Juvenile hormone regulates brain-reproduction tradeoff in bumble bees but not in honey bees.

Gonadotropic hormones coordinate processes in diverse tissues regulating animal reproductive physiology and behavior. Juvenile hormone (JH) is the ancient and most common gonadotropin in insects, but not in advanced eusocial honey bees and some ants. To start probing the evolutionary basis of this change, we combined endocrine manipulations, transcriptomics, and behavioral analyses to study JH regulated processes in a bumble bee showing a relatively simple level of eusociality. We found that in worker fat body, more JH-regulated genes were up- rather than down-regulated, and enriched for metabolic and biosynthetic pathways. This transcriptomic pattern is consistent with earlier evidence that JH is the major gonadotropin in bumble bees. In the brain, more JH-regulated genes were down- rather than up-regulated and enriched for protein turnover pathways. Brain ribosomal protein gene expression shows a similar trend of downregulation in dominant workers, which naturally have high JH titers. In other species, similar downregulation of protein turnover is found in aging brains or under stress, associated with compromised long-term memory and health. These findings suggest a previously unknown gonadotropin-mediated tradeoff. Analysis of published data reveals no such downregulation of protein turnover pathways in the brain of honey bee workers, which exhibit more complex eusociality and in which JH is not a gonadotropin but rather regulates division of labor. These results suggest that the evolution of complex eusociality in honey bees was associated with modifications in hormonal signalling supporting extended and extremely high fertility while reducing the ancient costs of high gonadotropin titers to the brain.

DETECTION OF COPATHOGENS IN FREE-RANGING EASTERN BOX TURTLES ( TERRAPENE CAROLINA CAROLINA) IN ILLINOIS AND TENNESSEE.

Conservation efforts are investigating the impact of diseases within a species of interest, including prevalence and transmission and morbidity and mortality rates. However, the majority of these studies focus solely on the characteristics of a single pathogen. Recently, the role of copathogens has been reported to impact disease susceptibility and mortality. To that effect, a survey was conducted including 318 eastern box turtles ( Terrapene carolina carolina) from populations in Illinois and Tennessee in 2014 and 2015. Blood samples and oral swabs were collected for quantitative polymerase chain reaction (qPCR) of 15 different pathogens performed in a multiplex format using Fluidigm array technology. Four pathogens were found with varying qPCR prevalence: ranavirus (FV3; n = 2, 0.6%), Terrapene herpesvirus 1 (TerHV1; n = 129, 40.7%), box turtle Mycoplasma sp. (BT Myco; n = 14, 4.6%), and box turtle adenovirus (BT Adv1; n = 18, 11%). Thirteen pathogens were not identified in any sample, including Mycoplasma agassizii, M. testudineum, Salmonella enteriditis, S. typhmirium, Borrelia burgdorferi, Anaplasma phagocyophilum, tortoise intranuclear coccidia, Ambystoma tigrinum virus, Bohle iridovirus, Epizootic hematopoietic necrosis virus, and testudinid herpesvirus 2. Copathogen occurrence was rare but was observed in eight individuals with TerHV1-BT Myco detection and two animals with TerHV1-Adv1. Significant differences were observed in pathogen detection across season (TerHV1, BT Adv1, BT Myco, and TerHV1-Myco) and year (TerHV1, BT Adv1, and TerHV1-Myco). The results of this survey highlight that a single pathogen model may not adequately explain pathogen dynamics and that conservation efforts need to be aimed at detecting multiple pathogens in order to fully characterize population health.

Whole-genome resequencing of two elite sires for the detection of haplotypes under selection in dairy cattle.

Using a combination of whole-genome resequencing and high-density genotyping arrays, genome-wide haplotypes were reconstructed for two of the most important bulls in the history of the dairy cattle industry, Pawnee Farm Arlinda Chief ("Chief") and his son Walkway Chief Mark ("Mark"), each accounting for ∼7% of all current genomes. We aligned 20.5 Gbp (∼7.3× coverage) and 37.9 Gbp (∼13.5× coverage) of the Chief and Mark genomic sequences, respectively. More than 1.3 million high-quality SNPs were detected in Chief and Mark sequences. The genome-wide haplotypes inherited by Mark from Chief were reconstructed using ∼1 million informative SNPs. Comparison of a set of 15,826 SNPs that overlapped in the sequence-based and BovineSNP50 SNPs showed the accuracy of the sequence-based haplotype reconstruction to be as high as 97%. By using the BovineSNP50 genotypes, the frequencies of Chief alleles on his two haplotypes then were determined in 1,149 of his descendants, and the distribution was compared with the frequencies that would be expected assuming no selection. We identified 49 chromosomal segments in which Chief alleles showed strong evidence of selection. Candidate polymorphisms for traits that have been under selection in the dairy cattle population then were identified by referencing Chief's DNA sequence within these selected chromosome blocks. Eleven candidate genes were identified with functions related to milk-production, fertility, and disease-resistance traits. These data demonstrate that haplotype reconstruction of an ancestral proband by whole-genome resequencing in combination with high-density SNP genotyping of descendants can be used for rapid, genome-wide identification of the ancestor's alleles that have been subjected to artificial selection.

Identification of male-specific amh duplication, sexually differentially expressed genes and microRNAs at early embryonic development of Nile tilapia (Oreochromis niloticus).

BACKGROUND: The probable influence of genes and the environment on sex determination in Nile tilapia suggests that it should be regarded as a complex trait. Detection of sex determination genes in tilapia has both scientific and commercial importance. The main objective was to detect genes and microRNAs that were differentially expressed by gender in early embryonic development. RESULTS: Artificial fertilization of Oreochromis niloticus XX females with either sex-reversed ΔXX males or genetically-modified YY 'supermales' resulted in all-female and all-male embryos, respectively. RNA of pools of all-female and all-male embryos at 2, 5 and 9 dpf were used as template for a custom Agilent eArray hybridization and next generation sequencing. Fifty-nine genes differentially expressed between genders were identified by a false discovery rate of p < 0.05. The most overexpressed genes were amh and tspan8 in males, and cr/20ß-hsd, gpa33, rtn4ipl and zp3 in females (p < 1 × 10-9). Validation of gene expression using qPCR in embryos and gonads indicated copy number variation in tspan8, gpa33, cr/20ß-hsd and amh. Sequencing of amh identified a male-specific duplication of this gene, denoted amhy, differing from the sequence of amh by a 233 bp deletion on exonVII, hence lacking the capability to encode the protein motif that binds to the transforming growth factor beta receptor (TGF-ß domain). amh and amhy segregated in the mapping family in full concordance with SD-linked marker on LG23 signifying the QTL for SD. We discovered 831 microRNAs in tilapia embryos of which nine had sexually dimorphic expression patterns by a false discovery rate of p < 0.05. An up-regulated microRNA in males, pma-mir-4585, was characterized with all six predicted target genes including cr/20ß-hsd, down-regulated in males. CONCLUSIONS: This study reports the first discovery of sexually differentially expressed genes and microRNAs at a very early stage of tilapia embryonic development, i.e. from 2 dpf. Genes with sexually differential expression patterns are enriched for copy number variation. A novel male-specific duplication of amh, denoted amhy, lacking the TGF-ß domain was identified and mapped to the QTL region on LG23 for SD, thus indicating its potential role in SD.

Transcriptional profiling of the spleen in progressive visceral leishmaniasis reveals mixed expression of type 1 and type 2 cytokine-responsive genes.

BACKGROUND: The Syrian golden hamster (Mesocricetus aureus) has been used as a model to study infections caused by a number of human pathogens. Studies of immunopathogenesis in hamster infection models are challenging because of the limited availability of reagents needed to define cellular and molecular determinants. RESULTS: We sequenced a hamster cDNA library and developed a first-generation custom cDNA microarray that included 5131 unique cDNAs enriched for immune response genes. We used this microarray to interrogate the hamster spleen response to Leishmania donovani, an intracellular protozoan that causes visceral leishmaniasis. The hamster model of visceral leishmaniasis is of particular interest because it recapitulates clinical and immunopathological features of human disease, including cachexia, massive splenomegaly, pancytopenia, immunosuppression, and ultimately death. In the microarray a differentially expressed transcript was identified as having at least a 2-fold change in expression between uninfected and infected groups and a False Discovery Rate of <5%. Following a relatively silent early phase of infection (at 7 and 14 days post-infection only 8 and 24 genes, respectively, were differentially expressed), there was dramatic upregulation of inflammatory and immune-related genes in the spleen (708 differentially expressed genes were evident at 28 days post-infection). The differentially expressed transcripts included genes involved in inflammation, immunity, and immune cell trafficking. Of particular interest there was concomitant upregulation of the IFN-γ and interleukin (IL)-4 signaling pathways, with increased expression of a battery of IFN-γ- and IL-4-responsive genes. The latter included genes characteristic of alternatively activated macrophages. CONCLUSIONS: Transcriptional profiling was accomplished in the Syrian golden hamster, for which a fully annotated genome is not available. In the hamster model of visceral leishmaniasis, a robust and functional IFN-γ response did not restrain parasite load and progression of disease. This supports the accumulating evidence that macrophages are ineffectively activated to kill the parasite. The concomitant expression of IL-4/IL-13 and their downstream target genes, some of which were characteristic of alternative macrophage activation, are likely to contribute to this. Further dissection of mechanisms that lead to polarization of macrophages toward a permissive state is needed to fully understand the pathogenesis of visceral leishmaniasis.

Molecular heterochrony and the evolution of sociality in bumblebees (Bombus terrestris).

Sibling care is a hallmark of social insects, but its evolution remains challenging to explain at the molecular level. The hypothesis that sibling care evolved from ancestral maternal care in primitively eusocial insects has been elaborated to involve heterochronic changes in gene expression. This elaboration leads to the prediction that workers in these species will show patterns of gene expression more similar to foundress queens, who express maternal care behaviour, than to established queens engaged solely in reproductive behaviour. We tested this idea in bumblebees (Bombus terrestris) using a microarray platform with approximately 4500 genes. Unlike the wasp Polistes metricus, in which support for the above prediction has been obtained, we found that patterns of brain gene expression in foundress and queen bumblebees were more similar to each other than to workers. Comparisons of differentially expressed genes derived from this study and gene lists from microarray studies in Polistes and the honeybee Apis mellifera yielded a shared set of genes involved in the regulation of related social behaviours across independent eusocial lineages. Together, these results suggest that multiple independent evolutions of eusociality in the insects might have involved different evolutionary routes, but nevertheless involved some similarities at the molecular level.

They live in the land down under: thyroid function and basal metabolic rate in the Blind Mole Rat, Spalax.

The Israeli blind subterranean mole rat (Spalax ehrenbergi superspecies) lives in sealed underground burrows under extreme, hypoxic conditions. The four Israeli Spalax allospecies have adapted to different climates, the cool-humid (Spalax galili, 2 n = 52 chromosomes), semihumid (S. golani, 2 n = 54) north regions, warm-humid (S. carmeli, 2 n = 58) central region and the warm-dry S. judaei, 2 n = 60) southern regions. A dramatic interspecies decline in basal metabolic rate (BMR) from north to south, even after years of captivity, indicates a genetic basis for this BMR trait. We examined the possibility that the genetically-conditioned interspecies BMR difference was expressed via circulating thyroid hormone. An unexpected north to south increase in serum free thyroxine (FT4) and total 3, 5, 3'-triiodo-L-thyronine (T3) (p < 0.02) correlated negatively with previously published BMR measurements. The increases in serum FT4 and T3 were symmetrical, so that the T3:FT4 ratio - interpretable as an index of conversion of T4 to T3 in nonthyroidal tissues - did not support relative decrease in production of T3 as a contributor to BMR. Increased north-to-south serum FT4 and T3 levels also correlated negatively with hemoglobin/hematocrit. North-to-south adaptations in spalacids include decreased BMR and hematocrit/hemoglobin in the face of increasing thyroid hormone levels, arguing for independent control of hormone secretion and BMR/hematocrit/hemoglobin. But the significant inverse relationship between thyroid hormone levels and BMR/hematocrit/hemoglobin is also consistent with a degree of cellular resistance to thyroid hormone action that protects against hormone-induced increase in oxygen consumption in a hostile, hypoxic environment.

Pronounced cancer resistance in a subterranean rodent, the blind mole-rat, Spalax: in vivo and in vitro evidence.

BACKGROUND: Subterranean blind mole rats (Spalax) are hypoxia tolerant (down to 3% O2), long lived (>20 years) rodents showing no clear signs of aging or aging related disorders. In 50 years of Spalax research, spontaneous tumors have never been recorded among thousands of individuals. Here we addressed the questions of (1) whether Spalax is resistant to chemically-induced tumorigenesis, and (2) whether normal fibroblasts isolated from Spalax possess tumor-suppressive activity. RESULTS: Treating animals with 3-Methylcholantrene (3MCA) and 7,12-Dimethylbenz(a) anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA), two potent carcinogens, confirmed Spalax high resistance to chemically induced cancers. While all mice and rats developed the expected tumors following treatment with both carcinogens, among Spalax no tumors were observed after DMBA/TPA treatment, while 3MCA induced benign fibroblastic proliferation in 2 Spalax individuals out of12, and only a single animal from the advanced age group developed malignancy 18 months post-treatment. The remaining animals are still healthy 30 months post-treatment. In vitro experiments showed an extraordinary ability of normal Spalax cultured fibroblasts to restrict malignant behavior in a broad spectrum of human-derived and in newly isolated Spalax 3MCA-induced cancer cell lines. Growth of cancer cells was inhibited by either direct interaction with Spalax fibroblasts or with soluble factors released into culture media and soft agar. This was accompanied by decreased cancer cell viability, reduced colony formation in soft agar, disturbed cell cycle progression, chromatin condensation and mitochondrial fragmentation. Cells from another cancer resistant subterranean mammal, the naked mole rat, were also tested for direct effect on cancer cells and, similar to Spalax, demonstrated anti-cancer activity. No effect on cancer cells was observed using fibroblasts from mouse, rat or Acomys. Spalax fibroblast conditioned media had no effect on proliferation of noncancerous cells. CONCLUSIONS: This report provides pioneering evidence that Spalax is not only resistant to spontaneous cancer but also to experimentally induced cancer, and shows the unique ability of Spalax normal fibroblasts to inhibit growth and kill cancer cells, but not normal cells, either through direct fibroblast-cancer cell interaction or via soluble factors. Obviously, along with adaptation to hypoxia, Spalax has evolved efficient anti-cancer mechanisms yet to be elucidated. Exploring the molecular mechanisms allowing Spalax to survive in extreme environments and to escape cancer as well as to kill homologous and heterologous cancer cells may hold the key for understanding the molecular nature of host resistance to cancer and identify new anti-cancer strategies for treating humans.

Gene expression profiling reveals the heterogeneous transcriptional activity of Oct3/4 and its possible interaction with Gli2 in mouse embryonic stem cells.

We examined the transcriptional activity of Oct3/4 (Pou5f1) in mouse embryonic stem cells (mESCs) maintained under standard culture conditions to gain a better understanding of self-renewal in mESCs. First, we built an expression vector in which the Oct3/4 promoter drives the monocistronic transcription of Venus and a puromycin-resistant gene via the foot-and-mouth disease virus self-cleaving peptide T2A. Then, a genetically-engineered mESC line with the stable integration of this vector was isolated and cultured in the presence or absence of puromycin. The cultures were subsequently subjected to Illumina expression microarray analysis. We identified approximately 4600 probes with statistically significant differential expression. The genes involved in nucleic acid synthesis were overrepresented in the probe set associated with mESCs maintained in the presence of puromycin. In contrast, the genes involved in cell differentiation were overrepresented in the probe set associated with mESCs maintained in the absence of puromycin. Therefore, it is suggested with these data that the transcriptional activity of Oct3/4 fluctuates in mESCs and that Oct3/4 plays an essential role in sustaining the basal transcriptional activities required for cell duplication in populations with equal differentiation potential. Heterogeneity in the transcriptional activity of Oct3/4 was dynamic. Interestingly, we found that genes involved in the hedgehog signaling pathway showed unique expression profiles in mESCs and validated this observation by RT-PCR analysis. The expression of Gli2, Ptch1 and Smo was consistently detected in other types of pluripotent stem cells examined in this study. Furthermore, the Gli2 protein was heterogeneously detected in mESC nuclei by immunofluorescence microscopy and this result correlated with the detection of the Oct3/4 protein. Finally, forced activation of Gli2 in mESCs increased their proliferation rate. Collectively, it is suggested with these results that Gli2 may play a novel role in the self-renewal of pluripotent stem cells.

Microbial populations vary between the upper and lower respiratory tract, but not within biogeographic regions of the lung of healthy horses.

Understanding normal microbial populations within areas of the respiratory tract is essential, as variable regional conditions create different niches for microbial flora, and proliferation of commensal microbes likely contributes to clinical respiratory disease. The objective was to describe microbial population variability between respiratory tract locations in healthy horses. Samples were collected from four healthy adult horses by nasopharyngeal lavage (NPL), transtracheal aspirate (TTA), and bronchoalveolar lavage (BAL) of six distinct regions within the lung. Full-length 16S ribosomal DNA sequencing and microbial profiling analysis was performed. There was a large amount of diversity, with over 1797 ASVs identified, reduced to 94 taxa after tip agglomeration and prevalence filtering. Number of taxa and diversity were highly variable across horses, sample types, and BAL locations. Firmicutes, proteobacteria, and actinobacteria were the predominant phyla. There was a significant difference in richness (Chao1, p = 0.02) and phylogenetic diversity (FaithPD, p = 0.01) between NPL, TTA, and BAL. Sample type (p = 0.03) and horse (p = 0.005) contributed significantly to Bray-Curtis compositional diversity, while Weighted Unifrac metric was only affected by simplified sample type (NPL and TTA vs BAL, p = 0.04). There was no significant effect of BAL locations within the lung with alpha or beta diversity statistical tests. Overall findings support diverse microbial populations that were variable between upper and lower respiratory tract locations, but with no apparent difference in microbial populations of the six biogeographic regions of the lung, suggesting that BAL fluid obtained blindly by standard clinical techniques may be sufficient for future studies in healthy horses.

Social regulation of maternal traits in nest-founding bumble bee (Bombus terrestris) queens.

During the nest-founding phase of the bumble bee colony cycle, queens undergo striking changes in maternal care behavior. Early in the founding phase, prior to the emergence of workers in the nest, queens are reproductive and also provision and feed their offspring. However, later in the founding phase, queens reduce their feeding of larvae and become specialized on reproduction. This transition is synchronized with the emergence of workers in the colony, who assume the task of feeding their siblings. Using a social manipulation experiment with the bumble bee Bombus terrestris, we tested the hypothesis that workers regulate the transition from feeding brood to specialization on reproduction in nest-founding bumble bee queens. Consistent with this hypothesis, we found that early-stage nest-founding queens with workers prematurely added to their nests reduce their brood-feeding behavior and increase egg laying, and likewise, late-stage nest-founding queens increase their brood-feeding behavior and decrease egg-laying when workers are removed from their nests. Further, brood-feeding and egg-laying behaviors were negatively correlated. We used Agilent microarrays designed from B. terrestris brain expressed sequenced tags (ESTs) to explore a second hypothesis, that workers alter brain gene expression in nest-founding queens. We found evidence that brain gene expression in nest-founding queens is altered by the presence of workers, with the effect being much stronger in late-stage founding queens. This study provides new insights into how the transition from feeding brood to specialization on reproduction in queen bumble bees is regulated during the nest initiation phase of the colony cycle.

Transcriptome analysis of the spalax hypoxia survival response includes suppression of apoptosis and tight control of angiogenesis.

BACKGROUND: The development of complex responses to hypoxia has played a key role in the evolution of mammals, as inadequate response to this condition is frequently associated with cardiovascular diseases, developmental disorders, and cancers. Though numerous studies have used mice and rats in order to explore mechanisms that contribute to hypoxia tolerance, these studies are limited due to the high sensitivity of most rodents to severe hypoxia. The blind subterranean mole rat Spalax is a hypoxia tolerant rodent, which exhibits unique longevity and therefore has invaluable potential in hypoxia and cancer research. RESULTS: Using microarrays, transcript abundance was measured in brain and muscle tissues from Spalax and rat individuals exposed to acute and chronic hypoxia for varying durations. We found that Spalax global gene expression response to hypoxia differs from that of rat and is characterized by the activation of functional groups of genes that have not been strongly associated with the response to hypoxia in hypoxia sensitive mammals. Using functional enrichment analysis of Spalax hypoxia induced genes we found highly significant overrepresentation of groups of genes involved in anti apoptosis, cancer, embryonic/sexual development, epidermal growth factor receptor binding, coordinated suppression and activation of distinct groups of transcription factors and membrane receptors, in addition to angiogenic related processes. We also detected hypoxia induced increases of different critical Spalax hub gene transcripts, including antiangiogenic genes associated with cancer tolerance in Down syndrome human individuals. CONCLUSIONS: This is the most comprehensive study of Spalax large scale gene expression response to hypoxia to date, and the first to use custom Spalax microarrays. Our work presents novel patterns that may underlie mechanisms with critical importance to the evolution of hypoxia tolerance, with special relevance to medical research.

Cross talk between S-nitrosylation and S-glutathionylation in control of the Na,K-ATPase regulation in hypoxic heart.

Oxygen-induced regulation of Na,K-ATPase was studied in rat myocardium. In rat heart, Na,K-ATPase responded to hypoxia with a dose-dependent inhibition in hydrolytic activity. Inhibition of Na,K-ATPase in hypoxic rat heart was associated with decrease in nitric oxide (NO) production and progressive oxidative stress. Accumulation of oxidized glutathione (GSSG) and decrease in NO availability in hypoxic rat heart were followed by a decrease in S-nitrosylation and upregulation of S-glutathionylation of the catalytic α-subunit of the Na,K-ATPase. Induction of S-glutathionylation of the α-subunit by treatment of tissue homogenate with GSSG resulted in complete inhibition of the enzyme in rat a myocardial tissue homogenate. Inhibitory effect of GSSG in rat sarcolemma could be significantly decreased upon activation of NO synthases. We have further tested whether oxidative stress and suppression of the Na,K-ATPase activity are observed in hypoxic heart of two subterranean hypoxia-tolerant blind mole species (Spalax galili and Spalax judaei). In both hypoxia-tolerant Spalax species activity of the enzyme and tissue redox state were maintained under hypoxic conditions. However, localization of cysteines within the catalytic subunit of the Na,K-ATPase was preserved and induction of S-glutathionylation by GSSG in tissue homogenate inhibited the Spalax ATPase as efficiently as in rat heart. The obtained data indicate that oxygen-induced regulation of the Na,K-ATPase in the heart is mediated by a switch between S-glutathionylation and S-nitrosylation of the regulatory thiol groups localized at the catalytic subunit of the enzyme.

Transcriptional regulation of brain gene expression in response to a territorial intrusion.

Aggressive behaviour associated with territorial defence is widespread and has fitness consequences. However, excess aggression can interfere with other important biological functions such as immunity and energy homeostasis. How the expression of complex behaviours such as aggression is regulated in the brain has long intrigued ethologists, but has only recently become amenable for molecular dissection in non-model organisms. We investigated the transcriptomic response to territorial intrusion in four brain regions in breeding male threespined sticklebacks using expression microarrays and quantitative polymerase chain reaction (qPCR). Each region of the brain had a distinct genomic response to a territorial challenge. We identified a set of genes that were upregulated in the diencephalon and downregulated in the cerebellum and the brain stem. Cis-regulatory network analysis suggested transcription factors that regulated or co-regulated genes that were consistently regulated in all brain regions and others that regulated gene expression in opposing directions across brain regions. Our results support the hypothesis that territorial animals respond to social challenges via transcriptional regulation of genes in different brain regions. Finally, we found a remarkably close association between gene expression and aggressive behaviour at the individual level. This study sheds light on the molecular mechanisms in the brain that underlie the response to social challenges.

Methionine sulfoxide reductases and methionine sulfoxide in the subterranean mole rat (Spalax): characterization of expression under various oxygen conditions.

The blind subterranean mole rat (Spalax ehrenbergi) exhibits a relatively long life span, which is attributed to an efficient antioxidant defense affording protection against accumulation of oxidative modifications of proteins. Methionine residues can be oxidized to methionine sulfoxide (MetO) and then enzymatically reduced by the methionine sulfoxide reductase (Msr) system. In the current study we have isolated the cDNA sequences of the Spalax Msr genes as well as 23 additional selenoproteins and monitored the activities of Msr enzymes in liver and brain of rat (Rattus norvegicus), Spalax galili, and Spalax judaei under normoxia, hypoxia, and hyperoxia. Under normoxia, the Msr activity was lower in S. galili in comparison to S. judaei and R. norvegicus especially in the brain. The pattern of Msr activity of the three species was similar throughout the tested conditions. However, exposure of the animals to hypoxia caused a significant enhancement of Msr activity, especially in S. galili. Hyperoxic exposure showed a highly significant induction of Msr activity compared with normoxic conditions for R. norvegicus and S. galili brain. It was concluded that among all species examined, S. galili appears to be more responsive to oxygen tension changes and that the Msr system is upregulated mainly by severe hypoxia.

Differential DNA methylation associated with multiple sclerosis and disease modifying treatments in an underrepresented minority population.

Black and Hispanic American patients frequently develop earlier onset of multiple sclerosis (MS) and a more severe disease course that can be resistant to disease modifying treatments. The objectives were to identify differential methylation of genomic DNA (gDNA) associated with disease susceptibility and treatment responses in a cohort of MS patients from underrepresented minority populations. Patients with MS and controls with non-inflammatory neurologic conditions were consented and enrolled under an IRB-approved protocol. Approximately 64% of donors identified as Black or African American and 30% as White, Hispanic-Latino. Infinium MethylationEPIC bead arrays were utilized to measure epigenome-wide gDNA methylation of whole blood. Data were analyzed in the presence and absence of adjustments for unknown covariates in the dataset, some of which corresponded to disease modifying treatments. Global patterns of differential methylation associated with MS were strongest for those probes that showed relative demethylation of loci with lower M values. Pathway analysis revealed unexpected associations with shigellosis and amoebiasis. Enrichment analysis revealed an over-representation of probes in enhancer regions and an under-representation in promoters. In the presence of adjustments for covariates that included disease modifying treatments, analysis revealed 10 differentially methylated regions (DMR's) with an FDR <1E-77. Five of these genes (ARID5B, BAZ2B, RABGAP1, SFRP2, WBP1L) are associated with cancer risk and cellular differentiation and have not been previously identified in MS studies. Hierarchical cluster and multi-dimensional scaling analysis of differential DNA methylation at 147 loci within those DMR's was sufficient to differentiate MS donors from controls. In the absence of corrections for disease modifying treatments, differential methylation in patients treated with dimethyl fumarate was associated with immune regulatory pathways that regulate cytokine and chemokine signaling, axon guidance, and adherens junctions. These results demonstrate possible associations of gastrointestinal pathogens and regulation of cellular differentiation with MS susceptibility in our patient cohort. This work further suggests that analyses can be performed in the presence and absence of corrections for immune therapies. Because of their high representation in our patient cohort, these results may be of specific relevance in the regulation of disease susceptibility and treatment responses in Black and Hispanic Americans.

Brain transcriptomic response of threespine sticklebacks to cues of a predator.

Predation pressure represents a strong selective force that influences the development and evolution of living organisms. An increasing number of studies have shown that both environmental and social factors, including exposure to predators, substantially shape the structure and function of the brain. However, our knowledge about the molecular mechanisms underlying the response of the brain to environmental stimuli is limited. In this study, we used whole-genome comparative oligonucleotide microarrays to investigate the brain transcriptomic response to cues of a predator in the threespine stickleback, Gasterosteus aculeatus. We found that repeated exposure to olfactory, visual and tactile cues of a predator (rainbow trout, Oncorrhynchus mykiss) for 6 days resulted in subtle but significant transcriptomic changes in the brain of sticklebacks. Gene functional analysis and gene ontology enrichment revealed that the majority of the transcripts differentially expressed between the fish exposed to cues of a predator and the control group were related to antigen processing and presentation involving the major histocompatibility complex, transmission of synaptic signals, brain metabolic processes, gene regulation and visual perception. The top four identified pathways were synaptic long-term depression, RAN signaling, relaxin signaling and phototransduction. Our study demonstrates that exposure of sticklebacks to cues of a predator results in the activation of a wide range of biological and molecular processes and lays the foundation for future investigations on the molecular factors that modulate the function and evolution of the brain in response to stressors.

The muscle ankyrin repeat proteins are hypoxia-sensitive: in vivo mRNA expression in the hypoxia-tolerant blind subterranean mole rat, Spalax ehrenbergi.

The muscle ankyrin repeat proteins (MARPs), also known as muscle stretch proteins, are members of a conserved family of genes known to be induced under stress conditions. The three primary members, cardiac ankyrin repeat protein (CARP), Ankyrin Repeat Domain 2 (ARPP), and diabetes-related ankyrin repeat protein (DARP) are expressed in cardiac and skeletal muscle, binding to the giant protein titin. In addition, both CARP and ARPP are proposed to have regulatory functions, shuttling to the nucleus and serving as a liaison between mechanical stress and the transcriptional response. In mouse and human models, CARP is induced during wound healing, denervation, neurogenesis, and angiogenesis; ARPP during an immobilized stretch; DARP is up-regulated in type 2 diabetes, as well as brown adipose tissue, suggesting a role in energy metabolism. Most animal models have focused on stretch response stress; however, little is known about the response of MARPs to hypoxic stress. The blind subterranean mole rat is a model for hypoxia tolerance with the ability to survive extremely hypoxic and hypercapnic underground conditions. Following observations that CARP is differentially expressed in the Spalax muscle in response to hypoxia, we have sequenced the Spalax orthologs of the MARP proteins and profiled expression patterns under varying levels of hypoxic stress among two Spalax species and Rattus. Results show expression patterns highly correlated to the degree of hypoxic tolerance among the three species. Understanding the differences in MARP expression further elucidates mechanisms of hypoxia tolerance with relevance to human ischemic disease.

Hypoxia-induced BNIP3 expression and mitophagy: in vivo comparison of the rat and the hypoxia-tolerant mole rat, Spalax ehrenbergi.

The blind subterranean mole rat of the Spalax ehrenbergi superspecies is an excellent animal model for hypoxic tolerance. Unique physiological, functional, and gene structure changes allow Spalax species to survive lower oxygen levels than most terrestrial animals. BNIP3, an HIF-1 dependent hypoxia-response gene, has a proapoptotic function; however, expression is suppressed in many types of cancers. Under hypoxic conditions, BNIP3 also functions as a mediator of mitochondrial autophagy, a survival adaptation to control ROS production and DNA damage. Using real-time PCR and Western blotting, we investigated the impact of hypoxia on BNIP3 expression and mitophagy, in the skeletal muscle and heart, of the Rattus and two Spalax species. BNIP3 transcript, as well as protein levels, increased to significantly higher levels under hypoxia in Rattus tissues, with smaller changes in Spalax. Mitophagy was correlated with BNIP3 expression in the heart with an inverse correlation to hypoxia tolerance. A dense network of vessels in Spalax muscle may offer protection from physiological hypoxia, while the response in Rattus reflects the increase of hypoxic stress. In Spalax tissues, as in many cancers, BNIP3 expression and mitophagy are significantly less affected by hypoxia. Similar mechanisms, beneficial to organisms adapted to stressful environments, may also confer malignant cells with survival features. Understanding the molecular basis of such adaptations may enhance development of new therapeutic modalities.