki-67 antigen suitable for double immunostaining of paraffin wax sections.

AIMS: To prepare a rabbit antiserum equivalent to MIB 1 to permit the simultaneous assessment of cell proliferation and other markers of interest using double labelling studies. METHODS: Rabbits were immunised with a synthetic peptide deduced from the cDNA sequence coding for the Ki-67 antigen. Serum samples were tested for immunoreactivity using different immunobiochemical methods. RESULTS: A polyclonal antiserum was derived which detects the native as well as recombinant parts of the Ki-67 antigen in different test systems. Furthermore, the antiserum stains the Ki-67 antigen in routinely processed, paraffin wax embedded material. CONCLUSIONS: After antigen unmasking by microwave treatment the antiserum described here represents a powerful tool for the determination of growth fractions even in archival material. It is especially suitable for double staining experiments in combination with monoclonal antibodies.

Short report epidemiologic studies on cutaneous leishmaniasis in eastern Panama.

The Panamanian Ministry of Health, through the Interamerican Development Bank, contracted the Gorgas Memorial Laboratory to conduct epidemiologic studies on leishmaniasis and malaria in eastern Panama from July 1984 through June 1985. Preliminary results of the biomedical and entomologic teams investigating the epidemiology of cutaneous leishmaniasis in the eastern part of the country are presented in this short report. The principal findings of the study revealed 1) a large disparity in the incidence and prevalence of the disease among the five communities investigated; 2) the appearance of self-cures without the benefit of effective treatment; 3) a relatively high percentage of subclinical cases; and 4) determination of the sandfly vector species for each community. Also reported here is a case of a double infection with two distinct species of Leishmania, L. mexicana and L. amazonensis, in a single individual.

Morphogenesis of larval Onchocerca volvulus in the Panamanian black fly, Simulium quadrivittatum.

Simulium quadrivittatum Loew (Diptera: Simuliidae), a man-biting black fly, was shown, for the first time, to be capable of supporting development of Onchocerca volvulus Leuckart (Nematoda: Filarioidea) from microfilariae to third-stage (infective) larvae. The black flies were collected in Chiriqui Province, Panama and transported alive to Guatemala, where they were allowed to feed on a human subject infected with O. volvulus. Samples of these flies were dissected over an 11-day period to assess morphogenesis of the parasite. Vigorously motile microfilariae were recovered from the mid-gut during the first 24 hours postfeeding; second-stage larvae were found in the thoracic musculature on day 4; and fully developed third-stage larvae were obtained from the cephalic capsule by day 10. This rate of larval development is similar to that observed in Guatemalan S. ochraceum. Onchocerciasis is not known to occur in Panama. The results of the present study direct attention to a potential public health hazard there and possibly elsewhere in Central America.

Isoenzyme patterns of Leishmania isolates from Colombia.

Promastigotes from four cutaneous leishmaniasis cases from Colombia were tested by cellulose acetate electrophoresis using nine enzyme systems. The isoenzyme profiles of the Colombian isolates were indistinguishable from each other and from Panamanian Leishmania braziliensis panamensis controls, but were distinct from an isolate of Leishmania braziliensis guyanensis from Brazil and three isolates from the Leishmania mexicana complex for the enzyme phosphogluconate dehydrogenase.

Transovarial transmission of Gamboa virus in a tropical mosquito, Aedeomyia squamipennis.

We report transovarial transmission of Gamboa virus (Bunyavirus) in Aedeomyia squamipennis, a tropical mosquito which is active and bloodfeeding throughout the year. Gamboa virus was isolated during each of the 28 months of the study from every mosquito stage, including eggs, demonstrating that vertical transmission is a maintenance mechanism of this virus. The overall minimum infection rate was 5.1/1,000 mosquitoes. Identification of the 567 isolates by neutralization indicated that greater than or equal to 2 serotypes or subtypes of Gamboa virus circulate at the study site.

Leishmania mexicana complex: human infections in the Republic of Panama.

Parasites of the genus Leishmania responsible for human cutaneous leishmaniasis in the New World form 2 major taxonomic divisions: the Leishmania braziliensis and the L. mexicana complexes. We report the isolation and characterization of the L. mexicana complex among humans in the Republic of Panama. Characterization was based on parasite morphology, pathogenesis in infected golden hamsters, cellulose acetate isoenzyme electrophoretic mobilities, and membrane-specific monoclonal antibodies using the radioimmune binding assay technique.

Isolation of HIV using T-lymphocyte 'panning': methodological aspects.

A technique for isolation of HIV using selective cultures of T4 cells obtained from peripheral blood by immunochemical separation was developed and optimized. Using this method infectious virus could be isolated in single isolation attempts from 89% of 35 HIV-infected patients in different stages of immunodeficiency. This isolation frequency was virtually independent of the stage of the disease, in contrast to the results obtained by the conventional isolation technique based on peripheral mononuclear cells (PMC). It is concluded that isolation of HIV from selected T4 cells is superior to other methods when isolation is attempted from healthy HIV-infected individuals.

Isolation of HIV from cultures of purified CD4+ lymphocytes.

Isolation of HIV from cultures of CD4+ lymphocytes purified from peripheral blood by indirect panning was optimized and evaluated. Infectious HIV was isolated by single isolation attempts in 98% of 102 HIV-antibody-positive patients (55 had AIDS or ARC and 47 were clinically healthy). The average culture time required for positive cultures was largely independent of the CD4 count of the patients and 87% of the positive isolation cultures from both groups of patients became positive within 14 days of culture. An evaluation of the possible influence of media additives on propagation of HIV showed that: amphotericin-B had a suppressive effect on HIV replication at concentrations recommended for anti-fungal activity; recombinant and human interleukin-2 were equally suitable for both isolation cultures and for propagation of HIV, and polybrene, at a concentration of 2 micrograms/ml in the culture medium had a beneficial effect.

Identification and biopsy of the sentinel lymph node in breast cancer.

AIMS: To examine the hypothesis that lymphatic dissemination in breast cancer occurs sequentially. METHODS: Thirty patients with clinically localized adenocarcinoma were studied. Patent blue dye was administered into the tumour at the beginning of a modified radical mastectomy or segmental mastectomy with en bloc axillary lymph-node dissection (ALND). In the removed specimen, blue-stained lymphatic channels were dissected from the primary tumour to the first draining lymph node(s) (sentinel node(s)). RESULTS: Identification of a sentinel node (SN) was successful in 26 patients (87%). In 10 patients the SN was tumour-positive. In six of these patients, the SN was the only tumour-positive node. There was no incidence of 'skip' metastasis. CONCLUSIONS: This study confirms the sequential nature of lymphatic dissemination. When confirmed in vivo, these data may lead to a substantial reduction of the need for ALND without compromising survival and regional control and without loss of prognostic and staging information.