RNA sequencing least shrew (Cryptotis parva) brainstem and gut transcripts following administration of a selective substance P neurokinin NK1 receptor agonist and antagonist expands genomics resources for emesis research.

The least shrew is among the subset of animals that are capable of vomiting and therefore serves as a valuable research model for investigating the biochemistry, molecular biology, pharmacology, and genomics of emesis. Both nausea and vomiting are associated with a variety of illnesses (bacterial/viral infections, bulimia, exposure to toxins, gall bladder disease), conditions (pregnancy, motion sickness, emotional stress, overeating) and reactions to drugs (chemotherapeutics, opiates). The severe discomfort and intense fear associated with the stressful symptoms of nausea and emesis are the major reason for patient non-compliance when being treated with cancer chemotherapeutics. Increased understanding of the physiology, pharmacology and pathophysiology underlying vomiting and nausea can accelerate progress for developing new antiemetics. As a major animal model for emesis, expanding genomic knowledge associated with emesis in the least shrew will further enhance the laboratory utility of this model. A key question is which genes mediate emesis, and are they expressed in response to emetics/antiemetics. To elucidate the mediators of emesis, in particular emetic receptors, their downstream signaling pathways, as well as the shared emetic signals, we carried out an RNA sequencing study focused on the central and peripheral emetic loci, the brainstem and gut. Thus, we sequenced RNA extracted from brainstem and gut tissues from different groups of least shrews treated with either a neurokinin NK1 receptor selective emetic agonist, GR73632 (5 mg/kg, i.p.), its corresponding selective antagonist netupitant (5 mg/kg, i.p.), a combination of these two agents, versus their corresponding vehicle-pretreated controls and drug naïve animals. The resulting sequences were processed using a de novo transcriptome assembly and used it to identify orthologs within human, dog, mouse, and ferret gene sets. We compared the least shrew to human and a veterinary species (dog) that may be treated with vomit-inducing chemotherapeutics, and the ferret, another well-established model organism for emesis research. The mouse was included because it does not vomit. In total, we identified a final set of 16,720 least shrew orthologs. We employed comparative genomics analyses as well as gene ontology enrichment, KEGG pathway enrichment and phenotype enrichment to better understand the molecular biology of genes implicated in vomiting.

Micromorphological study of the upper digestive tract of the Argentine tegu (Salvator merianae).

Argentine black and white tegus (Salvator merianae) are omnivorous lizards native to southeastern Brazil, Uruguay, eastern Paraguay and northern Argentina, and are invasive species in Florida and Georgia, USA. They are opportunistic feeders, which is what allow them to have such a diverse variety of foods. Tegus raised a particular concern due to their adaptive capability to different environments. Our goal was to provide a micromorphology baseline of oesophagus and stomach and correlate findings with their dietary and invasive capabilities. Four Argentine black and white tegus were used for this study. We collected and processed specimens from oesophagus and stomach using standard histological techniques and stained tissue sections using Haematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS), Alcian Blue (AB) and Verhoef's elastic stains. The oesophagus was lined with ciliated pseudostratified columnar epithelium (PSCE) with goblet cells. Gut-associated lymphoid tissues (GALT) were present occasionally in the oesophagus and more frequently in the stomach. Tunica muscularis (Tm) of the oesophageal-gastric junction had distinct smooth muscle which could function as a sphincter. The mucosa of the stomach was lined with simple columnar epithelium (SC). The glands had neck and dark oxyntico-peptic cells. The pyloric sphincter had more GALT and mucus cells than other regions. The Tm outer layer is thinner than the inner. Presence of large number of goblet cells would support faster transit of the bolus. The short digestive tract and the histological features observed are consistent with the ability of tegus consumption of large amount of food.

RNA sequencing demonstrates large-scale temporal dysregulation of gene expression in stimulated macrophages derived from MHC-defined chicken haplotypes.

Discovering genetic biomarkers associated with disease resistance and enhanced immunity is critical to developing advanced strategies for controlling viral and bacterial infections in different species. Macrophages, important cells of innate immunity, are directly involved in cellular interactions with pathogens, the release of cytokines activating other immune cells and antigen presentation to cells of the adaptive immune response. IFNγ is a potent activator of macrophages and increased production has been associated with disease resistance in several species. This study characterizes the molecular basis for dramatically different nitric oxide production and immune function between the B2 and the B19 haplotype chicken macrophages.A large-scale RNA sequencing approach was employed to sequence the RNA of purified macrophages from each haplotype group (B2 vs. B19) during differentiation and after stimulation. Our results demonstrate that a large number of genes exhibit divergent expression between B2 and B19 haplotype cells both prior and after stimulation. These differences in gene expression appear to be regulated by complex epigenetic mechanisms that need further investigation.

Bioinformatics Analysis of Chicken miRNAs Associated with Monocyte to Macrophage Differentiation and Subsequent IFNγ Stimulated Activation.

BACKGROUND: The goal of this project was to characterize the molecular and cellular roles of various gene targets regulated by miRNAs identified in differentiating and stimulating avian macrophages. Once a monocyte arrives to a site of infection, local signals induce a redistribution of resources into a macrophage phenotype. This may involve upregulating pathogen pattern recognizing receptors and increasing the efficiency of lysosomal biogenesis, while simultaneously recycling components involved in circulatory migration and leukocyte extravasation. a monocyte tooled with chemokine surface receptors and an internal cytoskeletal structure geared towards mobility may efficiently sense, react, and migrate toward a site of infection. METHODS: Peripheral blood derived monocytes were purified and cultured from young chickens. RNA sequencing was performed on both peripheral blood monocytes during differentiation into macrophages and on mature macrophages following stimulation with interferon gamma. A set of microRNAs were identified and investigated using bioinformatics methods to ascertain their potential role in avian macrophage biology. RESULTS: Among a number of miRNAs that are found to be expressed in avian macrophages, we focused on eight specific miRNAs (miR-1618, miR-1586, miR-1633, miR-1627, miR-1646, miR-1649, miR-1610, miR-1647) associated with macrophage differentiation and activation. Expression profiles of microRNAs were characterized during differentiation and activation. Candidate miRNA targets were implicated in processes including Wnt signaling, ubiquitination, PPAR mediated macrophage function, vesicle mediated cytokine trafficking, and WD40 domain protein functions. CONCLUSION: A global theme for macrophage function that may be modulated by microRNAs is the comprehensive redistribution of the cell's protein repertoire. This redistribution involves two processes: 1) the degradation and recycling of unneeded cytoplasmic and membrane components and 2) the mobilization of newly synthesized cellular components via vesicular trafficking. Generally, it appears that macrophages need to closely regulate gene expression for differentiation to be able to activate successfully in response to a pathogen. This is a process in which miRNAs participate by affecting several pathways critical for both, differentiation and activation.

In Silico Analysis of Gene Expression Network Components Underlying Pigmentation Phenotypes in the Python Identified Evolutionarily Conserved Clusters of Transcription Factor Binding Sites.

Color variation provides the opportunity to investigate the genetic basis of evolution and selection. Reptiles are less studied than mammals. Comparative genomics approaches allow for knowledge gained in one species to be leveraged for use in another species. We describe a comparative vertebrate analysis of conserved regulatory modules in pythons aimed at assessing bioinformatics evidence that transcription factors important in mammalian pigmentation phenotypes may also be important in python pigmentation phenotypes. We identified 23 python orthologs of mammalian genes associated with variation in coat color phenotypes for which we assessed the extent of pairwise protein sequence identity between pythons and mouse, dog, horse, cow, chicken, anole lizard, and garter snake. We next identified a set of melanocyte/pigment associated transcription factors (CREB, FOXD3, LEF-1, MITF, POU3F2, and USF-1) that exhibit relatively conserved sequence similarity within their DNA binding regions across species based on orthologous alignments across multiple species. Finally, we identified 27 evolutionarily conserved clusters of transcription factor binding sites within ~200-nucleotide intervals of the 1500-nucleotide upstream regions of AIM1, DCT, MC1R, MITF, MLANA, OA1, PMEL, RAB27A, and TYR from Python bivittatus. Our results provide insight into pigment phenotypes in pythons.

Integrating Genomic Data Sets for Knowledge Discovery: An Informed Approach to Management of Captive Endangered Species.

Many endangered captive populations exhibit reduced genetic diversity resulting in health issues that impact reproductive fitness and quality of life. Numerous cost effective genomic sequencing and genotyping technologies provide unparalleled opportunity for incorporating genomics knowledge in management of endangered species. Genomic data, such as sequence data, transcriptome data, and genotyping data, provide critical information about a captive population that, when leveraged correctly, can be utilized to maximize population genetic variation while simultaneously reducing unintended introduction or propagation of undesirable phenotypes. Current approaches aimed at managing endangered captive populations utilize species survival plans (SSPs) that rely upon mean kinship estimates to maximize genetic diversity while simultaneously avoiding artificial selection in the breeding program. However, as genomic resources increase for each endangered species, the potential knowledge available for management also increases. Unlike model organisms in which considerable scientific resources are used to experimentally validate genotype-phenotype relationships, endangered species typically lack the necessary sample sizes and economic resources required for such studies. Even so, in the absence of experimentally verified genetic discoveries, genomics data still provides value. In fact, bioinformatics and comparative genomics approaches offer mechanisms for translating these raw genomics data sets into integrated knowledge that enable an informed approach to endangered species management.

Leveraging Comparative Genomics to Identify and Functionally Characterize Genes Associated with Sperm Phenotypes in Python bivittatus (Burmese Python).

Comparative genomics approaches provide a means of leveraging functional genomics information from a highly annotated model organism's genome (such as the mouse genome) in order to make physiological inferences about the role of genes and proteins in a less characterized organism's genome (such as the Burmese python). We employed a comparative genomics approach to produce the functional annotation of Python bivittatus genes encoding proteins associated with sperm phenotypes. We identify 129 gene-phenotype relationships in the python which are implicated in 10 specific sperm phenotypes. Results obtained through our systematic analysis identified subsets of python genes exhibiting associations with gene ontology annotation terms. Functional annotation data was represented in a semantic scatter plot. Together, these newly annotated Python bivittatus genome resources provide a high resolution framework from which the biology relating to reptile spermatogenesis, fertility, and reproduction can be further investigated. Applications of our research include (1) production of genetic diagnostics for assessing fertility in domestic and wild reptiles; (2) enhanced assisted reproduction technology for endangered and captive reptiles; and (3) novel molecular targets for biotechnology-based approaches aimed at reducing fertility and reproduction of invasive reptiles. Additional enhancements to reptile genomic resources will further enhance their value.

Using the PubMatrix literature-mining resource to accelerate student-centered learning in a veterinary problem-based learning curriculum.

Problem-based learning (PBL) creates an atmosphere in which veterinary students must take responsibility for their own education. Unlike a traditional curriculum where students receive discipline-specific information by attending formal lectures, PBL is designed to elicit self-directed, student-centered learning such that each student determines (1) what he/she does not know (learning issues), (2) what he/she needs to learn, (3) how he/she will learn it, and (4) what resources he/she will use. One of the biggest challenges facing students in a PBL curriculum is efficient time management while pursuing learning issues. Bioinformatics resources, such as the PubMatrix literature-mining tool, allow access to tremendous amounts of information almost instantaneously. To accelerate student-centered learning it is necessary to include resources that enhance the rate at which students can process biomedical information. Unlike using the PubMed interface directly, the PubMatrix tool enables users to automate queries, allowing up to 1,000 distinct PubMed queries to be executed per single PubMatrix submission. Users may submit multiple PubMatrix queries per session, resulting in the ability to execute tens of thousands of PubMed queries in a single day. The intuitively organized results, which remain accessible from PubMatrix user accounts, enable students to rapidly assimilate and process hundreds of thousands of individual publication records as they relate to the student's specific learning issues and query terms. Subsequently, students can explore substantially more of the biomedical publication landscape per learning issue and spend a greater fraction of their time actively engaged in resolving their learning issues.