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Abstract Background The frequency of chromosomal rearrangements in Canadian breeding boars has been estimated at 0.91 to 1.64%. These abnormalities are widely recognized as a potential cause of subfertility in livestock production. Since artificial insemination is practiced in almost all intensive pig production systems, the use of elite boars carrying cytogenetic defects that have an impact on fertility can lead to major economic losses. To avoid keeping subfertile boars in artificial insemination centres and spreading chromosomal defects within populations, cytogenetic screening of boars is crucial. Different techniques are used for this purpose, but several issues are frequently encountered, i.e. environmental factors can influence the quality of results, the lack of genomic information outputted by these techniques, and the need for prior cytogenetic skills. The aim of this study was to develop a new pig karyotyping method based on fluorescent banding patterns. Results The use of 207,847 specific oligonucleotides generated 96 fluorescent bands that are distributed across the 18 autosomes and the sex chromosomes. Tested alongside conventional G-banding, this oligo-banding method allowed us to identify four chromosomal translocations and a rare unbalanced chromosomal rearrangement that was not detected by conventional banding. In addition, this method allowed us to investigate chromosomal imbalance in spermatozoa. Conclusions The use of oligo-banding was found to be appropriate for detecting chromosomal aberrations in a Canadian pig nucleus and its convenient design and use make it an interesting tool for livestock karyotyping and cytogenetic studies.

Abstract Background Canadienne cattle are the oldest breed of dairy cattle in North America. The Canadienne breed originates from cattle that were brought to America by the mid-seventeenth century French settlers. The herd book was established in 1886 and the current breed characteristics include dark coat color, small size compared to the modern Holstein breed, and overall rusticity shaped by the harsh environmental conditions that were prevalent during the settlement of North America. The Canadienne breed is an invaluable genetic resource due to its high resilience, longevity and fertility. However, it is heavily threatened with a current herd limited to an estimated 1200 registered animals, of which less than 300 are fullblood. To date, no effort has been made to document the genetic pool of this heritage breed in order to preserve it. Results In this project, we used genomic data, which allow a precise description of the genetic makeup of a population, to provide valuable information on the genetic diversity of this heritage breed and suggest management options for its long-term viability. Using a panel that includes 640,000 single nucleotide polymorphisms (SNPs), we genotyped 190 animals grouped into six purity ranges. Unsupervised clustering analyses revealed three genetically distinct groups among those with the higher levels of purity. The observed heterozygosity was higher than expected even in the 100% purebreds. Comparison with Holstein genotypes showed significantly shorter runs of homozygosity for the Canadienne breed, which was unexpected due to the high inbreeding value calculated from pedigree data. Conclusions Overall, our data indicate that the fullblood gene pool of the Canadienne breed is more diversified than expected and that bloodline management could promote breed sustainability. In its current state, the Canadienne is not a dead-end breed but remains highly vulnerable due to its small population size.

Abstract Background Most of the resources that support the early development of the embryo are stored in the oocyte. Clearing of maternal resources and activation of the embryonic genome to produce its own mRNA transcripts marks the maternal-to-embryo transition. Dependence on stored mRNA can last from a few hours to several days, depending on animal species. The mechanisms regulating stabilization and recruitment of stored maternal transcripts have not yet been described in full detail but are known to involve reversible polyadenylation and modulation of 3’UTR-mediated elements. RNA epigenetic modifications, new players in this field, have an important role in RNA regulation and stabilization. Results The objectives of this study were first to determine if some of post-transcriptional methylation of stored mRNA is greater in oocytes than in somatic cells. We found that m6A, known to be the most prevalent and involved in various aspects of RNA metabolism and physiological functions, is particularly abundant in porcine oocyte mRNA compared to liver used as a somatic tissue reference. The second objective was to compare the epitranscriptome machinery, such as methyltransferases (“writers”), binding proteins (“readers”) and demethylases (“erasers”) catalyzing the different process, in follicles and oocytes of different mammalian species by immunofluorescence and confocal microscopy. The expression and localization patterns of these proteins differ between mice, pigs and cows ovaries and oocytes. m5C-associated proteins were generally less abundant. In contrast, m6A-associated proteins were expressed strongly during the early and late stages of folliculogenesis. Transzonal projections were found to contain more granules bearing the m5C mark in mice but both m5C and m6A methylation marks in association with mature oocytes of pigs and cows. Eraser proteins showed the greatest interspecies diversity in terms of distribution in the germinal tissues. Conclusions So far, few studies have looked at the oocyte and ovarian epitranscriptomic profile. Our findings indicate that a hitherto unrecognized species-specific layer of transcript regulation occurs at the RNA level and might be consequential during the oocyte transcriptional silencing period.

Abstract Background Genome assembly into chromosomes facilitates several analyses including cytogenetics, genomics and phylogenetics. Despite rapid development in bioinformatics, however, assembly beyond scaffolds remains challenging, especially in species without closely related well-assembled and available reference genomes. So far, four draft genomes of Rangifer tarandus (caribou or reindeer, a circumpolar distributed cervid species) have been published, but none with chromosome-level assembly. This emblematic northern species is of high interest in ecological studies and conservation since most populations are declining. Results We have designed specific probes based on Oligopaint FISH technology to upgrade the latest published reindeer and caribou chromosome-level genomes. Using this oligonucleotide-based method, we found six mis-assembled scaffolds and physically mapped 68 of the largest scaffolds representing 78% of the most recent R. tarandus genome assembly. Combining physical mapping and comparative genomics, it was possible to document chromosomal evolution among Cervidae and closely related bovids. Conclusions Our results provide validation for the current chromosome-level genome assembly as well as resources to use chromosome banding in studies of Rangifer tarandus.

Abstract The loss of genetic diversity is a challenge many species are facing, with genomics being a potential tool to inform and prioritize decision‐making. Most caribou (Rangifer tarandus) populations have experienced significant recent declines throughout Québec, Canada, and are considered of concern, threatened or endangered. Here, we calculated the ancestral and contemporary patterns of genomic diversity of five representative caribou populations and applied a comparative population genomics framework to assess the interplay between demographic events and genomic diversity. We first calculated a caribou specific mutation rate, μ, by extracting orthologous genes from related ungulates and estimating the rate of synonymous mutations. Whole genome re‐sequencing was then completed on 67 caribou: from these data we calculated nucleotide diversity, θπ and estimated the coalescent or ancestral effective population size (Ne), which ranged from 12,030 to 15,513. When compared to the census size, NC, the endangered Gaspésie Mountain caribou population had the highest ancestral Ne:NC ratio which is consistent with recent work suggesting high ancestral Ne:NC is of conservation concern. In contrast, values of contemporary Ne, estimated from linkage‐disequilibrium, ranged from 11 to 162, with Gaspésie having among the highest contemporary Ne:NC ratio. Importantly, classic conservation genetics theory would predict this population to be of less concern based on this ratio. Interestingly, F varied only slightly between populations, and despite evidence of bottlenecks across the province, runs of homozygosity were not abundant in the genome. Tajima's D estimates mirrored the demographic models and current conservation status. Our study highlights how genomic patterns are nuanced and potentially misleading if viewed only through a contemporary lens; we argue a holistic conservation genomics view should integrate ancestral Ne and Tajima's D into management decisions.

Abstract Background Development of large single nucleotide polymorphism (SNP) arrays can make genomic data promptly available for conservation problematic. Medium and high-density panels can be designed with sufficient coverage to offer a genome-wide perspective and the generated genotypes can be used to assess different genetic metrics related to population structure, relatedness, or inbreeding. SNP genotyping could also permit sexing samples with unknown associated metadata as it is often the case when using non-invasive sampling methods favored for endangered species. Genome sequencing of wild species provides the necessary information to design such SNP arrays. We report here the development of a SNP-array for endangered Rangifer tarandus using a multi-platform sequencing approach from animals found in diverse populations representing the entire circumpolar distribution of the species. Results From a very large comprehensive catalog of SNPs detected over the entire sample set (N = 894), a total of 63,336 SNPs were selected. SNP selection accounted for SNPs evenly distributed across the entire genome (~ every 50Kb) with known minor alleles across populations world-wide. In addition, a subset of SNPs was selected to represent rare and local alleles found in Eastern Canada which could be used for ecotype and population assignments - information urgently needed for conservation planning. In addition, heterozygosity from SNPs located in the X-chromosome and genotyping call-rate of SNPs located into the SRY gene of the Y-chromosome yielded an accurate and robust sexing assessment. All SNPs were validated using a high-throughput SNP-genotyping chip. Conclusion This design is now integrated into the first genome-wide commercially available genotyping platform for Rangifer tarandus. This platform would pave the way to future genomic investigation of populations for this endangered species, including estimation of genetic diversity parameters, population assignments, as well as animal sexing from genetic SNP data for non-invasive samples.

Abstract Using a mouse model, studies by our group reveal that paternal preconception alcohol intake affects offspring fetal-placental growth, with long-lasting consequences on adult metabolism. Here, we tested the hypothesis that chronic preconception male alcohol exposure impacts histone enrichment in sperm and that these changes are associated with altered developmental programming in the placenta. Using chromatin immunoprecipitation, we find alcohol-induced increases in sperm histone H3 lysine 4 trimethylation (H3K4me3) that map to promoters and presumptive enhancer regions enriched in genes driving neurogenesis and craniofacial development. Given the colocalization of H3K4me3 with the chromatin binding factor CTCF across both sperm and embryos, we next examined CTCF localization in the placenta. We find global changes in CTCF binding within placentae derived from the male offspring of alcohol-exposed sires. Furthermore, altered CTCF localization correlates with dysregulated gene expression across multiple gene clusters; however, these transcriptional changes only occur in male offspring. Finally, we identified a correlation between genomic regions exhibiting alcohol-induced increases in sperm H3K4me3 and increased CTCF binding in male placentae. Collectively, our analysis demonstrates that the chromatin landscape of sperm is sensitive to chronic alcohol exposure and that a subset of these affected regions exhibits increased placental CTCF enrichment.

During the last decades, the emergence of Bibliometrics and the progress in Pain research have led to a proliferation of bibliometric studies on the medical and scientific literature of pain (B/P). This study charts the evolution of the B/P literature published during the last 30 years. Using various searching techniques, 189 B/P studies published from 1993 to August 2022 were collected for analysis—half were published since 2018. Most of the selected B/P publications use classic bibliometric analysis of Pain in toto, while some focus on specific types of Pain with Headache/Migraine, Low Back Pain, Chronic Pain, and Cancer Pain dominating. Each study is characterized by the origin (geographical, economical, institutional, …) and the medical/scientific context over a specified time span to provide a detailed landscape of the Pain research literature. Some B/P studies have been developed to pinpoint difficulties in appropriately identifying the Pain literature or to highlight some general publishing pitfalls. Having observed that most of the recent B/P studies have integrated newly emergent software visualization tools (SVTs), we found an increase of anomalies and suggest that readers exercise caution when interpreting results in the B/P literature details.

Long non-coding RNAs (lncRNAs) have been the subject of numerous studies over the past decade. First thought to come from aberrant transcriptional events, lncRNAs are now considered a crucial component of the genome with roles in multiple cellular functions. However, the functional annotation and characterization of bovine lncRNAs during early development remain limited. In this comprehensive analysis, we review lncRNAs expression in bovine ovarian follicles and early embryos, based on a unique database comprising 468 microarray hybridizations from a single platform designed to target 7,724 lncRNA transcripts, of which 5,272 are intergenic (lincRNA), 958 are intronic, and 1,524 are antisense (lncNAT). Compared to translated mRNA, lncRNAs have been shown to be more tissue-specific and expressed in low copy numbers. This analysis revealed that protein-coding genes and lncRNAs are both expressed more in oocytes. Differences between the oocyte and the 2-cell embryo are also more apparent in terms of lncRNAs than mRNAs. Co-expression network analysis using WGCNA generated 25 modules with differing proportions of lncRNAs. The modules exhibiting a higher proportion of lncRNAs were found to be associated with fewer annotated mRNAs and housekeeping functions. Functional annotation of co-expressed mRNAs allowed attribution of lncRNAs to a wide array of key cellular events such as meiosis, translation initiation, immune response, and mitochondrial related functions. We thus provide evidence that lncRNAs play diverse physiological roles that are tissue-specific and associated with key cellular functions alongside mRNAs in bovine ovarian follicles and early embryos. This contributes to add lncRNAs as active molecules in the complex regulatory networks driving folliculogenesis, oogenesis and early embryogenesis all of which are necessary for reproductive success.

Following their production in the testis, spermatozoa enter the epididymis where they gain their motility and fertilizing abilities. This post-testicular maturation coincides with sperm epigenetic profile changes that influence progeny outcome. While recent studies highlighted the dynamics of small non-coding RNAs in maturing spermatozoa, little is known regarding sperm methylation changes and their impact at the post-fertilization level. Fluorescence-activated cell sorting (FACS) was used to purify spermatozoa from the testis and different epididymal segments (i.e., caput, corpus and cauda) of CAG/su9-DsRed2; Acr3-EGFP transgenic mice in order to map out sperm methylome dynamics. Reduced representation bisulfite sequencing (RRBS-Seq) performed on DNA from these respective sperm populations indicated that high methylation changes were observed between spermatozoa from the caput vs. testis with 5,546 entries meeting our threshold values (q value

Controlling mating in the honeybee (Apis mellifera) is part of one of the greatest challenges for the beekeeping industry’s genetic selection programs due to specific characteristics of their reproduction. Several techniques for supervising honeybee mating with relative effective control have been developed over the years to allow honeybee selection. As part of this project, we compared the genetic gains for several colony performance traits, obtained using the BLUP-animal method, according to the selection pressure applied in controlled reproduction (directed fertilization versus instrumental insemination). Our results show similar genetic gains for hygienic behavior and honey production between colonies whether queens were fertilized naturally or via instrumental insemination, as well as similar or lower genetic gains for colonies with queens inseminated for spring development. In addition, we noticed greater fragility in queens following insemination. These findings show that instrumental insemination is an effective tool for reproductive control in genetic selection and for estimating breeding values more precisely. However, this technique does not result in queens of superior genetic quality for commercial purposes.

Abstract Background Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. Results The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes. Conclusion In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.

Abstract Background Assisted reproductive technologies (ART) are widely used to treat fertility issues in humans and for the production of embryos in mammalian livestock. The use of these techniques, however, is not without consequence as they are often associated with inauspicious pre- and postnatal outcomes including premature birth, intrauterine growth restriction and increased incidence of epigenetic disorders in human and large offspring syndrome in cattle. Here, global DNA methylation profiles in the trophectoderm and embryonic discs of in vitro produced (IVP), superovulation-derived (SOV) and unstimulated, synchronised control day 17 bovine conceptuses (herein referred to as AI) were interrogated using the EmbryoGENE DNA Methylation Array (EDMA). Pyrosequencing was used to validate four loci identified as differentially methylated on the array and to assess the differentially methylated regions (DMRs) of six imprinted genes in these conceptuses. The impact of embryo-production induced DNA methylation aberrations was determined using Ingenuity Pathway Analysis, shedding light on the potential functional consequences of these differences. Results Of the total number of differentially methylated loci identified (3140) 77.3 and 22.7% were attributable to SOV and IVP, respectively. Differential methylation was most prominent at intragenic sequences within the trophectoderm of IVP and SOV-derived conceptuses, almost a third (30.8%) of the differentially methylated loci mapped to intragenic regions. Very few differentially methylated loci were detected in embryonic discs (ED); 0.16 and 4.9% of the differentially methylated loci were located in the ED of SOV-derived and IVP conceptuses, respectively. The overall effects of SOV and IVP on the direction of methylation changes were associated with increased methylation; 70.6% of the differentially methylated loci in SOV-derived conceptuses and 57.9% of the loci in IVP-derived conceptuses were more methylated compared to AI-conceptuses. Ontology analysis of probes associated with intragenic sequences suggests enrichment for terms associated with cancer, cell morphology and growth. Conclusion By examining (1) the effects of superovulation and (2) the effects of an in vitro system (oocyte maturation, fertilisation and embryo culture) we have identified that the assisted reproduction process of superovulation alone has the largest impact on the DNA methylome of subsequent embryos.

Although supplementing the diet with zinc oxide and arginine is known to improve growth in weanling piglets, the mechanism of action is not well understood. We measured the antioxidant status and inflammatory response in 48 weanling castrated male piglets fed diets supplemented with or without zinc oxide (2,500 mg Zn oxide per kg) and arginine (1%) starting at the age of 20 days. The animals were injected with lipopolysaccharide (100 μg/kg) on day 5. Half of them received another injection on day 12. Blood samples were taken just before and 6, 24 and 48 h after injection and the mucosa lining the ileum was recovered following euthanizing on days 7 and 14. Zinc supplementation increased reduced and total glutathione (GSH) (reduced and total) during days 5 to 7 and arginine decreased oxidized GSH measured on days 5 and 12 and the ratio of total antioxidant capacity to total oxidative status during days 12 to 14. Zinc decreased plasma malondialdehyde measured on days 5 and 12 and serum haptoglobin measured on day 12 and increased both metallothionein-1 expression and total antioxidant capacity measured in the ileal mucosa on day 14. Tumour necrosis factor α concentration decreased from days 5 to 12 (all effects were significant at P

A multitude of model and non-model species studies have now taken full advantage of powerful high-throughput genotyping advances such as SNP arrays and genotyping-by-sequencing (GBS) technology to investigate the genetic basis of trait variation. However, due to incomplete genome coverage by these technologies, the identified SNPs are likely in linkage disequilibrium (LD) with the causal polymorphisms, rather than be causal themselves. In addition, researchers could benefit from annotations for the identified candidate SNPs and, simultaneously, for all neighboring genes in genetic linkage. In such case, LD extent estimation surrounding the candidate SNPs is required to determine the regions encompassing genes of interest. We describe here an automated pipeline, “LD-annot,” designed to delineate specific regions of interest for a given experiment and candidate polymorphisms on the basis of LD extent, and furthermore, provide annotations for all genes within such regions. LD-annot uses standard file formats, bioinformatics tools, and languages to provide identifiers, coordinates, and annotations for genes in genetic linkage with each candidate polymorphism. Although the focus lies upon SNP arrays and GBS data as they are being routinely deployed, this pipeline can be applied to a variety of datasets as long as genotypic data are available for a high number of polymorphisms and formatted into a vcf file. A checkpoint procedure in the pipeline allows to test several threshold values for linkage without having to rerun the entire pipeline, thus saving the user computational time and resources. We applied this new pipeline to four different sample sets: two breeding populations GBS datasets, one within-pedigree SNP set coming from whole genome sequencing (WGS), and a very large multi-varieties SNP dataset obtained from WGS, representing variable sample sizes, and numbers of polymorphisms. LD-annot performed within minutes, even when very high numbers of polymorphisms are investigated and thus will efficiently assist research efforts aimed at identifying biologically meaningful genetic polymorphisms underlying phenotypic variation. LD-annot tool is available under a GPL license from https://github.com/ArnaudDroitLab/LD-annot.

Genetic improvement programs have resulted in spectacular productivity gains for most animal species in recent years. The introduction of quantitative genetics and the use of statistical models have played a fundamental role in achieving these advances. For the honeybee (Apis mellifera), genetic improvement programs are still rare worldwide. Indeed, genetic and reproductive characteristics are more complex in honeybees than in other animal species, which presents additional challenges for access genetic selection. In recent years, advances in informatics have allowed statistical modelling of the honeybee, notably with the BLUP-animal model, and access to genetic selection for this species is possible now. The aim of this project was to present the genetic progress of several traits of interest to the Canadian beekeeping industry (hygienic behavior, honey production and spring development) achieved in our selection program since 2010. Our results show an improvement of 0.30% per year for hygienic behavior, 0.63 kg per year for honey production and 164 brood cells per year for spring development. These advances have opened a new era for our breeding program and sharing this superior genetic available to beekeepers will contribute to the sustainability and self-sufficiency of the beekeeping industry in Canada.

Genetic selection has led to spectacular advances in animal production in many domestic species. However, it is still little applied to honey bees (Apis mellifera), whose complex genetic and reproductive characteristics are a challenge to model statistically. Advances in informatics now enable creation of a statistical model consistent with honey bee genetics, and, consequently, genetic selection for this species. The aim of this project was to determine the genetic parameters of several traits important for Canadian beekeepers with a view to establishing a breeding program in a northern context. Our results show that the five traits measured (Varroa destructor infestation, spring development, honey production, winter consumption, and hygienic behavior) are heritable. Thus, the rate of V. destructor infestation has a high heritability (h2 = 0.44 ± 0.56), spring development and honey production have a medium heritability (respectively, h2 = 0.30 ± 0.14 and h2 = 0.20 ± 0.13), and winter consumption and hygienic behavior have a low heritability (respectively, h2 = 0.11 ± 0.09 and h2 = 0.18 ± 0.13). Furthermore, the genetic correlations between these traits are all positive or null, except between hygienic behavior and V. destructor infestation level. These genetic parameters will be instrumental to the development of a selection index that will be used to improve the capacity of honey bees to thrive in northern conditions.

Cryopreservation is known for its marked deleterious effects on embryonic health. Bovine compact morulae were vitrified or slow-frozen, and post-warm morulae were cultured to the expanded blastocyst stage. Blastocysts developed from vitrified and slow-frozen morulae were subjected to microarray analysis and compared with blastocysts developed from unfrozen control morulae for differential gene expression. Morula to blastocyst conversion rate was higher (P < 0.05) in control (72%) and vitrified (77%) than in slow-frozen (34%) morulae. Total 20 genes were upregulated and 44 genes were downregulated in blastocysts developed from vitrified morulae (fold change ≥ ± 2, P < 0.05) in comparison with blastocysts developed from control morulae. In blastocysts developed from slow-frozen morulae, 102 genes were upregulated and 63 genes were downregulated (fold change ≥ ± 1.5, P < 0.05). Blastocysts developed from vitrified morulae exhibited significant changes in gene expression mainly involving embryo implantation (PTGS2, CALB1), lipid peroxidation and reactive oxygen species generation (HSD3B1, AKR1B1, APOA1) and cell differentiation (KRT19, CLDN23). However, blastocysts developed from slow-frozen morulae showed changes in the expression of genes related to cell signaling (SPP1), cell structure and differentiation (DCLK2, JAM2 and VIM), and lipid metabolism (PLA2R1 and SMPD3). In silico comparison between blastocysts developed form vitrified and slow-frozen morulae revealed similar changes in gene expression as between blastocysts developed from vitrified and control morulae. In conclusion, blastocysts developed form vitrified morulae demonstrated better post-warming survival than blastocysts developed from slow-frozen morulae but their gene expression related to lipid metabolism, steroidogenesis, cell differentiation and placentation changed significantly (≥ 2 fold). Slow freezing method killed more morulae than vitrification but those which survived up to blastocyst stage did not express ≥ 2 fold change in their gene expression as compared with blastocysts from control morulae.

Local translation at the synapse plays key roles in neuron development and activity-dependent synaptic plasticity. mRNAs are translocated from the neuronal soma to the distant synapses as compacted ribonucleoparticles referred to as RNA granules. These contain many RNA-binding proteins, including the Fragile X Mental Retardation Protein (FMRP), the absence of which results in Fragile X Syndrome, the most common inherited form of intellectual disability and the leading genetic cause of autism. Using FMRP as a tracer, we purified a specific population of RNA granules from mouse brain homogenates. Protein composition analyses revealed a strong relationship between polyribosomes and RNA granules. However, the latter have distinct architectural and structural properties, since they are detected as close compact structures as observed by electron microscopy, and converging evidence point to the possibility that these structures emerge from stalled polyribosomes. Time-lapse video microscopy indicated that single granules merge to form cargoes that are transported from the soma to distal locations. Transcriptomic analyses showed that a subset of mRNAs involved in cytoskeleton remodelling and neural development is selectively enriched in RNA granules. One third of the putative mRNA targets described for FMRP appear to be transported in granules and FMRP is more abundant in granules than in polyribosomes. This observation supports a primary role for FMRP in granules biology. Our findings open new avenues for the study of RNA granule dysfunctions in animal models of nervous system disorders, such as Fragile X syndrome.

The heritability of schizophrenia and most personality traits has been well established, but the role of personality in susceptibility to schizophrenia remains uncertain. The aim of this study was to test for an association between personality traits and Neurological Soft Signs (NSS), a well-known biological marker of schizophrenia, in non-psychotic relatives of patients with schizophrenia. For this purpose, we evaluated the NSS scale and personality measured by the Temperament and Character inventory (TCI-R) in three groups of subjects: 29 patients with schizophrenia, 24 unaffected relatives and 37 controls. The results showed that patients with schizophrenia were more asocial (higher harm avoidance and lower reward dependence), more perseverative (higher persistence), and more schizotypal (lower self-directedness and cooperativeness, higher self-transcendence). The unaffected relatives showed higher harm avoidance, lower self-directedness and cooperativeness than the healthy controls. Higher NSS scores and sub-scores were found in patients and non-psychotic relatives compared with the controls. Among all the patients, total NSS scores were positively correlated with harm avoidance but negatively correlated with novelty seeking and persistence. Total NSS were also correlated with low scores on self-directedness and cooperativeness, which are indicators of personality disorder. Our results show that susceptibility to NSS and to schizophrenia are both related to individual differences in the temperament and character features in non-psychotic relatives of patients with schizophrenia. High harm avoidance, low persistence, low self-directedness and low cooperativeness contribute to both the risk of NSS and schizophrenia. These findings highlight the value of using both assessments to study high risk populations.

Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY), 4-cell (4C) or 16-cell (16C) were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP). Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO) using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic imprinting and chromosome segregation in IVP blastocyst groups. Furthermore, 1.6, 3.4, 3.9 and 9.4% of the differentially methylated regions that were overlapped to the transcriptome profile data were negatively correlated with the gene expression patterns in ZY, 4C, 16C and IVP blastocyst groups, respectively. Therefore, this finding indicated that suboptimal culture condition during preimplantation embryo development induced changes in the DNA methylation landscape of the resulting blastocysts in a stage dependent manner and the altered DNA methylation pattern was only partly explained the observed aberrant gene expression patterns of the blastocysts.

Gap junctional communication (GJC) plays a primordial role in oocyte maturation and meiotic resumption in mammals by directing the transfer of numerous molecules between cumulus cells and the oocyte. Gap junctions are made of connexins (Cx), proteins that regulate GJC in numerous ways. Understanding the dynamic regulation of connexin arrangements during in vitro maturation (IVM) could provide a powerful tool for controlling meiotic resumption and consequently in vitro development of fully competent oocytes. However, physiological events happening during the early hours of IVM may still be elucidated. The present study reports the dynamic regulation of connexin expression, degradation and localization during this stage. Cx43, Cx45 and Cx60 were identified as the main connexins expressed in swine COC. Cx43 and Cx45 transcripts were judged too static to be a regulator of GJC, while Cx43 protein expression was highly responsive to gonadotropins, suggesting that it might be the principal regulator of GJC. In addition, the degradation of Cx43 expressed after 4.5 h of IVM in response to equine chorionic gonadotropin appeared to involve the proteasomal complex. Cx43 localisation appeared to be associated with GJC. Taken together, these results show for the first time that gonadotropins regulate Cx43 protein expression, degradation and localisation in porcine COC during the first several hours of IVM. Regulation of Cx43 may in turn, via GJC, participate in the development of fully competent oocytes.

In this paper, we present two carefully documented cases of patients with sleep-related eating disorder (SRED), a parasomnia which is characterized by involuntary compulsive eating during the night and whose pathophysiology is not known. Using video-polysomnography and psychometric examination, we found that both patients present elevated novelty seeking and increased reward sensitivity on reward-related questionnaires. In light of new evidence on the mesolimbic dopaminergic implication in compulsive eating disorders, our findings suggest a role of an active reward system during sleep in the manifestation of SRED.

BACKGROUND:GATA4 is an essential transcription factor required for the development and function of multiple organs. Despite this important role, our knowledge of how the GATA4 gene is regulated remains limited. To better understand this regulation, we characterized the 5' region of the mouse, rat, and human GATA4 genes. METHODOLOGY/PRINCIPAL FINDINGS:Using 5' RACE, we identified novel transcription start sites in all three species. GATA4 is expressed as multiple transcripts with varying 5' ends encoded by alternative untranslated first exons. Two of these non-coding first exons are conserved between species: exon 1a located 3.5 kb upstream of the GATA4 ATG site in exon 2, and a second first exon (exon 1b) located 28 kb further upstream. Expression of both mRNA variants was found in all GATA4-expressing organs but with a preference for the exon 1a-containing transcript. The exception was the testis where exon 1a- and 1b-containing transcripts were similarly expressed. In some tissues such as the intestine, alternative transcript expression appears to be regionally regulated. Polysome analysis suggests that both mRNA variants contribute to GATA4 protein synthesis. CONCLUSIONS/SIGNIFICANCE:Taken together, our results indicate that the GATA4 gene closely resembles the other GATA family members in terms of gene structure where alternative first exon usage appears to be an important mechanism for regulating its tissue- and cell-specific expression.

In general relativity, the speed of light is considered as the only real invariant of the universe, while the flow of time would be an illusion. In quantum mechanics, conversely, time is constant, absolute, and universal. In this article, we support this concept and conclude that it is this flow of time which not only controls and limits the speed of light but also, through its progression, uncovers the universe as we know it. In fact, the flow of time is the real "Marshal" of the universe. [ABSTRACT FROM AUTHOR]

The myth of this "quasi-magical" gravity and its "effect at a distance" endures, and our brains remain "hardwired" to this perception. However, "quantum gravity" is increasingly closer to nullifying the importance of this mysterious force by treating it with the outmost contempt as a "negligible force." We concur with this interpretation, and more: We claim in this article that not only is gravity negligible but also explain how it can be completely eliminated as one of four fundamental forces of the universe. This article is, in fact, a continuation, a widening, and a confirmation of an article I wrote in 1993, titled "The proportional expansion of each and every celestial body as the cause of gravitation" [Phys. Essays 6, 473 (1993)]. There, I had argued that not only is empty space expanding in the classically understood sense, but that all celestial bodies are also subject to an accelerated expansion. According to Einstein's principle of equivalence, acceleration generates an opposite, "fictitious" force that is perceived as gravity. I had concluded there that "in terms of quantum mechanics, in this interpretation, the very existence of the predicted 'graviton' becomes extremely questionable." Thirty years have passed since then. And in spite of the development of the Large Hadron Collider and its impressive capabilities, the above claim has been confirmed, and the graviton has been quietly replaced by the Higgs boson in the nomenclature of the Standard Model of elementary particles. In this article, we revisit Galileo and Newton to show that the latter could not have arrived at any other conclusion than an "attractive" force of gravity. We also ask why Albert Einstein did not apply his own principle of equivalence to the Newtonian interpretation of gravity. At the same time, some very "peculiar" interpretations have arisen since my above-mentioned article from 1993. "Dark matter" and "dark energy," both very speculative conceptualizations, have been forced upon us to explain two phenomena: The anomaly discovered in the speed of rotation of the galaxies, and the accelerated expansion of the universe, despite the potentially shrinking effect of gravity. Of all the positive advances in physics, the most fundamental is the genesis of "quantum gravity," the tenets of which relegate gravity to the level of a "negligible" force. This is followed by the characterization of the Higgs boson, which has replaced the graviton in the Standard Model. This conceptually translates into the fact that the gravitational mass has possibly been replaced by the inertial mass of celestial bodies. The confirmation and measurements of the accelerated expansion of the universe, while there is still some controversies regarding the exact results of these measurements. Finally, the most important discovery in this regard was recently made in 2019, regarding the exceptionally high pressure inside protons and neutrons, (1035 Pa). This lends credence to the concept of the expansion of these subatomic particles. The multiplicity of these hadrons (3.26x1080) and their ubiquitous distribution in the baryonic matter of the entire universe explain its expansion. These hadrons are highly "diluted" in empty space but, on the contrary, are highly clustered and concentrated inside solid matter, where this explains the difference in density between these media and the massive dissimilarity in the magnitudes of their relative accelerated expansion. It also explains why what we call "gravity" is not an active force, but merely the consequence of the density-proportional accelerated radial expansion of each and every celestial body. As such, this baryonic expansion explains the expansion of the universe as a whole, and replaces gravity as a concept as well as its perceived effects. By dint of its ubiquitous nature, this notion of baryonic expansion provides a bridge between the microscopic and macroscopic universe. Quantum physics reconnects with general relativity in this formalism and clarifies the "what and how". [ABSTRACT FROM AUTHOR]