Genomic architecture constrained placental mammal X Chromosome evolution.

Susumu Ohno proposed that the gene content of the mammalian X Chromosome should remain highly conserved due to dosage compensation. X Chromosome linkage (gene order) conservation is widespread in placental mammals but does not fall within the scope of Ohno's prediction and may be an indirect result of selection on gene content or selection against rearrangements that might disrupt X-Chromosome inactivation (XCI). Previous comparisons between the human and mouse X Chromosome sequences have suggested that although single-copy X Chromosome genes are conserved between species, most ampliconic genes were independently acquired. To better understand the evolutionary and functional constraints on X-linked gene content and linkage conservation in placental mammals, we aligned a new, high-quality, long-read X Chromosome reference assembly from the domestic cat (incorporating 19.3 Mb of targeted BAC clone sequence) to the pig, human, and mouse assemblies. A comprehensive analysis of annotated X-linked orthologs in public databases demonstrated that the majority of ampliconic gene families were present on the ancestral placental X Chromosome. We generated a domestic cat Hi-C contact map from an F1 domestic cat/Asian leopard cat hybrid and demonstrated the formation of the bipartite structure found in primate and rodent inactivated X Chromosomes. Conservation of gene order and recombination patterns is attributable to strong selective constraints on three-dimensional genomic architecture necessary for superloop formation. Species with rearranged X Chromosomes retain the ancestral order and relative spacing of loci critical for superloop formation during XCI, with compensatory inversions evolving to maintain these long-range physical interactions.

A new domestic cat genome assembly based on long sequence reads empowers feline genomic medicine and identifies a novel gene for dwarfism.

The domestic cat (Felis catus) numbers over 94 million in the USA alone, occupies households as a companion animal, and, like humans, suffers from cancer and common and rare diseases. However, genome-wide sequence variant information is limited for this species. To empower trait analyses, a new cat genome reference assembly was developed from PacBio long sequence reads that significantly improve sequence representation and assembly contiguity. The whole genome sequences of 54 domestic cats were aligned to the reference to identify single nucleotide variants (SNVs) and structural variants (SVs). Across all cats, 16 SNVs predicted to have deleterious impacts and in a singleton state were identified as high priority candidates for causative mutations. One candidate was a stop gain in the tumor suppressor FBXW7. The SNV is found in cats segregating for feline mediastinal lymphoma and is a candidate for inherited cancer susceptibility. SV analysis revealed a complex deletion coupled with a nearby potential duplication event that was shared privately across three unrelated cats with dwarfism and is found within a known dwarfism associated region on cat chromosome B1. This SV interrupted UDP-glucose 6-dehydrogenase (UGDH), a gene involved in the biosynthesis of glycosaminoglycans. Importantly, UGDH has not yet been associated with human dwarfism and should be screened in undiagnosed patients. The new high-quality cat genome reference and the compilation of sequence variation demonstrate the importance of these resources when searching for disease causative alleles in the domestic cat and for identification of feline biomedical models.

mRatBN7.2: familiar and unfamiliar features of a new rat genome reference assembly.

Rat genomic tools have been slower to emerge than for those of humans and mice and have remained less thorough and comprehensive. The arrival of a new and improved rat reference genome, mRatBN7.2, in late 2020 is a welcome event. This assembly, like predecessor rat reference assemblies, is derived from an inbred Brown Norway rat. In this "user" survey we hope to provide other users of this assembly some insight into its characteristics and some assessment of its improvements as well as a few caveats that arise from the unique aspects of this assembly. mRatBN7.2 was generated by the Wellcome Sanger Institute as part of the large Vertebrate Genomes Project. This rat assembly has now joined human, mouse, chicken, and zebrafish in the National Center for Biotechnology Information (NCBI)'s Genome Reference Consortium, which provides ongoing curation of the assembly. Here we examine the technical procedures by which the assembly was created and assess how this assembly constitutes an improvement over its predecessor. We also indicate the technical limitations affecting the assembly, providing illustrations of how these limitations arise and the impact that results for this reference assembly.

Evolutionary conservation of Y Chromosome ampliconic gene families despite extensive structural variation.

Despite claims that the mammalian Y Chromosome is on a path to extinction, comparative sequence analysis of primate Y Chromosomes has shown the decay of the ancestral single-copy genes has all but ceased in this eutherian lineage. The suite of single-copy Y-linked genes is highly conserved among the majority of eutherian Y Chromosomes due to strong purifying selection to retain dosage-sensitive genes. In contrast, the ampliconic regions of the Y Chromosome, which contain testis-specific genes that encode the majority of the transcripts on eutherian Y Chromosomes, are rapidly evolving and are thought to undergo species-specific turnover. However, ampliconic genes are known from only a handful of species, limiting insights into their long-term evolutionary dynamics. We used a clone-based sequencing approach employing both long- and short-read sequencing technologies to assemble ∼2.4 Mb of representative ampliconic sequence dispersed across the domestic cat Y Chromosome, and identified the major ampliconic gene families and repeat units. We analyzed fluorescence in situ hybridization, qPCR, and whole-genome sequence data from 20 cat species and revealed that ampliconic gene families are conserved across the cat family Felidae but show high transcript diversity, copy number variation, and structural rearrangement. Our analysis of ampliconic gene evolution unveils a complex pattern of long-term gene content stability despite extensive structural variation on a nonrecombining background.

Mechanisms Underlying Mammalian Hybrid Sterility in Two Feline Interspecies Models.

The phenomenon of male sterility in interspecies hybrids has been observed for over a century, however, few genes influencing this recurrent phenotype have been identified. Genetic investigations have been primarily limited to a small number of model organisms, thus limiting our understanding of the underlying molecular basis of this well-documented "rule of speciation." We utilized two interspecies hybrid cat breeds in a genome-wide association study employing the Illumina 63 K single-nucleotide polymorphism array. Collectively, we identified eight autosomal genes/gene regions underlying associations with hybrid male sterility (HMS) involved in the function of the blood-testis barrier, gamete structural development, and transcriptional regulation. We also identified several candidate hybrid sterility regions on the X chromosome, with most residing in close proximity to complex duplicated regions. Differential gene expression analyses revealed significant chromosome-wide upregulation of X chromosome transcripts in testes of sterile hybrids, which were enriched for genes involved in chromatin regulation of gene expression. Our expression results parallel those reported in Mus hybrids, supporting the "Large X-Effect" in mammalian HMS and the potential epigenetic basis for this phenomenon. These results support the value of the interspecies feline model as a powerful tool for comparison to rodent models of HMS, demonstrating unique aspects and potential commonalities that underpin mammalian reproductive isolation.

An estrogen receptor α-derived peptide improves glucose homeostasis during obesity.

Estrogen receptor α (ERα) plays a crucial role in regulating glucose and energy homeostasis during type 2 diabetes mellitus (T2DM). However, the underlying mechanisms remain incompletely understood. Here we find a ligand-independent effect of ERα on the regulation of glucose homeostasis. Deficiency of ERα in the liver impairs glucose homeostasis in male, female, and ovariectomized (OVX) female mice. Mechanistic studies reveal that ERα promotes hepatic insulin sensitivity by suppressing ubiquitination-induced IRS1 degradation. The ERα 1-280 domain mediates the ligand-independent effect of ERα on insulin sensitivity. Furthermore, we identify a peptide based on ERα 1-280 domain and find that ERα-derived peptide increases IRS1 stability and enhances insulin sensitivity. Importantly, administration of ERα-derived peptide into obese mice significantly improves glucose homeostasis and serum lipid profiles. These findings pave the way for the therapeutic intervention of T2DM by targeting the ligand-independent effect of ERα and indicate that ERα-derived peptide is a potential insulin sensitizer for the treatment of T2DM.

ZnPP-laden nanoparticles improve glucose homeostasis and chronic inflammation during obesity.

BACKGROUND AND PURPOSE: Chronic inflammation plays a pivotal role in the development of Type 2 diabetes mellitus (T2DM). Previous studies have shown that haem oxygenase-1 (HO-1) plays a proinflammatory role during metabolic stress, suggesting that HO-1 inhibition could be an effective strategy to treat T2DM. However, the application of HO-1 inhibitors is restricted due to solubility-limited bioavailability. In this study, we encapsulated the HO-1 inhibitor, zinc protoporphyrin IX (ZnPP), within nanoparticles and investigated their role in regulating glucose homeostasis and chronic inflammation during obesity. EXPERIMENTAL APPROACH: We delivered DMSO-dissolved ZnPP (DMSO-ZnPP) and ZnPP-laden nanoparticles (Nano-ZnPP) to diet-induced obese male mice for 6 weeks. Glucose and insulin tolerance tests were carried out, liver and adipose tissue gene expression profiles analysed, and systemic inflammation analysed using flow cytometry. KEY RESULTS: Nanoparticles significantly increased the delivery efficiency of ZnPP in both cells and mice. In mice with diet-induced obesity, inhibition of HO-1 by Nano-ZnPP significantly decreased adiposity, increased insulin sensitivity, and improved glucose tolerance. Moreover, Nano-ZnPP treatment attenuated both local and systemic inflammatory levels during obesity. Mechanistically, Nano-ZnPP significantly attenuated glucagon, TNF, and fatty acid synthesis signalling pathways in the liver. In white adipose tissue, the oxidative phosphorylation signalling pathway was enhanced and the inflammation signalling pathway diminished by Nano-ZnPP. Our results show that Nano-ZnPP has better effects on the improvement of glucose homeostasis and attenuation of chronic inflammation, than those of DMSO-dissolved ZnPP. CONCLUSIONS AND IMPLICATIONS: These findings indicate that ZnPP-laden nanoparticles are potential therapeutic agents for treating T2DM.

Suppression of FOXO1 attenuates inflamm-aging and improves liver function during aging.

The liver is a key metabolic organ that maintains whole-body nutrient homeostasis. Aging-induced liver function alterations contribute to systemic susceptibility to aging-related diseases. However, the molecular mechanisms of liver aging remain insufficiently understood. In this study, we performed bulk RNA-Seq and single-cell RNA-Seq analyses to investigate the underlying mechanisms of the aging-induced liver function changes. We found that liver inflammation, glucose intolerance, and liver fat deposition were aggravated in old mice. Aging significantly increased pro-inflammation in hepatic macrophages. Furthermore, we found that Kupffer cells (KCs) were the major driver to induce pro-inflammation in hepatic macrophages during aging. In KCs, aging significantly increased pro-inflammatory levels; in monocyte-derived macrophages (MDMs), aging had a limited effect on pro-inflammation but led to a functional quiescence in antigen presentation and phagosome process. In addition, we identified an aging-responsive KC-specific (ARKC) gene set that potentially mediates aging-induced pro-inflammation in KCs. Interestingly, FOXO1 activity was significantly increased in the liver of old mice. FOXO1 inhibition by AS1842856 significantly alleviated glucose intolerance, hepatic steatosis, and systemic inflammation in old mice. FOXO1 inhibition significantly attenuated aging-induced pro-inflammation in KCs partially through downregulation of ARKC genes. However, FOXO1 inhibition had a limited effect on aging-induced functional quiescence in MDMs. These results indicate that aging induces pro-inflammation in liver mainly through targeting KCs and FOXO1 is a key player in aging-induced pro-inflammation in KCs. Thus, FOXO1 could be a potential therapeutic target for the treatment of age-associated chronic diseases.

The Assembled Genome of the Stroke-Prone Spontaneously Hypertensive Rat.

BACKGROUND: We report the creation and evaluation of a de novo assembly of the genome of the spontaneously hypertensive rat, the most widely used model of human cardiovascular disease. METHODS: The genome is assembled from long read sequencing (PacBio HiFi and continuous long read data [CLR]) and scaffolded with long-range structural information obtained from Bionano optical maps and proximity ligation sequencing proximity analysis of the genome. The genome assembly was polished with Illumina short reads. Completeness of the assembly was investigated using Benchmarking Universal Single Copy Orthologs analysis. The genome assembly was also evaluated with the rat reference gene set, using NCBI automated protocols. We also generated orthogonal single molecule transcript sequence reads (Iso-Seq) from 8 tissues and used them to validate the coding assembly, to annotate the assembly with RNA transcripts representing unique full length transcript isoforms for each gene and to determine whether divergences between RefSeq sequences and the assembly were attributable to assembly errors or polymorphisms. RESULTS: The assembly analysis indicates that this assembly is comparable in contiguity and completeness to the current rat reference assembly, while the use of HiFi sequencing yields an assembly that is more correct at the single base level. Synteny analysis was performed to uncover the extent of synteny and the presence and distribution of chromosomal rearrangements between the reference and this assembly. CONCLUSION: The resulting genome assembly is reference quality and captures significant structural variation.

Impact of E-cig aerosol vaping on fetal and neonatal respiratory development and function.

Electronic cigarette (e-cig) use has increased over the past decade, and exposure to e-cig aerosols during pregnancy raises concern for maternal and fetal health. The developing fetal lung is known to be sensitive to prenatal tobacco product exposure. Utilizing a 3-pronged approach, we examined the effects of prenatal e-cig aerosols with, and without nicotine on respiratory development in a murine model. RNAseq analysis of fetal lungs revealed extensive dysregulation in gene expression. Morphologic assessment of distal airspaces in neonatal lungs display an emphysematic phenotype. Respiratory mechanics of neonates display signs of increased respiratory workload, with increased resistance and decreased compliance. These data are novel and provide evidence that prenatal e-cig exposure may result in altered lung function or development of disease.

Case Report: Paucisymptomatic College-Age Population as a Reservoir for Potentially Neutralization-Resistant Severe Acute Respiratory Syndrome Coronavirus 2 Variants.

To better understand the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant lineage distribution in a college campus population, we carried out viral genome surveillance over a 7-week period from January to March 2021. Among the sequences were three novel viral variants: BV-1 with a B.1.1.7/20I genetic background and an additional spike mutation Q493R, associated with a mild but longer-than-usual COVID-19 case in a college-age person, BV-2 with a T478K mutation on a 20B genetic background, and BV-3, an apparent recombinant lineage. This work highlights the potential of an undervaccinated younger population as a reservoir for the spread and generation of novel variants. This also demonstrates the value of whole genome sequencing as a routine disease surveillance tool.