Immune and metabolic challenges induce changes in pain sensation and related pathways in the hypothalamus.

The hypothalamic molecular processes participate in the regulation of the neuro-immune-endocrine system, including hormone, metabolite, chemokine circulation, and corresponding physiological and behavioral responses. RNA-sequencing profiles were analyzed to understand the effect of juvenile immune and metabolic distress 100 days after virally elicited maternal immune activation during gestation in pigs. Over 1,300 genes exhibited significant additive or interacting effects of gestational immune activation, juvenile distress, and sex. One-third of these genes presented multiple effects, emphasizing the complex interplay of these factors. Key functional categories enriched among affected genes included sensory perception of pain, steroidogenesis, prolactin, neuropeptide, and inflammatory signaling. These categories underscore the intricate relationship between gestational immune activation during gestation, distress, and the response of hypothalamic pathways to insults. These effects were sex-dependent for many genes, such as Prdm12, Oprd1, Isg20, Prl, Oxt, and Vip. The prevalence of differentially expressed genes annotated to proinflammatory and cell cycle processes suggests potential implications for synaptic plasticity and neuronal survival. The gene profiles affected by immune activation, distress, and sex pointed to the action of transcription factors SHOX2, STAT1, and REST. These findings underscore the importance of considering sex and postnatal challenges when studying causes of neurodevelopmental disorders and highlight the complexity of the "two-hit" hypothesis in understanding their etiology. Our study furthers the understanding of the intricate molecular responses in the hypothalamus to gestational immune activation and subsequent distress, shedding light on the sex-specific effects and the potential long-lasting consequences on pain perception, neuroendocrine regulation, and inflammatory processes.NEW & NOTEWORTHY The interaction of infection during gestation and insults later in life influences the molecular mechanisms in the hypothalamus that participate in pain sensation. The response of the hypothalamic transcriptome varies between sexes and can also affect synapses and immune signals. The findings from this study assist in the identification of agonists or antagonists that can guide pretranslational studies to ameliorate the effects of gestational insults interacting with postnatal challenges on physiological or behavioral disorders.

Effect of estradiol and IGF1 on glycogen synthesis in bovine uterine epithelial cells.

In brief: Glucose is an important nutrient for the endometrium and embryo during pregnancy. This study shows that estradiol (E2)/IGF1 signaling stimulates glycogen synthesis in the uterine epithelium of cows, which could provide glucose when needed. Abstract: Glycogen storage in the uterine epithelium peaks near estrus and is a potential source of glucose for the endometrium and embryos. However, the hormonal regulation of glycogen synthesis in the uterine epithelium is poorly understood. Our objective was to evaluate the effect of E2 and insulin-like growth factor 1 (IGF1) on glycogenesis in immortalized bovine uterine epithelial (BUTE) cells. Treatment of BUTE cells with E2 (0.1-10 nM) did not increase glycogen levels. However, treatment of BUTE cells with IGF1 (50 or 100 ng/mL) resulted in a >2-fold increase in glycogen. To determine if the uterine stroma produced IGF1 in response to E2, bovine uterine fibroblasts were treated with E2, which increased IGF1 levels. Immunohistochemistry showed higher levels of IGF1 in the stroma on day 1 than on day 11, which coincides with higher glycogen levels in the uterine epithelium. Western blots revealed that IGF1 treatment increased the levels of phospho-AKT, phospho-GSKß, hexokinase 1, and glycogen synthase in BUTE cells. Metabolomic (GC-MS) analysis showed that IGF1 increased 3-phosphoglycerate and lactate, potentially indicative of increased flux through glycolysis. We also found higher levels of N-acetyl-glucosamine and protein glycosylation after IGF1 treatment, indicating increased hexosamine biosynthetic pathway activity. In conclusion, IGF1 is produced by uterine fibroblasts due to E2, and IGF1 increases glucose metabolism and glycogenesis in uterine epithelial cells. Glycogen stored in the uterine epithelium due to E2/IGF1 signaling at estrus could provide glucose to the endometrium or be secreted into the uterine lumen as a component of histotroph.

Effects of maternal immune activation in porcine transcript isoforms of neuropeptide and receptor genes.

The prolonged effects of maternal immune activation in response stressors during gestation on the offspring's molecular pathways after birth are beginning to be understood. An association between maternal immune activation and neurodevelopmental and behavior disorders such as autism and schizophrenia spectrum disorders has been detected in long-term gene dysregulation. The incidence of alternative splicing among neuropeptides and neuropeptide receptor genes, critical cell-cell signaling molecules, associated with behavior may compromise the replicability of reported maternal immune activation effects at the gene level. This study aims to advance the understanding of the effect of maternal immune activation on transcript isoforms of the neuropeptide system (including neuropeptide, receptor and connecting pathway genes) underlying behavior disorders later in life. Recognizing the wide range of bioactive peptides and functional receptors stemming from alternative splicing, we studied the effects of maternal immune activation at the transcript isoform level on the hippocampus and amygdala of three-week-old pigs exposed to maternal immune activation due to viral infection during gestation. In the hippocampus and amygdala, 29 and 9 transcript isoforms, respectively, had maternal immune activation effects (P-value < 0.01). We demonstrated that the study of the effect of maternal immune activation on neuropeptide systems at the isoform level is necessary to expose opposite effects among transcript isoforms from the same gene. Genes were maternal immune activation effects have also been associated with neurodevelopmental and behavior disorders. The characterization of maternal immune activation effects at the transcript isoform level advances the understanding of neurodevelopmental disorders and identifies precise therapeutic targets.

Jersey steer ruminal papillae histology and nutrigenomics with diet changes.

The transition from a high forage to a high concentrate diet is an important milestone for beef cattle moving from a stocker system to the feedlot. However, little is known about how this transition affects the rumen epithelial gene expression. This study assessed the effects of the transition from a high forage to a high concentrate diet as well as the transition from a high concentrate to a high forage diet on a variety of genes as well as ruminal papillae morphology in rumen fistulated Jersey steers. Jersey steers (n = 5) were fed either a high forage diet (80% forage and 20% grain) and transitioned to a high concentrate diet (20% forage and 80% grain) or a high concentrate diet (40% forage and 60% grain) and transitioned to a high forage diet (100% forage). Papillae from the rumen were collected for histology and RT-qPCR analysis. Body weight had a tendency for significant difference (p = .08). Histological analysis did not show changes in papillae length or width in steers transitioning from a high forage to a high concentrate diet or vice versa (p > .05). Genes related to cell membrane structure (CLDN1, CLDN4, DSG1), fatty acid metabolism (CPT1A, ACADSB), glycolysis (PFKL), ketogenesis (HMGCL, HMGCS2, ACAT1), lactate/pyruvate (LDHA), oxidative stress (NQO1), tissue growth (AKT3, EGFR, EREG, IGFBP5, IRS1) and the urea cycle (SLC14A1) were considered in this study. Overall, genes related to fatty acid metabolism (ACADSB) and growth and development (AKT3 and IGFBP5) had a tendency for a treatment × day on trial interaction effect. These profiles may be indicators of rumen epithelial adaptations in response to changes in diet. In conclusion, these results indicate that changes in the composition of the diet can alter the expression of genes with specific functions in rumen epithelial metabolism.

Identification of prohormones and pituitary neuropeptides in the African cichlid, Astatotilapia burtoni.

BACKGROUND: Cichlid fishes have evolved remarkably diverse reproductive, social, and feeding behaviors. Cell-to-cell signaling molecules, notably neuropeptides and peptide hormones, are known to regulate these behaviors across vertebrates. This class of signaling molecules derives from prohormone genes that have undergone multiple duplications and losses in fishes. Whether and how subfunctionalization, neofunctionalization, or losses of neuropeptides and peptide hormones have contributed to fish behavioral diversity is largely unknown. Information on fish prohormones has been limited and is complicated by the whole genome duplication of the teleost ancestor. We combined bioinformatics, mass spectrometry-enabled peptidomics, and molecular techniques to identify the suite of neuropeptide prohormones and pituitary peptide products in Astatotilapia burtoni, a well-studied member of the diverse African cichlid clade. RESULTS: Utilizing the A. burtoni genome, we identified 148 prohormone genes, with 21 identified as a single copy and 39 with at least 2 duplicated copies. Retention of prohormone duplicates was therefore 41 %, which is markedly above previous reports for the genome-wide average in teleosts. Beyond the expected whole genome duplication, differences between cichlids and mammals can be attributed to gene loss in tetrapods and additional duplication after divergence. Mass spectrometric analysis of the pituitary identified 620 unique peptide sequences that were matched to 120 unique proteins. Finally, we used in situ hybridization to localize the expression of galanin, a prohormone with exceptional sequence divergence in cichlids, as well as the expression of a proopiomelanocortin, prohormone that has undergone an additional duplication in some bony fish lineages. CONCLUSION: We characterized the A. burtoni prohormone complement. Two thirds of prohormone families contain duplications either from the teleost whole genome duplication or a more recent duplication. Our bioinformatic and mass spectrometric findings provide information on a major vertebrate clade that will further our understanding of the functional ramifications of these prohormone losses, duplications, and sequence changes across vertebrate evolution. In the context of the cichlid radiation, these findings will also facilitate the exploration of neuropeptide and peptide hormone function in behavioral diversity both within A. burtoni and across cichlid and other fish species.

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken-the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

Advancing the understanding of behaviors associated with Bacille Calmette Guérin infection using multivariate analysis.

Behavioral indicators in the murine Bacille Calmette Guérin (BCG) model of inflammation have been studied individually; however, the variability of the behaviors across BCG levels and the mouse-to-mouse variation within BCG-treatment group are only partially understood. The objectives of this study were: (1) to gain a comprehensive understanding of sickness and depression-like behaviors in a BCG model of inflammation using multivariate approaches, and (2) to explore behavioral differences between BCG-treatment groups and among mice within group. Adult mice were challenged with either 0mg (saline), 5mg or 10mg of BCG (BCG-treatment groups: BCG0, BCG5, or BCG10, respectively) at Day 0 of the experiment. Sickness indicators included body weight changes between Day 0 and Day 2 and between Day 2 and Day 5, and horizontal locomotor activity and vertical activity (rearing) measured at Day 6. Depression-like indicators included duration of immobility in the forced swim test and in the tail suspension test at Day 6 and sucrose consumption in the sucrose preference test at Day 7. The simultaneous consideration of complementary sickness and depression-like indicators enabled a more precise characterization of behavioral changes associated with BCG-treatment and of mouse-to-mouse variation, relative to the analysis of indicators individually. Univariate and multivariate analyses confirmed differences between BCG-treatment groups in weight change early on the trial. Significant differences between BCG-treatment groups in depression-like behaviors were still measurable after Day 5. The potential for multivariate models to account for the correlation between behavioral indicators and to augment the analytical precision relative to univariate models was demonstrated both for sickness and for depression-like indicators. Unsupervised learning approaches revealed the complementary information provided by the sickness and depression-like indicators considered. Supervised learning approaches using cross-validation confirmed subtle differences between BCG-treatment groups and among mice within group identified by the consideration of sickness and depression-like indicators. These findings support the recommendation for multivariate and multidimensional analyses of sickness and depression-like indicators to augment the systemic understanding of the behavioral changes associated with infection.

Diet and endocrine effects on behavioral maturation-related gene expression in the pars intercerebralis of the honey bee brain.

Nervous and neuroendocrine systems mediate environmental conditions to control a variety of life history traits. Our goal was to provide mechanistic insights as to how neurosecretory signals mediate division of labor in the honey bee (Apis mellifera). Worker division of labor is based on a process of behavioral maturation by individual bees, which involves performing in-hive tasks early in adulthood, then transitioning to foraging for food outside the hive. Social and nutritional cues converge on endocrine factors to regulate behavioral maturation, but whether neurosecretory systems are central to this process is not known. To explore this, we performed transcriptomic profiling of a neurosecretory region of the brain, the pars intercerebralis (PI). We first compared PI transcriptional profiles for bees performing in-hive tasks and bees engaged in foraging. Using these results as a baseline, we then performed manipulative experiments to test whether the PI is responsive to dietary changes and/or changes in juvenile hormone (JH) levels. Results reveal a robust molecular signature of behavioral maturation in the PI, with a subset of gene expression changes consistent with changes elicited by JH treatment. In contrast, dietary changes did not induce transcriptomic changes in the PI consistent with behavioral maturation or JH treatment. Based on these results, we propose a new verbal model of the regulation of division of labor in honey bees in which the relationship between diet and nutritional physiology is attenuated, and in its place is a relationship between social signals and nutritional physiology that is mediated by JH.

Comparing label-free quantitative peptidomics approaches to characterize diurnal variation of peptides in the rat suprachiasmatic nucleus.

Mammalian circadian rhythm is maintained by the suprachiasmatic nucleus (SCN) via an intricate set of neuropeptides and other signaling molecules. In this work, peptidomic analyses from two times of day were examined to characterize variation in SCN peptides using three different label-free quantitation approaches: spectral count, spectra index and SIEVE. Of the 448 identified peptides, 207 peptides were analyzed by two label-free methods, spectral count and spectral index. There were 24 peptides with significant (adjusted p-value < 0.01) differential peptide abundances between daytime and nighttime, including multiple peptides derived from secretogranin II, cocaine and amphetamine regulated transcript, and proprotein convertase subtilisin/kexin type 1 inhibitor. Interestingly, more peptides were analyzable and had significantly different abundances between the two time points using the spectral count and spectral index methods than with a prior analysis using the SIEVE method with the same data. The results of this study reveal the importance of using the appropriate data analysis approaches for label-free relative quantitation of peptides. The detection of significant changes in so rich a set of neuropeptides reflects the dynamic nature of the SCN and the number of influences such as feeding behavior on circadian rhythm. Using spectral count and spectral index, peptide level changes are correlated to time of day, suggesting their key role in circadian function.

Comparative brain transcriptomic analyses of scouting across distinct behavioural and ecological contexts in honeybees.

Individual differences in behaviour are often consistent across time and contexts, but it is not clear whether such consistency is reflected at the molecular level. We explored this issue by studying scouting in honeybees in two different behavioural and ecological contexts: finding new sources of floral food resources and finding a new nest site. Brain gene expression profiles in food-source and nest-site scouts showed a significant overlap, despite large expression differences associated with the two different contexts. Class prediction and 'leave-one-out' cross-validation analyses revealed that a bee's role as a scout in either context could be predicted with 92.5% success using 89 genes at minimum. We also found that genes related to four neurotransmitter systems were part of a shared brain molecular signature in both types of scouts, and the two types of scouts were more similar for genes related to glutamate and GABA than catecholamine or acetylcholine signalling. These results indicate that consistent behavioural tendencies across different ecological contexts involve a mixture of similarities and differences in brain gene expression.

Bioinformatics for Prohormone and Neuropeptide Discovery.

Neuropeptides and peptide hormones are signaling molecules produced via complex posttranslational modifications of precursor proteins known as prohormones. Neuropeptides activate specific receptors and are associated with the regulation of physiological systems and behaviors. The identification of prohormones-and the neuropeptides created by these prohormones-from genomic assemblies has become essential to support the annotation and use of the rapidly growing number of sequenced genomes. Here we describe a well-validated methodology for identifying the prohormone complement from genomic assemblies that employs widely available public toolsets and databases. The uncovered prohormone sequences can then be screened for putative neuropeptides to enable accurate proteomic discovery and validation.

Epigenetic disruptions in the offspring hypothalamus in response to maternal infection.

DNA methylation is an epigenetic modification that can alter gene expression, and the incidence can vary across developmental stages, inflammatory conditions, and sexes. The effects of viral maternal viral infection and sex on the DNA methylation patterns were studied in the hypothalamus of a pig model of immune activation during development. DNA methylation at single-base resolution in regions of high CpG density was measured on 24 individual hypothalamus samples using reduced representation bisulfite sequencing. Differential over- and under-methylated sites were identified and annotated to proximal genes and corresponding biological processes. A total of 120 sites were differentially methylated (FDR-adjusted p-value < 0.05) between maternal infection or sex groups. Among the 66 sites differentially methylated between groups exposed to inflammatory signals and control, most sites were over-methylated in the challenged group and included sites in the promoter regions of genes SIRT3 and NRBP1. Among the 54 differentially methylated sites between females and males, most sites were over-methylated in females and included sites in the promoter region of genes TNC and EIF4G1. The analysis of the genes proximal to the differentially methylated sites suggested that biological processes potentially impacted include immune response, neuron migration and ensheathment, peptide signaling, adaptive thermogenesis, and tissue development. These results suggest that translational studies should consider that the prolonged effect of maternal infection during gestation may be enacted through epigenetic regulatory mechanisms that may differ between sexes.

Quantitative peptidomics for discovery of circadian-related peptides from the rat suprachiasmatic nucleus.

In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, 10 endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (adjusted p-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥ 30% increases at ZT 18, and the VGRPEWWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN.

Single nucleotide polymorphisms and haplotypes associated with feed efficiency in beef cattle.

BACKGROUND: General, breed- and diet-dependent associations between feed efficiency in beef cattle and single nucleotide polymorphisms (SNPs) or haplotypes were identified on a population of 1321 steers using a 50 K SNP panel. Genomic associations with traditional two-step indicators of feed efficiency - residual feed intake (RFI), residual average daily gain (RADG), and residual intake gain (RIG) - were compared to associations with two complementary one-step indicators of feed efficiency: efficiency of intake (EI) and efficiency of gain (EG). Associations uncovered in a training data set were evaluated on independent validation data set. A multi-SNP model was developed to predict feed efficiency. Functional analysis of genes harboring SNPs significantly associated with feed efficiency and network visualization aided in the interpretation of the results. RESULTS: For the five feed efficiency indicators, the numbers of general, breed-dependent, and diet-dependent associations with SNPs (P-value < 0.0001) were 31, 40, and 25, and with haplotypes were six, ten, and nine, respectively. Of these, 20 SNP and six haplotype associations overlapped between RFI and EI, and five SNP and one haplotype associations overlapped between RADG and EG. This result confirms the complementary value of the one and two-step indicators. The multi-SNP models included 89 SNPs and offered a precise prediction of the five feed efficiency indicators. The associations of 17 SNPs and 7 haplotypes with feed efficiency were confirmed on the validation data set. Nine clusters of Gene Ontology and KEGG pathway categories (mean P-value < 0.001) including, 9nucleotide binding; ion transport, phosphorous metabolic process, and the MAPK signaling pathway were overrepresented among the genes harboring the SNPs associated with feed efficiency. CONCLUSIONS: The general SNP associations suggest that a single panel of genomic variants can be used regardless of breed and diet. The breed- and diet-dependent associations between SNPs and feed efficiency suggest that further refinement of variant panels require the consideration of the breed and management practices. The unique genomic variants associated with the one- and two-step indicators suggest that both types of indicators offer complementary description of feed efficiency that can be exploited for genome-enabled selection purposes.

Influence of Maternal Immune Activation and Stressors on the Hippocampal Metabolome.

Prenatal stress often results in maternal immune activation (MIA) that can impact prenatal brain development, molecular processes, and substrates and products of metabolism that participate in physiological processes at later stages of life. Postnatal metabolic and immunological stressors can affect brain metabolites later in life, independently or in combination with prenatal stressors. The effects of prenatal and postnatal stressors on hippocampal metabolites were studied using a pig model of viral MIA exposed to immunological and metabolic stressors at 60 days of age using gas chromatography mass spectrometry. Postnatal stress and MIA elicited effects (FDR-adjusted p-value < 0.1) on fifty-nine metabolites, while eight metabolites exhibited an interaction effect. The hippocampal metabolites impacted by MIA or postnatal stress include 4-aminobutanoate (GABA), adenine, fumarate, glutamate, guanine, inosine, ornithine, putrescine, pyruvate, and xanthine. Metabolites affected by MIA or postnatal stress encompassed eight significantly (FDR-adjusted p-value < 0.1) enriched Kyoto Encyclopedia of Genes and Genomes Database (KEGG) pathways. The enriched arginine biosynthesis and glutathione metabolism pathways included metabolites that are also annotated for the urea cycle and polyamine biosynthesis pathways. Notably, the prenatal and postnatal challenges were associated with disruption of the glutathione metabolism pathway and changes in the levels of glutamic acid, glutamate, and purine nucleotide metabolites that resemble patterns elicited by drugs of abuse and may underlie neuroinflammatory processes. The combination of MIA and postnatal stressors also supported the double-hit hypothesis, where MIA amplifies the impact of stressors later in life, sensitizing the hippocampus of the offspring to future challenges. The metabolites and pathways characterized in this study offer evidence of the role of immunometabolism in understanding the impact of MIA and stressors later in life on memory, spatial navigation, neuropsychiatric disorders, and behavioral disorders influenced by the hippocampus.

Genes Participating in the Ensheathment of Neurons Are Affected by Postnatal Stress and Maternal Immune Activation in the Pituitary Gland.

Immune challenges during gestation are associated with neurodevelopmental disorders and can interact with stress later in life. The pituitary gland participates in endocrine- and immune-related processes that influence development, growth, and reproduction and can modulate physiological and behavioral responses to challenges. The objective of this study was to investigate the effect of stressors at different time points on the molecular mechanisms of the pituitary gland and detect sex differences. RNA sequencing was used to profile the pituitary glands of female and male pigs exposed to weaning stress and virally induced maternal immune activation (MIA), relative to unchallenged groups. Significant effects (FDR-adjusted p-value < 0.05) of MIA and weaning stress were detected in 1829 and 1014 genes, respectively. Of these, 1090 genes presented significant interactions between stressors and sex. The gene ontology biological process of the ensheathment of neurons (GO:0007272), substance abuse, and immuno-related pathways, including the measles disease (ssc05162), encompasses many genes with profiles impacted by MIA and weaning stress. A gene network analysis highlighted the under-expression of myelin protein zero (Mpz) and inhibitors of DNA binding 4 (Id4) among the non-stressed males exposed to MIA, relative to the control and non-MIA males exposed to weaning stress, relative to non-stressed pigs. The detection of changes in the molecular mechanisms of the pituitary gland could advance our understanding of disruptions in the formation of the myelin sheath and the transmission of neuron-to-neuron signals in behavioral disorders associated with maternal immune activation and stress.

Evaluation of database search programs for accurate detection of neuropeptides in tandem mass spectrometry experiments.

Neuropeptide identification in mass spectrometry experiments using database search programs developed for proteins is challenging. Unlike proteins, the detection of the complete sequence using a single spectrum is required to identify neuropeptides or prohormone peptides. This study compared the performance of three open-source programs used to identify proteins, OMSSA, X!Tandem and Crux, to identify prohormone peptides. From a target database of 7850 prohormone peptides, 23550 query spectra were simulated across different scenarios. Crux was the only program that correctly matched all peptides regardless of p-value and at p-value<1×10(-2), 33%, 64%, and >75%, of the 5, 6, and ≥7 amino acid-peptides were detected. Crux also had the best performance in the identification of peptides from chimera spectra and in a variety of missing ion scenarios. OMSSA, X!Tandem and Crux correctly detected 98.9% (99.9%), 93.9% (97.4%) and 88.7% (98.3%) of the peptides at E- or p-value<1×10(-6) (<1×10(-2)), respectively. OMSSA and X!Tandem outperformed the other programs in significance level and computational speed, respectively. A consensus approach is not recommended because some prohormone peptides were only identified by one program.

First survey and functional annotation of prohormone and convertase genes in the pig.

BACKGROUND: The pig is a biomedical model to study human and livestock traits. Many of these traits are controlled by neuropeptides that result from the cleavage of prohormones by prohormone convertases. Only 45 prohormones have been confirmed in the pig. Sequence homology can be ineffective to annotate prohormone genes in sequenced species like the pig due to the multifactorial nature of the prohormone processing. The goal of this study is to undertake the first complete survey of prohormone and prohormone convertases genes in the pig genome. These genes were functionally annotated based on 35 gene expression microarray experiments. The cleavage sites of prohormone sequences into potentially active neuropeptides were predicted. RESULTS: We identified 95 unique prohormone genes, 2 alternative calcitonin-related sequences, 8 prohormone convertases and 1 cleavage facilitator in the pig genome 10.2 assembly and trace archives. Of these, 11 pig prohormone genes have not been reported in the UniProt, UniGene or Gene databases. These genes are intermedin, cortistatin, insulin-like 5, orexigenic neuropeptide QRFP, prokineticin 2, prolactin-releasing peptide, parathyroid hormone 2, urocortin, urocortin 2, urocortin 3, and urotensin 2-related peptide. In addition, a novel neuropeptide S was identified in the pig genome correcting the previously reported pig sequence that is identical to the rabbit sequence. Most differentially expressed prohormone genes were under-expressed in pigs experiencing immune challenge relative to the un-challenged controls, in non-pregnant relative to pregnant sows, in old relative to young embryos, and in non-neural relative to neural tissues. The cleavage prediction based on human sequences had the best performance with a correct classification rate of cleaved and non-cleaved sites of 92% suggesting that the processing of prohormones in pigs is similar to humans. The cleavage prediction models did not find conclusive evidence supporting the production of the bioactive neuropeptides urocortin 2, urocortin 3, torsin family 2 member A, tachykinin 4, islet amyloid polypeptide, and calcitonin receptor-stimulating peptide 2 in the pig. CONCLUSIONS: The present genomic and functional characterization supports the use of the pig as an effective animal model to gain a deeper understanding of prohormones, prohormone convertases and neuropeptides in biomedical and agricultural research.

Quantitative peptidomics reveal brain peptide signatures of behavior.

The honey bee genome predicts approximately 100 peptides from 36 prohormones, but the functions of many of these peptides are unknown. We used differential isotope labeling combined with mass spectrometric analysis to quantify approximately 50% of known bee brain peptides in the context of foraging, with 8 showing robust and dynamic regulation. Some showed differences in brain abundance as a function of experience; specifically, nectar and pollen collection led to quick changes in abundance. These changes were related to the act of food collection, not ingestion, because foragers bring food back to the hive for storage rather than eating it themselves. Other peptide differences in brain abundance were seen in bees that either flew to a nectar feeder or a pollen feeder, but did not yet collect any food. These differences likely reflect well-known predispositions of some bees to collect either nectar or pollen, but not both. Tachykinin, PBAN, and sNPF were among the peptides with the strongest changes in association with nectar and pollen foraging. These peptides are known to be involved in regulating food intake in solitary insects, suggesting an evolutionary connection between that behavior and social foraging. These results demonstrate that it is now possible to use quantitative peptidomics to help determine which brain peptides are bioactive and to elucidate their function in the regulation of behavior.

Honey bee aggression supports a link between gene regulation and behavioral evolution.

A prominent theory states that animal phenotypes arise by evolutionary changes in gene regulation, but the extent to which this theory holds true for behavioral evolution is not known. Because "nature and nurture" are now understood to involve hereditary and environmental influences on gene expression, we studied whether environmental influences on a behavioral phenotype, i.e., aggression, could have evolved into inherited differences via changes in gene expression. Here, with microarray analysis of honey bees, we show that aggression-related genes with inherited patterns of brain expression are also environmentally regulated. There were expression differences in the brain for hundreds of genes between the highly aggressive Africanized honey bee compared with European honey bee (EHB) subspecies. Similar results were obtained for EHB in response to exposure to alarm pheromone (which provokes aggression) and when comparing old and young bees (aggressive tendencies increase with age). There was significant overlap of the gene lists generated from these three microarray experiments. Moreover, there was statistical enrichment of several of the same cis regulatory motifs in promoters of genes on all three gene lists. Aggression shows a remarkably robust brain molecular signature regardless of whether it occurs because of inherited, age-related, or environmental (social) factors. It appears that one element in the evolution of different degrees of aggressive behavior in honey bees involved changes in regulation of genes that mediate the response to alarm pheromone.