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Acute intoxication with cholinesterase inhibiting organophosphates (OP) can produce life-threatening cholinergic crisis and status epilepticus (SE). Survivors often develop long-term neurological consequences, including spontaneous recurrent seizures (SRS) and impaired cognition. Numerous studies implicate OP-induced neuroinflammation as a pathogenic mechanism contributing to these chronic sequelae; however, little is known about the inflammatory phenotype of innate immune cells in the brain following acute OP intoxication. Thus, the aim of this study was to characterize the natural history of microglial and astrocytic inflammatory phenotypes following acute intoxication with the OP, diisopropylfluorophosphate (DFP). Adult male and female Sprague-Dawley rats were administered a single dose of DFP (4 mg/kg, sc) followed by standard medical countermeasures. Within minutes, animals developed benzodiazepine-resistant SE as determined by monitoring seizures using a modified Racine scale. At 1, 3, 7, 14, and 28 d post-exposure (DPE), neuroinflammation was assessed using translocator protein (TSPO) positron emission tomography (PET) and magnetic resonance imaging (MRI). In both sexes, we observed consistently elevated radiotracer uptake across all examined brain regions and time points. A separate group of animals was euthanized at these same time points to collect tissues for immunohistochemical analyses. Colocalization of IBA-1, a marker for microglia, with iNOS or Arg1 was used to identify pro- and anti-inflammatory microglia, respectively; colocalization of GFAP, a marker for astrocytes, with C3 or S100A10, pro- and anti-inflammatory astrocytes, respectively. We observed shifts in the inflammatory profiles of microglia and astrocyte populations during the first month post-intoxication, largely in hyperintense inflammatory lesions in the piriform cortex and amygdala regions. In these areas, iNOS+ proinflammatory microglial cell density peaked at 3 and 7 DPE, while anti-inflammatory Arg1+ microglia cell density peaked at 14 DPE. Pro- and anti-inflammatory astrocytes emerged within 7 DPE, and roughly equal ratios of C3+ pro-inflammatory and S100A10+ anti-inflammatory astrocytes persisted at 28 DPE. In summary, microglia and astrocytes adopted mixed inflammatory phenotypes post-OP intoxication, which evolved over one month post exposure. These activated cell populations were most prominent in the piriform and amygdala areas and were more abundant in males compared to females. The temporal relationship between microglial and astrocytic responses suggests that initial microglial activity may influence delayed, persistent astrocytic responses. Further, our findings identify putative windows for inhibition of OP-induced neuroinflammatory responses in both sexes to evaluate the therapeutic benefit of anti-inflammation in this context.

Advances in sequencing technologies have enabled the comparison of high-quality genomes of diverse primate species, revealing vast amounts of divergence due to structural variation. Given their large size, structural variants (SVs) can simultaneously alter the function and regulation of multiple genes. Studies estimate that collectively more than 3.5% of the genome is divergent in humans versus other great apes, impacting thousands of genes. Functional genomics and gene-editing tools in various model systems recently emerged as an exciting frontier - investigating the wide-ranging impacts of SVs on molecular, cellular, and systems-level phenotypes. This review examines existing research and identifies future directions to broaden our understanding of the functional roles of SVs on phenotypic innovations and diversity impacting uniquely human features, ranging from cognition to metabolic adaptations.

The International Mouse Phenotyping Consortium (IMPC) is a worldwide effort producing and phenotyping knockout mouse lines to expose the pathophysiological roles of all genes in human diseases and make mice and data available and accessible to the global research community. It has created new knowledge on the function of thousands of genes for which little to anything was known. This new knowledge has informed the genetic basis of rare diseases, posited gene product influences on common diseases, influenced research on targeted therapies, revealed functional pleiotropy, essentiality, and sexual dimorphism, and many more insights into the role of genes in health and disease. Its scientific contributions have been many and widespread, however there remain thousands of "dark" genes yet to be illuminated. Nearing the end of its current funding cycle, IMPC is at a crossroads. The vision forward is clear, the path to proceed less so.

The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps - generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

The Greying with age phenotype in horses involves loss of hair pigmentation whereas skin pigmentation is not reduced, and a predisposition to melanoma. The causal mutation was initially reported as a duplication of a 4.6 kb intronic sequence in Syntaxin 17. The speed of greying varies considerably among Grey horses. Here we demonstrate the presence of two different Grey alleles, G2 carrying two tandem copies of the duplicated sequence and G3 carrying three. The latter is by far the most common allele, probably due to strong selection for the striking white phenotype. Our results reveal a remarkable dosage effect where the G3 allele is associated with fast greying and high incidence of melanoma whereas G2 is associated with slow greying and low incidence of melanoma. The copy number expansion transforms a weak enhancer to a strong melanocyte-specific enhancer that underlies hair greying (G2 and G3) and a drastically elevated risk of melanoma (G3 only). Our direct pedigree-based observation of the origin of a G2 allele from a G3 allele by copy number contraction demonstrates the dynamic evolution of this locus and provides the ultimate evidence for causality of the copy number variation of the 4.6 kb intronic sequence.

The Mouse Metabolic Phenotyping Center (MMPC)Live Program was established in 2023 by the National Institute for Diabetes, Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH) to advance biomedical research by providing the scientific community with standardized, high quality phenotyping services for mouse models of diabetes and obesity. Emerging as the next iteration of the MMPC Program which served the biomedical research community for 20 years (2001-2021), MMPCLive is designed as an outwardly-facing consortium of service cores that collaborate to provide reduced-cost consultation and metabolic, physiologic, and behavioral phenotyping tests on live mice for U.S. biomedical researchers. Four MMPCLive Centers located at universities around the country perform complex and often unique procedures in vivo on a fee for service basis, typically on mice shipped from the client or directly from a repository or vendor. Current areas of expertise include energy balance and body composition, insulin action and secretion, whole body carbohydrate and lipid metabolism, cardiovascular and renal function, food intake and behavior, microbiome and xenometabolism, and metabolic pathway kinetics. Additionally, an opportunity arose to reduce barriers to access and expand the diversity of the biomedical research workforce by establishing the VIBRANT Program. Directed at researchers historically underrepresented in the biomedical sciences, VIBRANT-eligible investigators have access to testing services, travel and career development awards, expert advice and experimental design consultation, and short internships to learn test technologies. Data derived from experiments run by the Centers belongs to the researchers submitting mice for testing which can be made publicly available and accessible from the MMPCLive database following publication. In addition to services, MMPCLive staff provide expertise and advice to researchers, develop and refine test protocols, engage in outreach activities, publish scientific and technical papers, and conduct educational workshops and training sessions to aid researchers in unraveling the heterogeneity of diabetes and obesity.

BACKGROUND: The pleiotropic effects of the melanocortin system show promise in overcoming limitations associated with large variations in opioid analgesic effectiveness observed in equine practice. Of particular interest is variation in the melanocortin-1-receptor (MC1R) gene, which dictates pigment type expression through its epistatic interaction with the agouti signalling protein (ASIP) gene. MC1R has previously been implicated in opioid efficacy in other species; however, this relationship is yet to be explored in horses. In this study, analgesic effectiveness was scored (1-3) based on noted response to dura penetration during the performance of cerebrospinal fluid centisis after sedation and tested for association with known genetic regions responsible for pigmentation variation in horses. RESULTS: The chestnut phenotype was statistically significant (P < 0.05) in lowering analgesic effectiveness when compared to the bay base coat colour. The 11bp indel in ASIP known to cause the black base coat colour was not significant (P>0.05); however, six single nucleotide polymorphisms (SNPs) within the genomic region encoding the ASIP gene and one within MC1R were identified as being nominally significant (P

OBJECTIVE: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DESIGN: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. RESULTS: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. CONCLUSIONS: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.

Anisogamy, different-sized male and female gametes, sits at the heart of sexual selection and conflict between the sexes. Sperm producers (males) and egg producers (females) of the same species generally share most, if not all, of the same genome, but selection frequently favours different trait values in each sex for traits common to both. The extent to which this conflict might be resolved, and the potential mechanisms by which this can occur, have been widely debated. Here, we summarize recent findings and emphasize that once the sexes evolve, sexual selection is ongoing, and therefore new conflict is always possible. In addition, sexual conflict is largely a multivariate problem, involving trait combinations underpinned by networks of interconnected genes. Although these complexities can hinder conflict resolution, they also provide multiple possible routes to decouple male and female phenotypes and permit sex-specific evolution. Finally, we highlight difficulty in the study of sexual conflict over shared traits and promising directions for future research.

Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding of how developmental programs react to these challenges remains limited. Here, we have examined the phenotypic consequences of disturbing the bicoid regulatory network in early Drosophila embryos. We generated flies with two extra copies of bicoid, which causes a posterior shift of the networks regulatory outputs and a decrease in fitness. We subjected these flies to EMS mutagenesis, followed by experimental evolution. After only 8-15 generations, experimental populations have normalized patterns of gene expression and increased survival. Using a phenomics approach, we find that populations were normalized through rapid increases in embryo size driven by maternal changes in metabolism and ovariole development. We extend our results to additional populations of flies, demonstrating predictability. Together, our results necessitate a broader view of regulatory network evolution at the systems level.

PurposeMutations in the genes encoding type IV collagen alpha 1 (COL4A1) and alpha 2 (COL4A2) cause a multisystem disorder that includes ocular anterior segment dysgenesis (ASD) and glaucoma. We previously showed that transforming growth factor beta (TGFβ) signaling was elevated in developing anterior segments from Col4a1 mutant mice and that reducing TGFβ signaling ameliorated ASD, supporting a role for the TGFβ pathway in disease pathogenesis. Here, we tested whether altered TGFβ signaling also contributes to glaucoma-related phenotypes in Col4a1 mutant mice.MethodsTo test the role of TGFβ signaling in glaucoma-relevant phenotypes, we genetically reduced TGFβ signaling using mice with mutated Tgfbr2, which encodes the common receptor for all TGFβ ligands in Col4a1+/G1344D mice. We performed slit-lamp biomicroscopy and optical coherence tomography for qualitative and quantitative analyses of anterior and posterior ocular segments, histological analyses of ocular tissues and optic nerves, and intraocular pressure assessments using rebound tonometry.ResultsCol4a1+/G1344D mice showed defects of the ocular drainage structures, including iridocorneal adhesions, and phenotypes consistent with glaucomatous neurodegeneration, including thinning of the nerve fiber layer, retinal ganglion cell loss, optic nerve head excavation, and optic nerve degeneration. We found that reducing TGFβ receptor 2 (TGFBR2) was protective for ASD, ameliorated ocular drainage structure defects, and protected against glaucomatous neurodegeneration in Col4a1+/G1344D mice.ConclusionsOur results suggest that elevated TGFβ signaling contributes to glaucomatous neurodegeneration in Col4a1 mutant mice.

Endurance exercise training is known to reduce risk for a range of complex diseases. However, the molecular basis of this effect has been challenging to study and largely restricted to analyses of either few or easily biopsied tissues. Extensive transcriptome data collected across 15 tissues during exercise training in rats as part of the Molecular Transducers of Physical Activity Consortium has provided a unique opportunity to clarify how exercise can affect tissue-specific gene expression and further suggest how exercise adaptation may impact complex disease-associated genes. To build this map, we integrate this multi-tissue atlas of gene expression changes with gene-disease targets, genetic regulation of expression, and trait relationship data in humans. Consensus from multiple approaches prioritizes specific tissues and genes where endurance exercise impacts disease-relevant gene expression. Specifically, we identify a total of 5523 trait-tissue-gene triplets to serve as a valuable starting point for future investigations [Exercise; Transcription; Human Phenotypic Variation].

Phenotypic variation among species is a product of evolutionary changes to developmental programs1,2. However, how these changes generate novel morphological traits remains largely unclear. Here we studied the genomic and developmental basis of the mammalian gliding membrane, or patagium-an adaptative trait that has repeatedly evolved in different lineages, including in closely related marsupial species. Through comparative genomic analysis of 15 marsupial genomes, both from gliding and non-gliding species, we find that the Emx2 locus experienced lineage-specific patterns of accelerated cis-regulatory evolution in gliding species. By combining epigenomics, transcriptomics and in-pouch marsupial transgenics, we show that Emx2 is a critical upstream regulator of patagium development. Moreover, we identify different cis-regulatory elements that may be responsible for driving increased Emx2 expression levels in gliding species. Lastly, using mouse functional experiments, we find evidence that Emx2 expression patterns in gliders may have been modified from a pre-existing program found in all mammals. Together, our results suggest that patagia repeatedly originated through a process of convergent genomic evolution, whereby regulation of Emx2 was altered by distinct cis-regulatory elements in independently evolved species. Thus, different regulatory elements targeting the same key developmental gene may constitute an effective strategy by which natural selection has harnessed regulatory evolution in marsupial genomes to generate phenotypic novelty.

In animal models, Nipbl deficiency phenocopies gene expression changes and birth defects seen in Cornelia de Lange syndrome, the most common cause of which is Nipbl haploinsufficiency. Previous studies in Nipbl+/- mice suggested that heart development is abnormal as soon as cardiogenic tissue is formed. To investigate this, we performed single-cell RNA sequencing on wild-type and Nipbl+/- mouse embryos at gastrulation and early cardiac crescent stages. Nipbl+/- embryos had fewer mesoderm cells than wild-type and altered proportions of mesodermal cell subpopulations. These findings were associated with underexpression of genes implicated in driving specific mesodermal lineages. In addition, Nanog was found to be overexpressed in all germ layers, and many gene expression changes observed in Nipbl+/- embryos could be attributed to Nanog overexpression. These findings establish a link between Nipbl deficiency, Nanog overexpression, and gene expression dysregulation/lineage misallocation, which ultimately manifest as birth defects in Nipbl+/- animals and Cornelia de Lange syndrome.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

ObjectiveThis study was undertaken to assess reproducibility of the epilepsy outcome and phenotype in a lateral fluid percussion model of posttraumatic epilepsy (PTE) across three study sites.MethodsA total of 525 adult male Sprague Dawley rats were randomized to lateral fluid percussion-induced brain injury (FPI) or sham operation. Of these, 264 were assigned to magnetic resonance imaging (MRI cohort, 43 sham, 221 traumatic brain injury [TBI]) and 261 to electrophysiological follow-up (EEG cohort, 41 sham, 220 TBI). A major effort was made to harmonize the rats, materials, equipment, procedures, and monitoring systems. On the 7th post-TBI month, rats were video-EEG monitored for epilepsy diagnosis.ResultsA total of 245 rats were video-EEG phenotyped for epilepsy on the 7th postinjury month (121 in MRI cohort, 124 in EEG cohort). In the whole cohort (n = 245), the prevalence of PTE in rats with TBI was 22%, being 27% in the MRI and 18% in the EEG cohort (p > .05). Prevalence of PTE did not differ between the three study sites (p > .05). The average seizure frequency was .317 ± .725 seizures/day at University of Eastern Finland (UEF; Finland), .085 ± .067 at Monash University (Monash; Australia), and .299 ± .266 at University of California, Los Angeles (UCLA; USA; p  .05). Of the 219 seizures, 53% occurred as part of a seizure cluster (≥3 seizures/24 h; p >.05 between the study sites). Of the 209 seizures, 56% occurred during lights-on period and 44% during lights-off period (p > .05 between the study sites).SignificanceThe PTE phenotype induced by lateral FPI is reproducible in a multicenter design. Our study supports the feasibility of performing preclinical multicenter trials in PTE to increase statistical power and experimental rigor to produce clinically translatable data to combat epileptogenesis after TBI.

The high incidence of epithelial malignancies in HIV-1 infected individuals is associated with co-infection with oncogenic viruses, such as high-risk human papillomaviruses (HR HPVs), mostly HPV16. The molecular mechanisms underlying the HIV-1-associated increase in epithelial malignancies are not fully understood. A collaboration between HIV-1 and HR HPVs in the malignant transformation of epithelial cells has long been anticipated. Here, we delineated the effects of HIV-1 reverse transcriptase on the in vitro and in vivo properties of HPV16-infected cervical cancer cells. A human cervical carcinoma cell line infected with HPV16 (Ca Ski) was made to express HIV-1 reverse transcriptase (RT) by lentiviral transduction. The levels of the mRNA of the E6 isoforms and of the factors characteristic to the epithelial/mesenchymal transition were assessed by real-time RT-PCR. The parameters of glycolysis and mitochondrial respiration were determined using Seahorse technology. RT expressing Ca Ski subclones were assessed for the capacity to form tumors in nude mice. RT expression increased the expression of the E6*I isoform, modulated the expression of E-CADHERIN and VIMENTIN, indicating the presence of a hybrid epithelial/mesenchymal phenotype, enhanced glycolysis, and inhibited mitochondrial respiration. In addition, the expression of RT induced phenotypic alterations impacting cell motility, clonogenic activity, and the capacity of Ca Ski cells to form tumors in nude mice. These findings suggest that HIV-RT, a multifunctional protein, affects HPV16-induced oncogenesis, which is achieved through modulation of the expression of the E6 oncoprotein. These results highlight a complex interplay between HIV antigens and HPV oncoproteins potentiating the malignant transformation of epithelial cells.

BACKGROUND: How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum have long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. RESULTS: The final assembled and filtered Berghia genome is comparable to other high-quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes) and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. CONCLUSIONS: Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

Disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability (SRID). Without functional SynGAP1 protein, individuals are developmentally delayed and have prominent features of intellectual disability (ID), motor impairments, and epilepsy. Over the past two decades, there have been numerous discoveries indicating the critical role of Syngap1. Several rodent models with a loss of Syngap1 have been engineered, identifying precise roles in neuronal structure and function, as well as key biochemical pathways key for synapse integrity. Homozygous loss of SYNGAP1/Syngap1 is lethal. Heterozygous mutations of Syngap1 result in a broad range of behavioral phenotypes. Our in vivo functional data, using the original mouse model from the Huganir laboratory, corroborated behaviors including robust hyperactivity and deficits in learning and memory in young adults. Furthermore, we described impairments in the domain of sleep, characterized using neurophysiological data that was collected with wireless, telemetric electroencephalography (EEG). Syngap1+/- mice exhibited elevated spiking events and spike trains, in addition to elevated power, most notably in the delta power frequency. For the first time, we illustrated that primary neurons from Syngap1+/- mice displayed: 1) increased network firing activity, 2) greater bursts, 3) and shorter inter-burst intervals between peaks, by utilizing high density microelectrode arrays (HD-MEA). Our work bridges in vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate quantitative, translational biomarkers in vivo and in vitro that can be utilized for the development and efficacy assessment of targeted treatments for SRID.

This study aimed to characterise three Ghanaian local chicken ecotypes, namely, Interior Savannah, Forest, and Coastal Savannah, based on morphological data and single nucleotide polymorphism (SNP) genotypes. Morphological data including body weight, shank length, body girth, back length, thigh length, beak length, comb length, and wattle length were collected from 250 local chickens. DNA isolated from blood of 1,440 local chickens was used for SNP genotyping with the Affymetrix chicken 600k SNP chip. Principal component analysis showed that Forest and Coastal Savannah birds were closely related. Generally, all three ecotypes exhibited high genetic diversity, especially birds from the Interior Savannah zone. Morphological characterisation showed that ecotype (p = 0.016) and sex (p = 0.000) had significant effects on body weight. Birds of the Interior Savannah ecotype were the heaviest (p = 0.004), with mean weights of 1.23 kg for females and 1.40 kg for males. Sex also had a strong significant effect on most of the morphological measurements, but the sex * ecotype interaction effect was not significant. Very few of the feather phenotypes previously reported to be associated with heat resistance-frizzle (2%) and naked neck (1.6%)-were found in the studied populations. It is concluded that the three local ecotypes are genetically diverse but with similar morphological features and the information provided would be useful for future selection decisions.

In various organisms, sequencing of selectively bred lines at apparent selection limits has demonstrated that genetic variation can remain at many loci, implying that evolution at the genetic level may continue even if the population mean phenotype remains constant. We compared selection signatures at generations 22 and 61 of the High Runner mouse experiment, which includes 4 replicate lines bred for voluntary wheel-running behavior (HR) and 4 non-selected control (C) lines. Previously, we reported multiple regions of differentiation between the HR and C lines, based on whole-genome sequence data for 10 mice from each line at generation 61, which was >31 generations after selection limits had been reached in all HR lines. Here, we analyzed pooled sequencing data from ~20 mice for each of the 8 lines at generation 22, around when HR lines were reaching limits. Differentiation analyses of allele frequencies at ~4.4 million SNP loci used the regularized T-test and detected 258 differentiated regions with FDR = 0.01. Comparable analyses involving pooling generation 61 individual mouse genotypes into allele frequencies by line produced only 11 such regions, with almost no overlap among the largest and most statistically significant peaks between the two generations. These results implicate a sort of genetic churn that continues at loci relevant for running. Simulations indicate that loss of statistical power due to random genetic drift and sampling error are insufficient to explain the differences in selection signatures. The 13 differentiated regions at generation 22 with strict culling measures include 79 genes related to a wide variety of functions. Gene ontology identified pathways related to olfaction and vomeronasal pathways as being overrepresented, consistent with generation 61 analyses, despite those specific regions differing between generations. Genes Dspp and Rbm24 are also identified as potentially explaining known bone and skeletal muscle differences, respectively, between the linetypes.

BACKGROUND: Detailed descriptions of clinical signs associated with radiological findings of the caudal cervical vertebral column are not available. OBJECTIVES/HYPOTHESES: Describe the clinical features associated with neck pain or stiffness, neck-related thoracic limb lameness, proprioceptive ataxia consistent with a cervicothoracic spinal cord or nerve lesion, and their frequency of occurrence compared with control horses. ANIMALS: A total of 223 Warmblood horses. METHODS: Case-control study. Controls and cases were recruited prospectively. All horses underwent predetermined lameness and neurologic examinations. The frequency of occurrence of each clinical feature was compared between cases and controls and relative risk (RR) were calculated. RESULTS: Ninety-six cases and 127 controls were included. Forty-seven (49%) of the cases were classified as neurologic, 31 (32.3%) had thoracic limb lameness, and 18 (18.7%) had neck stiffness or pain or both. Focal caudal cervical muscle atrophy (46, 47.9%), hypoesthesia (38, 39.6%), patchy sweating (16, 16.7%), hyperesthesia (11, 11.5%), and pain upon firm pressure applied over the caudal cervical articular process joints and transverse processes (58, 60.4%) were only observed in cases (P

Understanding the evolutionary mechanisms underlying the maintenance of individual differences in behavior and physiology is a fundamental goal in ecology and evolution. The pace-of-life syndrome hypothesis is often invoked to explain the maintenance of such within-population variation. This hypothesis predicts that behavioral traits are part of a suite of correlated traits that collectively determine an individual's propensity to prioritize reproduction or survival. A key assumption of this hypothesis is that these traits are underpinned by genetic trade-offs among life-history traits: genetic variants that increase fertility, reproduction and growth might also reduce lifespan. We performed a systematic literature review and meta-analysis to summarize the evidence for the existence of genetic trade-offs between five key life-history traits: survival, growth rate, body size, maturation rate, and fertility. Counter to our predictions, we found an overall positive genetic correlation between survival and other life-history traits and no evidence for any genetic correlations between the non-survival life-history traits. This finding was generally consistent across pairs of life-history traits, sexes, life stages, lab vs. field studies, and narrow- vs. broad-sense correlation estimates. Our study highlights that genetic trade-offs may not be as common, or at least not as easily quantifiable, in animals as often assumed.

Understanding and treating human diseases require valid animal models. Leveraging the genetic diversity in rhesus macaque populations across eight primate centers in the United States, we conduct targeted-sequencing on 1845 individuals for 374 genes linked to inherited human retinal and neurodevelopmental diseases. We identify over 47,000 single nucleotide variants, a substantial proportion of which are shared with human populations. By combining rhesus and human allele frequencies with established variant prediction methods, we develop a machine learning-based score that outperforms established methods in predicting missense variant pathogenicity. Remarkably, we find a marked number of loss-of-function variants and putative deleterious variants, which may lead to the development of rhesus disease models. Through phenotyping of macaques carrying a pathogenic OPA1:p.A8S variant, we identify a genetic model of autosomal dominant optic atrophy. Finally, we present a public website housing variant and genotype data from over two thousand rhesus macaques.

BACKGROUND: Schistosomiasis is a common cause of pulmonary hypertension (PH) worldwide. Type 2 inflammation contributes to the development of Schistosoma-induced PH. Specifically, interstitial macrophages (IMs) derived from monocytes play a pivotal role by producing thrombospondin-1 (TSP-1), which in turn activates TGF-β, thereby driving the pathology of PH. Resident and recruited IM subpopulations have recently been identified. We hypothesized that in Schistosoma-PH, one IM subpopulation expresses monocyte recruitment factors, whereas recruited monocytes become a separate IM subpopulation that expresses TSP-1. METHODS: Mice were intraperitoneally sensitized and then intravenously challenged with S. mansoni eggs. Flow cytometry on lungs and blood was performed on wildtype and reporter mice to identify IM subpopulations and protein expression. Single-cell RNA sequencing (scRNAseq) was performed on flow-sorted IMs from unexposed and at day 1, 3 and 7 following Schistosoma exposure to complement flow cytometry based IM characterization and identify gene expression. RESULTS: Flow cytometry and scRNAseq both identified 3 IM subpopulations, characterized by CCR2, MHCII, and FOLR2 expression. Following Schistosoma exposure, the CCR2+ IM subpopulation expanded, suggestive of circulating monocyte recruitment. Schistosoma exposure caused increased monocyte-recruitment ligand CCL2 expression in the resident FOLR2+ IM subpopulation. In contrast, the vascular pathology-driving protein TSP-1 was greatest in the CCR2+ IM subpopulation. CONCLUSION: Schistosoma-induced PH involves crosstalk between IM subpopulations, with increased expression of monocyte recruitment ligands by resident FOLR2+ IMs, and the recruitment of CCR2+ IMs which express TSP-1 that activates TGF-β and causes PH.

Candida auris has become a serious threat to public health. The mechanisms of how this fungal pathogen adapts to the mammalian host are poorly understood. Here we report the rapid evolution of an adaptive C. auris multicellular aggregative morphology in the murine host during systemic infection. C. auris aggregative cells accumulate in the brain and exhibit obvious advantages over the single-celled yeast-form cells during systemic infection. Genetic mutations, specifically de novo point mutations in genes associated with cell division or budding processes, underlie the rapid evolution of this aggregative phenotype. Most mutated C. auris genes are associated with the regulation of cell wall integrity, cytokinesis, cytoskeletal properties, and cellular polarization. Moreover, the multicellular aggregates are notably more recalcitrant to the host antimicrobial peptides LL-37 and PACAP relative to the single-celled yeast-form cells. Overall, to survive in the host, C. auris can rapidly evolve a multicellular aggregative morphology via genetic mutations.

Acute myeloid leukemia (AML) is prevalent in both adult and pediatric patients. Despite advances in patient categorization, the heterogeneity of AML remains a challenge. Recent studies have explored the use of gene expression data to enhance AML diagnosis and prognosis, however, alternative approaches rooted in physics and chemistry may provide another level of insight into AML transformation. Utilizing publicly available databases, we analyze 884 human and mouse blood and bone marrow samples. We employ a personalized medicine strategy, combining state-transition theory and surprisal analysis, to assess the RNA transcriptome of individual patients. The transcriptome is transformed into physical parameters that represent each sample's steady state and the free energy change (FEC) from that steady state, which is the state with the lowest free energy.We found the transcriptome steady state was invariant across normal and AML samples. FEC, representing active molecular processes, varied significantly between samples and was used to create patient-specific barcodes to characterize the biology of the disease. We discovered that AML samples that were in a transition state had the highest FEC. This disease state may be characterized as the most unstable and hence the most therapeutically targetable since a change in free energy is a thermodynamic requirement for disease progression. We also found that distinct sets of ongoing processes may be at the root of otherwise similar clinical phenotypes, implying that our integrated analysis of transcriptome profiles may facilitate a personalized medicine approach to cure AML and restore a steady state in each patient.

The Farm Animal Genotype-Tissue Expression (FarmGTEx) project has been established to develop a public resource of genetic regulatory variants in livestock, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biological discovery and exploitation in animal breeding and human biomedicine. Here we show results from the pilot phase of PigGTEx by processing 5,457 RNA-sequencing and 1,602 whole-genome sequencing samples passing quality control from pigs. We build a pig genotype imputation panel and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We evaluate tissue specificity of regulatory effects and elucidate molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying 207 pig complex phenotypes and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, supporting the importance of pigs as a human biomedical model.

Despite great progress in understanding lipoprotein physiology, there is still much to be learned about the genetic drivers of lipoprotein abundance, composition, and function. We used ion mobility spectrometry to survey 16 plasma lipoprotein subfractions in 500 Diversity Outbred mice maintained on a Western-style diet. We identified 21 quantitative trait loci (QTL) affecting lipoprotein abundance. To refine the QTL and link them to disease risk in humans, we asked if the human homologs of genes located at each QTL were associated with lipid traits in human genome-wide association studies. Integration of mouse QTL with human genome-wide association studies yielded candidate gene drivers for 18 of the 21 QTL. This approach enabled us to nominate the gene encoding the neutral ceramidase, Asah2, as a novel candidate driver at a QTL on chromosome 19 for large HDL particles (HDL-2b). To experimentally validate Asah2, we surveyed lipoproteins in Asah2-/- mice. Compared to wild-type mice, female Asah2-/- mice showed an increase in several lipoproteins, including HDL. Our results provide insights into the genetic regulation of circulating lipoproteins, as well as mechanisms by which lipoprotein subfractions may affect cardiovascular disease risk in humans.

The composite material-like extracellular matrix (ECM) in the sinoatrial node (SAN) supports the native pacemaking cardiomyocytes (PCMs). To test the roles of SAN ECM in the PCM phenotype and function, we engineered reconstructed-SAN heart tissues (rSANHTs) by recellularizing porcine SAN ECMs with hiPSC-derived PCMs. The hiPSC-PCMs in rSANHTs self-organized into clusters resembling the native SAN and displayed higher expression of pacemaker-specific genes and a faster automaticity compared with PCMs in reconstructed-left ventricular heart tissues (rLVHTs). To test the protective nature of SAN ECMs under strain, rSANHTs and rLVHTs were transplanted onto the murine thoracic diaphragm to undergo constant cyclic strain. All strained-rSANHTs preserved automaticity, whereas 66% of strained-rLVHTs lost their automaticity. In contrast to the strained-rLVHTs, PCMs in strained-rSANHTs maintained high expression of key pacemaker genes (HCN4, TBX3, and TBX18). These findings highlight the promotive and protective roles of the composite SAN ECM and provide valuable insights for pacemaking tissue engineering.

Dynamic lower airway obstruction is the primary component of canine bronchomalacia, but the ventilatory function remains underinvestigated. This prospective study analyzed tidal breathing characteristics in 28 dogs, comprising 14 with severe bronchomalacia diagnosed by bronchoscopy versus 14 without respiratory disease. Spirometry was conducted in all dogs. Bronchoscopy with bronchoalveolar lavage or brush under anesthesia was performed in 14 dogs with cough and expiratory effort. Severe bronchomalacia was defined by the severity of collapse and total number of bronchi affected. Ventilatory characteristics were compared between groups. Results revealed that dogs with severe bronchomalacia had lower minute volume (218 vs 338 mL/kg, p = .039) and greater expiratory-to-inspiratory time ratio (1.55 vs 1.35, p = .01) compared to control dogs. The tidal breathing pattern of dogs with bronchomalacia was different from that of normal dogs, and the pattern differed from the concave or flat expiratory curves typical of lower airway obstruction. Compared to control dogs, dogs with severe bronchomalacia had a significantly prolonged low-flow expiratory phase (p < .001) on the flow-time plot and a more exponential shape of the expiratory curve (p < .001) on the volume-time plot. Flow-time index ExpLF/Te (>0.14) and volume-time index Vt-AUCexp (≤31%) had a high ROC-AUC (1.00, 95% confidence interval 0.88 to 1.00) in predicting severe bronchomalacia. In conclusion, the tidal breathing pattern identified here indicates abnormal and complicated ventilatory mechanics in dogs with severe bronchomalacia. The role of this pulmonary functional phenotype should be investigated for disease progression and therapeutic monitoring in canine bronchomalacia.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

Tephritid fruit fly pests pose an increasing threat to the agricultural industry due to their global dispersion and a highly invasive nature. Here we showcase the feasibility of an early-detection SEPARATOR sex sorting approach through using the non-model Tephritid pest, Ceratitis capitata. This system relies on female-only fluorescent marker expression, accomplished through the use of a sex-specific intron of the highly-conserved transformer gene from C. capitata and Anastrepha ludens. The herein characterized strains have 100% desired phenotype outcomes, allowing accurate male-female separation during early development. Overall, we describe an antibiotic and temperature-independent sex-sorting system in C. capitata, which, moving forward, may be implemented in other non-model Tephritid pest species. This strategy can facilitate the establishment of genetic sexing systems with endogenous elements exclusively, which, on a wider scale, can improve pest population control strategies like sterile insect technique.

Aging is a nearly inescapable trait among organisms yet lifespan varies tremendously across different species and spans several orders of magnitude in vertebrates alone. This vast phenotypic diversity is driven by distinct evolutionary trajectories and tradeoffs that are reflected in patterns of diversification and constraint in organismal genomes. Age-specific impacts of selection also shape allele frequencies in populations, thus impacting disease susceptibility and environment-specific mortality risk. Further, the mutational processes that spawn this genetic diversity in both germline and somatic cells are strongly influenced by age and life history. We discuss recent advances in our understanding of the evolution of aging and lifespan at organismal, population, and cellular scales, and highlight outstanding questions that remain unanswered.

Long-term management of inflammatory skin diseases is challenging because of side effects from repeated use of systemic treatments or topical corticosteroids. This study sought to identify the mechanisms and developmental therapeutics for these diseases using genetic models and pharmacological approaches. We found that mice overexpressing SMAD7 in keratinocytes but not mice overexpressing the N-terminal domain of SMAD7 (i.e., N-SMAD7) were resistant to imiquimod-induced T helper 1/17- and T helper 2-type inflammation. We generated a Tat-PYC-SMAD7 (truncated SMAD7 protein encompassing C-terminal SMAD7 and PY motif fused with cell-penetrating Tat peptide). Topically applied Tat-PYC-SMAD7 to inflamed skin entered cells upon contact and attenuated imiquimod-, 2,4-dinitrofluorobenzene-, and tape-stripping-induced inflammation. RNA-sequencing analyses of mouse skin exposed to these insults showed that in addition to inhibiting TGFβ/NF-κB, SMAD7 blunted IL-22/signal transducer and activator of transcription 3 activation and associated pathogenesis, which is due to SMAD7 transcriptionally upregulating IL-22 antagonist IL-22RA2. Mechanistically, SMAD7 facilitated nuclear translocation and DNA binding of C/EBPβ to IL22RA2 promoter for IL22RA2 transactivation. Consistent with the observations in mice mentioned earlier, transcript levels of IL22RA2 were increased in human atopic dermatitis and psoriasis lesions with clinical remission. Our study identified the anti-inflammation functional domain of SMAD7 and suggests the mechanism and feasibility for developing SMAD7-based biologics as a topical therapy for skin inflammatory disorders.

Mucopolysaccharidosis III (MPSIII, Sanfilippo syndrome) is a devastating lysosomal storage disease that primarily affects the central nervous system. MPSIIIA is caused by loss-of-function mutations in the gene coding for sulfamidase (N-sulfoglucosamine sulfohydrolase/SGSH) resulting in SGSH enzyme deficiency, a buildup of heparin sulfate and subsequent neurodegeneration. There is currently no cure or disease modifying treatment for MPSIIIA. A mouse model for MPSIIIA was characterized in 1999 and later backcrossed onto the C57BL/6 background. In the present study, a novel immune deficient MPSIIIA mouse model (MPSIIIA-TKO) was created by backcrossing the immune competent, C57BL/6 MPSIIIA mouse to an immune deficient mouse model lacking Rag2, CD47 and Il2rg genes. The resulting mouse model has undetectable SGSH activity, exhibits histological changes consistent with MPSIIIA and lacks T cells, B cells and NK cells. This new mouse model has the potential to be extremely useful in testing human cellular therapies in an animal model as it retains the MPSIIIA disease phenotype while tolerating xenotransplantation.

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using in situ hybridization chain reaction (HCR). We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2α phosphorylation (p-eIF2α), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2α is mediated via activation of mTORC1. Genetic reduction of p-eIF2α only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, restoration of p-eIF2α solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS.

The miR-15/16 family targets a large network of genes in T cells to restrict their cell cycle, memory formation, and survival. Upon T cell activation, miR-15/16 are downregulated, allowing rapid expansion of differentiated effector T cells to mediate a sustained response. Here, we used conditional deletion of miR-15/16 in regulatory T cells (Tregs) to identify immune functions of the miR-15/16 family in T cells. miR-15/16 are indispensable to maintain peripheral tolerance by securing efficient suppression by a limited number of Tregs. miR-15/16 deficiency alters expression of critical Treg proteins and results in accumulation of functionally impaired FOXP3loCD25loCD127hi Tregs. Excessive proliferation in the absence of miR-15/16 shifts Treg fate and produces an effector Treg phenotype. These Tregs fail to control immune activation, leading to spontaneous multi-organ inflammation and increased allergic inflammation in a mouse model of asthma. Together, our results demonstrate that miR-15/16 expression in Tregs is essential to maintain immune tolerance.

Bystander activation of memory T cells occurs via cytokine signaling alone in the absence of T cell receptor (TCR) signaling and provides a means of amplifying T cell effector responses in an antigen-nonspecific manner. While the role of Programmed Cell Death Protein 1 (PD-1) on antigen-specific T cell responses is extensively characterized, its role in bystander T cell responses is less clear. We examined the role of the PD-1 pathway during human and mouse non-antigen-specific memory T cell bystander activation and observed that PD-1+ T cells demonstrated less activation and proliferation than activated PD-1- populations in vitro. Higher activation and proliferative responses were also observed in the PD-1- memory population in both mice and patients with cancer receiving high-dose IL-2, mirroring the in vitro phenotypes. This inhibitory effect of PD-1 could be reversed by PD-1 blockade in vivo or observed using memory T cells from PD-1-/- mice. Interestingly, increased activation through abrogation of PD-1 signaling in bystander-activated T cells also resulted in increased apoptosis due to activation-induced cell death (AICD) and eventual T cell loss in vivo. These results demonstrate that the PD-1/PD-Ligand 1 (PD-L1) pathway inhibited bystander-activated memory T cell responses but also protected cells from AICD.

Exercise changes the tumor microenvironment by remodeling blood vessels and increasing infiltration by cytotoxic immune cells. The mechanisms driving these changes remain unclear. Herein, we demonstrate that exercise normalizes tumor vasculature and upregulates endothelial expression of VCAM1 in YUMMER 1.7 and B16F10 murine models of melanoma but differentially regulates tumor growth, hypoxia, and the immune response. We found that exercise suppressed tumor growth and increased CD8+ T-cell infiltration in YUMMER but not in B16F10 tumors. Single-cell RNA sequencing and flow cytometry revealed exercise modulated the number and phenotype of tumor-infiltrating CD8+ T cells and myeloid cells. Specifically, exercise caused a phenotypic shift in the tumor-associated macrophage population and increased the expression of MHC class II transcripts. We further demonstrated that ERK5 S496A knock-in mice, which are phosphorylation deficient at the S496 residue, mimicked the exercise effect when unexercised, yet when exercised, these mice displayed a reversal in the effect of exercise on tumor growth and macrophage polarization compared with wild-type mice. Taken together, our results reveal tumor-specific differences in the immune response to exercise and show that ERK5 signaling via the S496 residue plays a crucial role in exercise-induced tumor microenvironment changes. See related Spotlight by Betof Warner, p. 1158.

Craniosynostosis is a group of disorders of premature calvarial suture fusion. The identity of the calvarial stem cells (CSCs) that produce fusion-driving osteoblasts in craniosynostosis remains poorly understood. Here we show that both physiologic calvarial mineralization and pathologic calvarial fusion in craniosynostosis reflect the interaction of two separate stem cell lineages; a previously identified cathepsin K (CTSK) lineage CSC1 (CTSK+ CSC) and a separate discoidin domain-containing receptor 2 (DDR2) lineage stem cell (DDR2+ CSC) that we identified in this study. Deletion of Twist1, a gene associated with craniosynostosis in humans2,3, solely in CTSK+ CSCs is sufficient to drive craniosynostosis in mice, but the sites that are destined to fuse exhibit an unexpected depletion of CTSK+ CSCs and a corresponding expansion of DDR2+ CSCs, with DDR2+ CSC expansion being a direct maladaptive response to CTSK+ CSC depletion. DDR2+ CSCs display full stemness features, and our results establish the presence of two distinct stem cell lineages in the sutures, with both populations contributing to physiologic calvarial mineralization. DDR2+ CSCs mediate a distinct form of endochondral ossification without the typical haematopoietic marrow formation. Implantation of DDR2+ CSCs into suture sites is sufficient to induce fusion, and this phenotype was prevented by co-transplantation of CTSK+ CSCs. Finally, the human counterparts of DDR2+ CSCs and CTSK+ CSCs display conserved functional properties in xenograft assays. The interaction between these two stem cell populations provides a new biologic interface for the modulation of calvarial mineralization and suture patency.

Large-effect loci-those statistically significant loci discovered by genome-wide association studies or linkage mapping-associated with key traits segregate amidst a background of minor, often undetectable, genetic effects in wild and domesticated plants and animals. Accurately attributing mean differences and variance explained to the correct components in the linear mixed model analysis is vital for selecting superior progeny and parents in plant and animal breeding, gene therapy, and medical genetics in humans. Marker-assisted prediction and its successor, genomic prediction, have many advantages for selecting superior individuals and understanding disease risk. However, these two approaches are less often integrated to study complex traits with different genetic architectures. This simulation study demonstrates that the average semivariance can be applied to models incorporating Mendelian, oligogenic, and polygenic terms simultaneously and yields accurate estimates of the variance explained for all relevant variables. Our previous research focused on large-effect loci and polygenic variance separately. This work aims to synthesize and expand the average semivariance framework to various genetic architectures and the corresponding mixed models. This framework independently accounts for the effects of large-effect loci and the polygenic genetic background and is universally applicable to genetics studies in humans, plants, animals, and microbes.

Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex trait with a polygenic component that is mainly unidentified. We propose that levels of intermediate molecular phenotypes (IMPs) in the myocardium associated with histopathological damage could explain CDA susceptibility, so variants of genes encoding these IMPs could identify patients susceptible to this complication. Thus, a genetically heterogeneous cohort of mice (n = 165) generated by backcrossing were treated with doxorubicin and docetaxel. We quantified heart fibrosis using an Ariol slide scanner and intramyocardial levels of IMPs using multiplex bead arrays and QPCR. We identified quantitative trait loci linked to IMPs (ipQTLs) and cdaQTLs via linkage analysis. In three cancer patient cohorts, CDA was quantified using echocardiography or Cardiac Magnetic Resonance. CDA behaves as a complex trait in the mouse cohort. IMP levels in the myocardium were associated with CDA. ipQTLs integrated into genetic models with cdaQTLs account for more CDA phenotypic variation than that explained by cda-QTLs alone. Allelic forms of genes encoding IMPs associated with CDA in mice, including AKT1, MAPK14, MAPK8, STAT3, CAS3, and TP53, are genetic determinants of CDA in patients. Two genetic risk scores for pediatric patients (n = 71) and women with breast cancer (n = 420) were generated using machine-learning Least Absolute Shrinkage and Selection Operator (LASSO) regression. Thus, IMPs associated with heart damage identify genetic markers of CDA risk, thereby allowing more personalized patient management.

BACKGROUND: The harlequin ladybird Harmonia axyridis (Coleoptera: Coccinellidae), native to Asia, has been introduced to other major continents where it has caused serious negative impacts on local biodiversity. Though notable advances to understand its invasion success have been made during the past decade, especially with then newer molecular tools, the conclusions reached remain to be confirmed with more advanced genomic analyses and especially using more samples from larger geographical regions across the native range. Furthermore, although H. axyridis is one of the best studied invasive insect species with respect to life history traits (often comparing invasive and native populations), the traits responsible for its colonization success in non-native areas warrant more research. RESULTS: Our analyses of genome-wide nuclear population structure indicated that an eastern Chinese population could be the source of all non-native populations and revealed several putatively adaptive candidate genomic loci involved in body color variation, visual perception, and hemolymph synthesis. Our estimates of evolutionary history indicate (1) asymmetric migration with varying population sizes across its native and non-native range, (2) a recent admixture between eastern Chinese and American populations in Europe, (3) signatures of a large progressive, historical bottleneck in the common ancestors of both populations and smaller effective sizes of the non-native population, and (4) the southwest origin and subsequent dispersal routes within its native range in China. In addition, we found that while two mitochondrial haplotypes-Hap1 and Hap2 were dominant in the native range, Hap1 was the only dominant haplotype in the non-native range. Our laboratory observations in both China and USA found statistical yet slight differences between Hap1 and Hap2 in some of life history traits. CONCLUSIONS: Our study on H. axyridis provides new insights into its invasion processes into other major continents from its native Asian range, reconstructs a geographic range evolution across its native region China, and tentatively suggests that its invasiveness may differ between mitochondrial haplotypes.

Coloration facilitates evolutionary investigations in nature because the interaction between genotype, phenotype and environment is relatively accessible. In a landmark set of studies, Endler addressed this complexity by demonstrating that the evolution of male Trinidadian guppy coloration is shaped by the local balance between selection for mate attractiveness versus crypsis. This became a textbook paradigm for how antagonistic selective pressures may determine evolutionary trajectories in nature. However, recent studies have challenged the generality of this paradigm. Here, we respond to these challenges by reviewing five important yet underappreciated factors that contribute to colour pattern evolution: (i) among-population variation in female preference and correlated variation in male coloration, (ii) differences in how predators versus conspecifics view males, (iii) biased assessment of pigmentary versus structural coloration, (iv) the importance of accounting for multi-species predator communities, and (v) the importance of considering the multivariate genetic architecture and multivariate context of selection and how sexual selection encourages polymorphic divergence. We elaborate these issues using two challenging papers. Our purpose is not to criticize but to point out the potential pitfalls in colour research and to emphasize the depth of consideration necessary for testing evolutionary hypotheses using complex multi-trait phenotypes such as guppy colour patterns.

The idea that changing environmental conditions drive adaptive evolution is a pillar of evolutionary ecology. But, the opposite-that adaptive evolution alters ecological processes-has received far less attention yet is critical for eco-evolutionary dynamics. We assessed the ecological impact of divergent values in a key adaptive trait using 16 populations of the brown anole lizard (Anolis sagrei). Mirroring natural variation, we established islands with short- or long-limbed lizards at both low and high densities. We then monitored changes in lower trophic levels, finding that on islands with a high density of short-limbed lizards, web-spider densities decreased and plants grew more via an indirect positive effect, likely through an herbivore-mediated trophic cascade. Our experiment provides strong support for evolution-to-ecology connections in nature, likely closing an otherwise well-characterized eco-evolutionary feedback loop.

Campylobacter species are the leading cause of bacterial diarrhoea worldwide and consumption of contaminated chicken meat is the most common route of infection. Chickens can be infected with multiple strains of Campylobacter and during the infection cycle this pathogen must survive a wide variety of environments. Numerous studies have reported a high degree of genetic variability in this pathogen that can use antigenic and phase variation to alter the expression of key phenotypes. In this study the phenotypic profile of isolates from freshly slaughtered chickens, chicken products in the supermarket and stool samples from infected patients were compared to identify phenotypic changes during the passage of the bacteria through the infection cycle. Isolates from different stages of the infection cycle had altered phenotypic profiles with isolates from human stool samples showing a lower ability to form a biofilm and an increased ability to associate with epithelial cells in vitro. Resistance to fluoroquinolones was found in all cohorts but at a much higher occurrence (94%) in isolates from supermarket chicken. Isolates displaying high levels of resistance to fluoroquinolones also were more likely to display a higher tolerance to growth in the presence of oxygen. In conclusion, isolates with specific phenotypes appear to be overly represented at different stages of the infection cycle suggesting that environmental stresses may be enriched for strains with these phenotypes.

X-linked spinal and bulbar muscular atrophy (SBMA; Kennedy's disease) is a rare neuromuscular disorder characterized by adult-onset proximal muscle weakness and lower motor neuron degeneration. SBMA was the first human disease found to be caused by a repeat expansion mutation, as affected patients possess an expanded tract of CAG repeats, encoding polyglutamine, in the androgen receptor (AR) gene. We previously developed a conditional BAC fxAR121 transgenic mouse model of SBMA and used it to define a primary role for skeletal muscle expression of polyglutamine-expanded AR in causing the motor neuron degeneration. Here we sought to extend our understanding of SBMA disease pathophysiology and cellular basis by detailed examination and directed experimentation with the BAC fxAR121 mice. First, we evaluated BAC fxAR121 mice for non-neurological disease phenotypes recently described in human SBMA patients, and documented prominent non-alcoholic fatty liver disease, cardiomegaly, and ventricular heart wall thinning in aged male BAC fxAR121 mice. Our discovery of significant hepatic and cardiac abnormalities in SBMA mice underscores the need to evaluate human SBMA patients for signs of liver and heart disease. To directly examine the contribution of motor neuron-expressed polyQ-AR protein to SBMA neurodegeneration, we crossed BAC fxAR121 mice with two different lines of transgenic mice expressing Cre recombinase in motor neurons, and after updating characterization of SBMA phenotypes in our current BAC fxAR121 colony, we found that excision of mutant AR from motor neurons did not rescue neuromuscular or systemic disease. These findings further validate a primary role for skeletal muscle as the driver of SBMA motor neuronopathy and indicate that therapies being developed to treat patients should be delivered peripherally.

Transcriptome analysis of several animal clades suggests that male reproductive tract gene expression evolves quickly. However, the factors influencing the abundance and distribution of within-species variation, the ultimate source of interspecific divergence, are poorly known. Drosophila melanogaster, an ancestrally African species that has recently spread throughout the world and colonized the Americas in the last roughly 100 years, exhibits phenotypic and genetic latitudinal clines on multiple continents, consistent with a role for spatially varying selection in shaping its biology. Nevertheless, geographic expression variation in the Americas is poorly described, as is its relationship to African expression variation. Here, we investigate these issues through the analysis of two male reproductive tissue transcriptomes [testis and accessory gland (AG)] in samples from Maine (USA), Panama, and Zambia. We find dramatic differences between these tissues in differential expression between Maine and Panama, with the accessory glands exhibiting abundant expression differentiation and the testis exhibiting very little. Latitudinal expression differentiation appears to be influenced by the selection of Panama expression phenotypes. While the testis shows little latitudinal expression differentiation, it exhibits much greater differentiation than the accessory gland in Zambia vs American population comparisons. Expression differentiation for both tissues is non-randomly distributed across the genome on a chromosome arm scale. Interspecific expression divergence between D. melanogaster and D. simulans is discordant with rates of differentiation between D. melanogaster populations. Strongly heterogeneous expression differentiation across tissues and timescales suggests a complex evolutionary process involving major temporal changes in the way selection influences expression evolution in these organs.