A hydrolyzed lipid blend diet promotes myelination in neonatal piglets in a region and concentration-dependent manner.

The impact of early life nutrition on myelin development is of interest given that cognitive and behavioral function depends on proper myelination. Evidence shows that myelination can be altered by dietary lipid, but most of these studies have been performed in the context of disease or impairment. Here, we assessed the effects of lipid blends containing various levels of a hydrolyzed fat (HF) system on myelination in healthy piglets. Piglets were sow-reared, fed a control diet, or a diet containing 12%, 25%, or 53% HF consisting of cholesterol, fatty acids, monoglycerides, and phospholipid from lecithin. At postnatal day 28/29, magnetic resonance imaging (MRI) was performed to assess changes to brain development, followed by brain collection for microscopic analyses of myelin in targeted regions using CLARITY tissue clearing, immunohistochemistry, and electron microscopy techniques. Sow-reared piglets exhibited the highest overall brain white matter volume by MRI. However, a 25% HF diet resulted in the greatest total myelin density in the prefrontal cortex based on 3D modeling analysis of myelinated filaments. Nodal gap length and g-ratio were inversely correlated with percentage of HF in the corpus callosum, as well as in the PFC and internal capsule for g-ratio, indicating that a 53% HF diet resulted in the thickest myelin per axon and a 0% HF control diet the thinnest in specific brain regions. These findings indicate that HF promoted myelination in the neonatal piglet in a region- and concentration-dependent manner.

Impact of neonatal iron deficiency on hippocampal DNA methylation and gene transcription in a porcine biomedical model of cognitive development.

BACKGROUND: Iron deficiency is a common childhood micronutrient deficiency that results in altered hippocampal function and cognitive disorders. However, little is known about the mechanisms through which neonatal iron deficiency results in long lasting alterations in hippocampal gene expression and function. DNA methylation is an epigenetic mark involved in gene regulation and altered by environmental factors. In this study, hippocampal DNA methylation and gene expression were assessed via reduced representation bisulfite sequencing and RNA-seq on samples from a previous study reporting reduced hippocampal-based learning and memory in a porcine biomedical model of neonatal iron deficiency. RESULTS: In total 192 differentially expressed genes (DEGs) were identified between the iron deficient and control groups. GO term and pathway enrichment analysis identified DEGs associated with hypoxia, angiogenesis, increased blood brain barrier (BBB) permeability, and altered neurodevelopment and function. Of particular interest are genes previously implicated in cognitive deficits and behavioral disorders in humans and mice, including HTR2A, HTR2C, PAK3, PRSS12, and NETO1. Altered genome-wide DNA methylation was observed across 0.5 million CpG and 2.4 million non-CpG sites. In total 853 differentially methylated (DM) CpG and 99 DM non-CpG sites were identified between groups. Samples clustered by group when comparing DM non-CpG sites, suggesting high conservation of non-CpG methylation in response to neonatal environment. In total 12 DM sites were associated with 9 DEGs, including genes involved in angiogenesis, neurodevelopment, and neuronal function. CONCLUSIONS: Neonatal iron deficiency leads to altered hippocampal DNA methylation and gene regulation involved in hypoxia, angiogenesis, increased BBB permeability, and altered neurodevelopment and function. Together, these results provide new insights into the mechanisms through which neonatal iron deficiency results in long lasting reductions in cognitive development in humans.

Adult porcine genome-wide DNA methylation patterns support pigs as a biomedical model.

BACKGROUND: Pigs (Sus scrofa) provide relevant biomedical models to dissect complex diseases due to their anatomical, genetic, and physiological similarities with humans. Aberrant DNA methylation has been linked to many of these diseases and is associated with gene expression; however, the functional similarities and differences between porcine and human DNA methylation patterns are largely unknown. METHODS: DNA and RNA was isolated from eight tissue samples (fat, heart, kidney, liver, lung, lymph node, muscle, and spleen) from the adult female Duroc utilized for the pig genome sequencing project. Reduced representation bisulfite sequencing (RRBS) and RNA-seq were performed on an Illumina HiSeq2000. RRBS reads were aligned using BSseeker2, and only sites with a minimum depth of 10 reads were used for methylation analysis. RNA-seq reads were aligned using Tophat, and expression analysis was performed using Cufflinks. In addition, SNP calling was performed using GATK for targeted control and whole genome sequencing reads for CpG site validation and allelic expression analysis, respectively. RESULTS: Analysis on the influence of DNA variation in methylation calling revealed a reduced effectiveness of WGS datasets in covering CpG rich regions, as well as the usefulness of a targeted control library for SNP detection. Analysis of over 500,000 CpG sites demonstrated genome wide methylation patterns similar to those observed in humans, including reduced methylation within CpG islands and at transcription start sites (TSS), X chromosome inactivation, and anticorrelation of TSS CpG methylation with gene expression. In addition, a positive correlation between TSS CpG density and expression, and a negative correlation between TSS TpG density and expression were demonstrated. Low but non-random non-CpG methylation (<1%) was also detected in all non-neuronal somatic tissues, with differences in tissue clustering observed based on CpG and non-CpG methylation patterns. Finally, allele specific expression analysis revealed enrichment of genes involved in metabolic and regulatory processes. DISCUSSION: These results provide transcriptional and DNA methylation datasets for the biomedical community that are directly relatable to current genomic resources. In addition, the correlation between TSS CpG density and expression suggests increased mutation rates at CpG sites play a significant role in adaptive evolution by reducing CpG density at TSS over time, resulting in higher methylation levels in these regions and more permanent changes to lower gene expression. This is proposed to occur predominantly through deamination of 5-methylcytosine to thymidine, resulting in the replacement of CpG with TpG sites in these regions, as indicated by the increased TSS TpG density observed in non-expressed genes, resulting in a negative correlation between expression and TSS TpG density. CONCLUSIONS: This study provides baseline methylation and gene transcription profiles for a healthy adult pig, reports similar patterns to those observed in humans, and supports future porcine studies related to human disease and development. Additionally, the observed reduced CpG and increased TpG density at TSS of lowly expressed genes suggests DNA methylation plays a significant role in adaptive evolution through more permanent changes to lower gene expression.

Oncopig bladder cancer cells recapitulate human bladder cancer treatment responses in vitro.

Introduction: Bladder cancer is a common neoplasia of the urinary tract that holds the highest cost of lifelong treatment per patient, highlighting the need for a continuous search for new therapies for the disease. Current bladder cancer models are either imperfect in their ability to translate results to clinical practice (mouse models), or rare and not inducible (canine models). Swine models are an attractive alternative to model the disease due to their similarities with humans on several levels. The Oncopig Cancer Model has been shown to develop tumors that closely resemble human tumors. However, urothelial carcinoma has not yet been studied in this platform. Methods: We aimed to develop novel Oncopig bladder cancer cell line (BCCL) and investigate whether these urothelial swine cells mimic human bladder cancer cell line (5637 and T24) treatment-responses to cisplatin, doxorubicin, and gemcitabine in vitro. Results: Results demonstrated consistent treatment responses between Oncopig and human cells in most concentrations tested (p>0.05). Overall, Oncopig cells were more predictive of T24 than 5637 cell therapeutic responses. Microarray analysis also demonstrated similar alterations in expression of apoptotic (GADD45B and TP53INP1) and cytoskeleton-related genes (ZMYM6 and RND1) following gemcitabine exposure between 5637 (human) and Oncopig BCCL cells, indicating apoptosis may be triggered through similar signaling pathways. Molecular docking results indicated that swine and humans had similar Dg values between the chemotherapeutics and their target proteins. Discussion: Taken together, these results suggest the Oncopig could be an attractive animal to model urothelial carcinoma due to similarities in in vitro therapeutic responses compared to human cells.

Effects of an inulin fiber diet on the gut microbiome, colon, and inflammatory biomarkers in aged mice.

Due to the increasing human life expectancy and limited supply of healthcare resources, strategies to promote healthy aging and reduce associated functional deficits are of public health importance. The gut microbiota, which remodels with age, has been identified as a significant contributor to the aging process that is modifiable by diet. Since prebiotic dietary components such as inulin have been shown to impart positive benefits with regards to aging, this study used C57Bl6 mice to investigate whether 8 weeks on a 2.5 % inulin enhanced AIN-93M 1 % cellulose diet could offset age-associated changes in gut microbiome composition and markers of colon health and systemic inflammation in comparison to a AIN 93M 1 % cellulose diet with 0 % inulin. Our results demonstrated that, in both age groups, dietary inulin significantly increased production of butyrate in the cecum and induced changes in the community structure of the gut microbiome but did not significantly affect systemic inflammation or other markers of gastrointestinal health. Aged mice had different and less diverse microbiomes when compared to adult mice and were less sensitive to inulin-induced microbiome community shifts, evidenced by longitudinal differences in differentially abundant taxa and beta diversity. In aged mice, inulin restored potentially beneficial taxa including Bifidobacterium and key butyrate producing genera (e.g. Faecalibaculum). Despite inducing notable taxonomic changes, however, the 2.5 % inulin diet reduced alpha diversity in both age groups and failed to reduce overall community compositional differences between age groups. In conclusion, a 2.5 % inulin enhanced diet altered gut microbiome α and ß diversity, composition, and butyrate production in both adult and aged mice, with more potent effects on ß diversity and greater number of taxa significantly altered in adult mice. However, significant benefits in age-associated changes in systemic inflammation or intestinal outcomes were not detected.

Association of the porcine transforming growth factor beta type I receptor (TGFBR1) gene with growth and carcass traits.

BACKGROUND: Growth and carcass traits are of great economic importance in livestock production. A large number of quantitative trait loci (QTL) have been identified for growth and carcass traits on porcine chromosome one (SSC1). A key positional candidate for this chromosomal region is TGFBR1 (transforming growth factor beta type I receptor). This gene plays a key role in inherited disorders at cardiovascular, craniofacial, neurocognitive, and skeletal development in mammals. RESULTS: In this study, 27 polymorphic SNPs in the porcine TGFBR1 gene were identified on the University of Illinois Yorkshire × Meishan resource population. Three SNPs (SNP3, SNP43, SNP64) representing major polymorphic patterns of the 27 SNPs in F1 and F0 individuals of the Illinois population were selected for analyses of QTL association and genetic diversity. An association analysis for growth and carcass traits was completed using these three representative SNPs in the Illinois population with 298 F2 individuals and a large commercial population of 1008 animals. The results indicate that the TGFBR1 gene polymorphism (SNP64) is significantly associated (p < 0.05) with growth rates including average daily gains between birth and 56 kg (p = 0.049), between 5.5 and 56 kg (p = 0.024), between 35 and 56 kg (p = 0.021). Significant associations (p < 0.05) were also identified between TGFBR1 gene polymorphisms (SNP3/SNP43) and carcass traits including loin-eye-area (p = 0.022) in the Illinois population, and back-fat thickness (p = 0.0009), lean percentage (p = 0.0023) and muscle color (p = 0.021) in the commercial population. These three SNPs were also used to genotype a diverse panel of 130 animals representing 11 pig breeds. Alleles SNP3_T and SNP43_G were fixed in Pietrain and Sinclair pig breeds. SNP64_G allele was uniquely identified in Chinese Meishan pigs. Strong evidence of association (p < 0.01) between both SNP3 and SNP64 alleles and reproductive traits including gestation length and number of corpora lutea were also observed in the Illinois population. CONCLUSION: This study gives the first evidence of association between the porcine TGFBR1 gene and traits of economic importance and provides support for using TGFBR1 markers for pig breeding and selection programs. The genetic diversities in different pig breeds would be helpful to understand the genetic background and migration of the porcine TGFBR1 gene.

Terpenoid Backbone Biosynthesis among Pig Hippocampal Pathways Impacted by Stressors.

Neurogenomic changes induced by maternal immune activation (MIA) during gestation and the social stress of weaning can alter brain plasticity in the hippocampus of offspring. The present study furthers the understanding of how these stressors impact hippocampus gene networks. The hippocampus transcriptome was profiled in pigs that were either exposed to MIA or not and were weaned or nursed. Overall, 1576 genes were differentially expressed (FDR-adjusted p-value < 0.05 and |log2 (fold change between pig groups)| > 1.2) in response to the main and interacting effects of MIA, weaning, and sex. Functional analysis identified 17 enriched immunological and neurological pathways in the Kyoto Encyclopedia of Genes and Genomes database. The enrichment of the terpenoid backbone biosynthesis pathway was characterized by genes under-expressed in MIA relative to non-MIA exposed, males relative to females, and weaned relative to nursed pigs. On the other hand, the enrichment of drug addiction pathways was characterized by gene over-expression in MIA relative to non-exposed pigs. Our results indicate that weaning and sex can modify the effects of MIA on the offspring hippocampus. This knowledge can aid in precise identification of molecular targets to reduce the prolonged effects of pre- and postnatal stressors.

Hippocampal Changes Elicited by Metabolic and Inflammatory Stressors following Prenatal Maternal Infection.

The hippocampus participates in spatial navigation and behavioral processes, displays molecular plasticity in response to environmental challenges, and can play a role in neuropsychiatric diseases. The combined effects of inflammatory prenatal and postnatal challenges can disrupt the hippocampal gene networks and regulatory mechanisms. Using a proven pig model of viral maternal immune activation (MIA) matched to controls and an RNA-sequencing approach, the hippocampal transcriptome was profiled on two-month-old female and male offspring assigned to fasting, mimetic viral, or saline treatments. More than 2600 genes presented single or combined effects (FDR-adjusted p-value < 0.05) of MIA, postnatal stress, or sex. Biological processes and pathways encompassing messenger cyclic adenosine 3',5'-monophosphate (cAMP) signaling were enriched with genes including gastric inhibitory polypeptide receptor (GIPR) predominantly over-expressed in the MIA-exposed fasting males relative to groups that differed in sex, prenatal or postnatal challenge. While this pattern was amplified in fasting offspring, the postnatal inflammatory challenge appeared to cancel out the effects of the prenatal challenge. The transcription factors C-terminal binding protein 2 (CTBP2), RE1 silencing transcription factor (REST), signal transducer and activator of transcription 1 (STAT1), and SUZ12 polycomb repressive complex 2 subunit were over-represented among the genes impacted by the prenatal and postnatal factors studied. Our results indicate that one environmental challenge can influence the effect of another challenge on the hippocampal transcriptome. These findings can assist in the identification of molecular targets to ameliorate the effects of pre-and post-natal stressors on hippocampal-associated physiology and behavior.

Prefrontal Cortex Response to Prenatal Insult and Postnatal Opioid Exposure.

The influence of proinflammatory challenges, such as maternal immune activation (MIA) or postnatal exposure to drugs of abuse, on brain molecular pathways has been reported. On the other hand, the simultaneous effects of MIA and drugs of abuse have been less studied and sometimes offered inconsistent results. The effects of morphine exposure on a pig model of viral-elicited MIA were characterized in the prefrontal cortex of males and females using RNA-sequencing and gene network analysis. Interacting and main effects of morphine, MIA, and sex were detected in approximately 2000 genes (false discovery rate-adjusted p-value < 0.05). Among the enriched molecular categories (false discovery rate-adjusted p-value < 0.05 and −1.5 > normalized enrichment score > 1.5) were the cell adhesion molecule pathways associated with inflammation and neuronal development and the long-term depression pathway associated with synaptic strength. Gene networks that integrate gene connectivity and expression profiles displayed the impact of morphine-by-MIA interaction effects on the pathways. The cell adhesion molecules and long-term depression networks presented an antagonistic effect between morphine and MIA. The differential expression between the double-challenged group and the baseline saline-treated Controls was less extreme than the individual challenges. The previous findings advance the knowledge about the effects of prenatal MIA and postnatal morphine exposure on the prefrontal cortex pathways.

Label-free screening of brain tissue myelin content using phase imaging with computational specificity (PICS).

Inadequate myelination in the central nervous system is associated with neurodevelopmental complications. Thus, quantitative, high spatial resolution measurements of myelin levels are highly desirable. We used spatial light interference microcopy (SLIM), a highly sensitive quantitative phase imaging (QPI) technique, to correlate the dry mass content of myelin in piglet brain tissue with dietary changes and gestational size. We combined SLIM micrographs with an artificial intelligence (AI) classifying model that allows us to discern subtle disparities in myelin distributions with high accuracy. This concept of combining QPI label-free data with AI for the purpose of extracting molecular specificity has recently been introduced by our laboratory as phase imaging with computational specificity. Training on 8000 SLIM images of piglet brain tissue with the 71-layer transfer learning model Xception, we created a two-parameter classification to differentiate gestational size and diet type with an accuracy of 82% and 80%, respectively. To our knowledge, this type of evaluation is impossible to perform by an expert pathologist or other techniques.

The Combined Effect of Weaning Stress and Immune Activation during Pig Gestation on Serum Cytokine and Analyte Concentrations.

Weaning stress can elicit changes in the metabolic, hormone and immune systems of pigs and interact with prolonged disruptions stemming from maternal immune activation (MIA) during gestation. The present study advances the characterization of the combined effects of weaning stress and MIA on blood chemistry, immune and hormone indicators that inform on the health of pigs. Three-week-old female and male offspring of control gilts or gilts infected with the porcine reproductive and respiratory syndrome virus were allocated to weaned or nursed groups. The anion gap and bilirubin profiles suggest that MIA enhances tolerance to the effects of weaning stress. Interleukin 1 beta and interleukin 2 were highest among weaned MIA females, and cortisol was higher among weaned relative to nursed pigs across sexes. Canonical discriminant analysis demonstrated that weaned and nursed pigs have distinct chemistry profiles, whereas MIA and control pigs have distinct cytokine profiles. The results from this study can guide management practices that recognize the effects of the interaction between MIA and weaning stress on the performance and health of pigs.

Copper-Binding Peptides Attenuate Microglia Inflammation through Suppression of NF-kB Pathway.

SCOPE: Activation of microglia, the resident immune cells of the central nervous system, has been related to the etiology and progression of neurodegenerative diseases; thus, finding novel approaches to suppress the neuroinflammatory process is of utmost relevance. METHODS AND RESULTS: The anti-inflammatory activity of whey Cu-, Fe-, and Zn-binding peptides and their possible underlying mechanism of action were evaluated in microglia. Whey metal-binding peptides decreased nitric oxide production and tumor necrosis factor α (TNF-α) at mRNA and protein levels by stimulated BV-2 microglia in comparison to the control with no peptide treatment. The hydrophobicity, specific sequences, and possible synergistic effects seem to play a role. Cu-binding peptides (Cu-bp) presented anti-inflammatory activity both in BV-2 and primary microglia cultures. These peptides exert their action by suppressing nuclear factor kappa B (NF-kB) pathway since nuclear translocation of NF-kB p65 is decreased by roughly 30% upon Cu-bp treatment. Specific sequences identified in Cu-bp showed high affinity to bind NF-kB p65 by molecular docking (up to -8.8 kcal mol-1 ), corroborating the immunofluorescence studies. CONCLUSION: Cu-bp represent food-derived peptides that may be useful for neuroprotective purposes. Chelation of copper excess in the CNS and the bioavailability of such peptides, as well as their behavior in in vivo models, deserve further research for future applications.

Impact of Weaning and Maternal Immune Activation on the Metabolism of Pigs.

Weaning wields environmental, social, and nutritional stresses that are detectable in the blood metabolite levels of the offspring. Prenatal stress in the form of maternal immune activation (MIA) in response to infection, which is associated with health and behavior disorders, also elicits prolonged changes in blood and brain cytokine and metabolite levels of the offspring. The goal of this study was to investigate the effects of weaning and MIA on the offspring's liver function to advance the understanding of the impact of stressors on peripheral and central nervous systems, physiology, and health. Gas chromatography-mass spectrometry analysis was used to compare the level of hepatic metabolites from 22-day-old pigs (n = 48) evenly distributed among weaning (nursed or weaned), viral MIA exposure (yes or no), and sexes. Weaning effects were detected on 38 metabolites at p-value < 0.05 (28 metabolites at FDR p-value < 0.05), and sex-dependent MIA effects were detected on 11 metabolites. Multiple intermediate and final products of the enriched (FDR p-value < 0.05) glycolysis and gluconeogenesis and pentose phosphate pathways were over-abundant in nursed relative to weaned pigs. The enriched pathways confirm the impact of weaning on hepatic metabolic shift, oxidative stress, and inflammation. Higher levels of the glucogenic amino acid histidine are observed in pigs exposed to MIA relative to controls, suggesting that the role of this metabolite in modulating inflammation may supersede the role of this amino acid as an energy source. The lower levels of cholesterol detected in MIA pigs are consistent with hypocholesterolemia profiles detected in individuals with MIA-related behavior disorders. Our findings underline the impact of weaning and MIA stressors on hepatic metabolites that can influence peripheral and central nervous system metabolic products associated with health and behavior disorders.

Biochemistry and Immune Biomarkers Indicate Interacting Effects of Pre- and Postnatal Stressors in Pigs across Sexes.

The effects of maternal immune activation (MIA) elicited by a prenatal stressor and postnatal metabolic or immune stressors on chemical and inflammatory biomarkers were studied in male and female pigs. Pigs exposed to MIA elicited by porcine reproductive and respiratory syndrome virus and matching controls were assigned at two months of age to fasting stress, immune stress, or a saline group. The serum levels of over 30 chemistry and immune analytes were studied. Significantly low levels of blood urea nitrogen were detected in females exposed to MIA, while the highest creatinine levels were identified in fasting females exposed to MIA. The levels of interferon gamma and interleukin 8 were highest in pigs exposed to postnatal immune challenge. The profiles suggest that MIA may sensitize pigs to postnatal stressors for some indicators while making them more tolerant of other stressors. Effectiveness of practices to ameliorate the impact of postnatal stressors on the physiology of the pig could be enhanced by considering the prenatal stress circumstances.

Interacting impact of maternal inflammatory response and stress on the amygdala transcriptome of pigs.

Changes at the molecular level capacitate the plasticity displayed by the brain in response to stress stimuli. Weaning stress can trigger molecular changes that influence the physiology of the offspring. Likewise, maternal immune activation (MIA) during gestation has been associated with behavior disorders and molecular changes in the amygdala of the offspring. This study advances the understanding of the effects of pre- and postnatal stressors in amygdala gene networks. The amygdala transcriptome was profiled on female and male pigs that were either exposed to viral-elicited MIA or not and were weaned or nursed. Overall, 111 genes presented interacting or independent effects of weaning, MIA, or sex (FDR-adjusted P-value <0.05). PIGY upstream reading frame and orthodenticle homeobox 2 are genes associated with MIA-related neurological disorders, and presented significant under-expression in weaned relative to nursed pigs exposed to MIA, with a moderate pattern observed in non-MIA pigs. Enriched among the genes presenting highly over- or under-expression profiles were 24 Kyoto Encyclopedia of Genes and Genomes pathways including inflammation, and neurological disorders. Our results indicate that MIA and sex can modulate the effect of weaning stress on the molecular mechanisms in the developing brain. Our findings can help identify molecular targets to ameliorate the effects of pre- and postnatal stressors on behaviors regulated by the amygdala such as aggression and feeding.

Quantifying myelin content in brain tissue using color Spatial Light Interference Microscopy (cSLIM).

Deficient myelination of the brain is associated with neurodevelopmental delays, particularly in high-risk infants, such as those born small in relation to their gestational age (SGA). New methods are needed to further study this condition. Here, we employ Color Spatial Light Interference Microscopy (cSLIM), which uses a brightfield objective and RGB camera to generate pathlength-maps with nanoscale sensitivity in conjunction with a regular brightfield image. Using tissue sections stained with Luxol Fast Blue, the myelin structures were segmented from a brightfield image. Using a binary mask, those portions were quantitatively analyzed in the corresponding phase maps. We first used the CLARITY method to remove tissue lipids and validate the sensitivity of cSLIM to lipid content. We then applied cSLIM to brain histology slices. These specimens are from a previous MRI study, which demonstrated that appropriate for gestational age (AGA) piglets have increased internal capsule myelination (ICM) compared to small for gestational age (SGA) piglets and that a hydrolyzed fat diet improved ICM in both. The identity of samples was blinded until after statistical analyses.

Altered Hippocampal Epigenetic Regulation Underlying Reduced Cognitive Development in Response to Early Life Environmental Insults.

The hippocampus is involved in learning and memory and undergoes significant growth and maturation during the neonatal period. Environmental insults during this developmental timeframe can have lasting effects on brain structure and function. This study assessed hippocampal DNA methylation and gene transcription from two independent studies reporting reduced cognitive development stemming from early life environmental insults (iron deficiency and porcine reproductive and respiratory syndrome virus (PRRSv) infection) using porcine biomedical models. In total, 420 differentially expressed genes (DEGs) were identified between the reduced cognition and control groups, including genes involved in neurodevelopment and function. Gene ontology (GO) terms enriched for DEGs were associated with immune responses, angiogenesis, and cellular development. In addition, 116 differentially methylated regions (DMRs) were identified, which overlapped 125 genes. While no GO terms were enriched for genes overlapping DMRs, many of these genes are known to be involved in neurodevelopment and function, angiogenesis, and immunity. The observed altered methylation and expression of genes involved in neurological function suggest reduced cognition in response to early life environmental insults is due to altered cholinergic signaling and calcium regulation. Finally, two DMRs overlapped with two DEGs, VWF and LRRC32, which are associated with blood brain barrier permeability and regulatory T-cell activation, respectively. These results support the role of altered hippocampal DNA methylation and gene expression in early life environmentally-induced reductions in cognitive development across independent studies.

Lasting and Sex-Dependent Impact of Maternal Immune Activation on Molecular Pathways of the Amygdala.

The prolonged and sex-dependent impact of maternal immune activation (MIA) during gestation on the molecular pathways of the amygdala, a brain region that influences social, emotional, and other behaviors, is only partially understood. To address this gap, we investigated the effects of viral-elicited MIA during gestation on the amygdala transcriptome of pigs, a species of high molecular and developmental homology to humans. Gene expression levels were measured using RNA-Seq on the amygdala for 3-week-old female and male offspring from MIA and control groups. Among the 403 genes that exhibited significant MIA effect, a prevalence of differentially expressed genes annotated to the neuroactive ligand-receptor pathway, glutamatergic functions, neuropeptide systems, and cilium morphogenesis were uncovered. Genes in these categories included corticotropin-releasing hormone receptor 2, glutamate metabotropic receptor 4, glycoprotein hormones, alpha polypeptide, parathyroid hormone 1 receptor, vasointestinal peptide receptor 2, neurotensin, proenkephalin, and gastrin-releasing peptide. These categories and genes have been associated with the MIA-related human neurodevelopmental disorders, including schizophrenia and autism spectrum disorders. Gene network reconstruction highlighted differential vulnerability to MIA effects between sexes. Our results advance the understanding necessary for the development of multifactorial therapies targeting immune modulation and neurochemical dysfunction that can ameliorate the effects of MIA on offspring behavior later in life.

Long-Lasting Impact of Maternal Immune Activation and Interaction With a Second Immune Challenge on Pig Behavior.

The combined effects on pig behavior of maternal immune challenge during gestation followed by a second immune challenge later in life have not been studied. Porcine reproductive and respiratory syndrome virus (PRRSV) infection during gestation can elicit maternal immune activation (MIA) yet the interactions with the offspring response to a second immune challenge after birth remains unexplored. Knowledge on the response to viral challenges in rodents has been gained through the use of the viral mimetic polyinosinic-polycytidylic acid (Poly(I:C)), yet the effects of this immune stimulant on pig behavior have not been assessed. This study advances the understanding of the combined effect of MIA and a second immune challenge later in life on female and male pig behavior. Three complementary experiments enabled the development of an effective Poly(I:C) challenge in pigs, and testing the interaction between PRRSV-elicited MIA, Poly(I:C) challenge at 60 days of age, and sex on behaviors. Individual-level observations on sickness, locomotor, and social behaviors were measured 1-3 h after Poly(I:C) challenge. Vomiting, panting, lethargy, walking, laying, playing, and touching behaviors were analyzed using generalized linear mixed effect models. Results indicated that a Poly(I:C) dose of 1 mg/kg within 1 h after injection increased the incidence of laying and sickness behavior. The Poly(I:C) challenge decreased the incidence of locomotor behaviors and activity levels. Pigs exposed to MIA had lower rates of social behaviors such as playing. The combined effect of PRRSV-elicited MIA and Poly(I:C) immune challenge further sensitized the pigs to behavior disruption across sexes including changes in sternal and lateral laying, walking, lethargy, and touching incidence. Notably, the effects of Poly(I:C) immune challenge alone on behaviors tended to be more extreme in males, whereas the effects of Poly(I:C) following MIA tended to be more extreme in females. Our findings demonstrate that MIA and Poly(I:C) affected behaviors, and the viral mimetic effects shortly after injection can offer insights into the prolonged effect of postnatal viral infections on feeding, social interactions and health status. Management practices that reduce the likelihood of gestational diseases and accommodate for behavioral disruptions in the offspring can minimize the impact of MIA.

An improved pig reference genome sequence to enable pig genetics and genomics research.

BACKGROUND: The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model given its similarity in size, anatomy, physiology, metabolism, pathology, and pharmacology to humans. The draft reference genome (Sscrofa10.2) of a purebred Duroc female pig established using older clone-based sequencing methods was incomplete, and unresolved redundancies, short-range order and orientation errors, and associated misassembled genes limited its utility. RESULTS: We present 2 annotated highly contiguous chromosome-level genome assemblies created with more recent long-read technologies and a whole-genome shotgun strategy, 1 for the same Duroc female (Sscrofa11.1) and 1 for an outbred, composite-breed male (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy than Sscrofa10.2. CONCLUSIONS: These highly contiguous assemblies plus annotation of a further 11 short-read assemblies provide an unprecedented view of the genetic make-up of this important agricultural and biomedical model species. We propose that the improved Duroc assembly (Sscrofa11.1) become the reference genome for genomic research in pigs.