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In October 2023, a partially engorged female Ixodes tick was removed from a dog in Bozeman, Montana, USA, that had recently spent time in eastern Montana. The tick was identified as I. scapularis according to morphologic characteristics and genomic sequencing, suggesting an expanded geographic distribution requiring continued public health surveillance.

Background: Vascular leakage is a deadly complication of severe infections, ranging from bacterial sepsis to malaria. Worldwide, septicemia is among the top 10 causes of lethality because of the shock and multiorgan dysfunction that arise from the host vascular response. In the monoclonal gammopathy-associated capillary leak syndrome (MG-CLS), even otherwise mundane infections induce recurrent septic-like episodes of profound microvascular hyperpermeability and shock. There are no defined genetic risk factors for MG-CLS or effective treatments for acute crises., Methods: We characterized predicted loss-of-function mutations in PARP15 (poly[ADP-ribose] polymerase 15), a protein of unknown function that is absent in mice, in patients with MG-CLS. We analyzed barrier function in PARP15-deficient vascular endothelial cells and vascular leakage in mice engineered to express wild-type or loss-of-function variant human PARP15., Results: We discovered several loss-of-function PARP15 variants associated with MG-CLS. These mutations severely reduced PARP15 enzymatic function. The presence of the most frequently detected variant (G628R) correlated with clinical markers of severe vascular leakage. In human microvascular endothelial cells, PARP15 suppressed cytokine-induced barrier disruption by ADP-ribosylating the scaffold protein JIP3 (c-Jun N-terminal kinase-interacting protein 3) and inhibiting p38 MAP (mitogen-activated protein) kinase activation. Mice expressing enzymatically inactive human PARP15(G628R) were significantly more prone to inflammation-associated vascular leakage than mice expressing wild-type PARP15 in a p38-dependent fashion., Conclusions: PARP15 represents a previously unrecognized genetic susceptibility factor for MG-CLS. PARP15-mediated ADP ribosylation is an essential and genetically determined mechanism of the human vascular response to inflammation., Competing Interests: None.

While inputs regulating CD4 + T helper cell (Th) differentiation are well-defined, the integration of downstream signaling with transcriptional and epigenetic programs that define Th-lineage identity remain unresolved. PI3K signaling is a critical regulator of T cell function; activating mutations affecting PI3Kδ result in an immunodeficiency with multiple T cell defects. Using mice expressing activated-PI3Kδ, we found aberrant expression of proinflammatory Th1-signature genes under Th2-inducing conditions, both in vivo and in vitro . This dysregulation was driven by a robust PI3Kδ-IL-2-Foxo1 signaling loop, fueling Foxo1-inactivation, loss of Th2-lineage restriction, altered chromatin accessibility and global impairment of CTCF-DNA interactions. Surprisingly, ablation of Fasl , a Foxo1-repressed gene, restored normal Th2 differentiation, TCR signaling and CTCF expression. BioID revealed Fas interactions with TCR-signaling components, which were supported by Fas-mediated potentiation of TCR signaling. Our results highlight Fas-FasL signaling as a critical intermediate in phenotypes driven by activated-PI3Kδ, thereby linking two key pathways of immune dysregulation., Competing Interests: Declaration of Interests The authors declare no competing interests.

X-linked chronic granulomatous disease (X-CGD) is an inborn error of immunity (IEI) resulting from genetic mutations in the cytochrome b-245 beta chain ( CYBB ) gene. The applicability of base editors (BEs) to correct mutations that cause X-CGD is constrained by the requirement of Cas enzymes to recognize specific protospacer adjacent motifs (PAMs). Our recently engineered PAMless Cas enzyme, SpRY, can overcome the PAM limitation. However, the efficiency, specificity, and applicability of SpRY-based BEs to correct mutations in human hematopoietic stem and progenitor cells (HSPCs) have not been thoroughly examined. Here, we demonstrated that the adenine BE ABE8e-SpRY can access a range of target sites in HSPCs to correct mutations causative of X-CGD. For the prototypical X-CGD mutation CYBB c.676C>T, ABE8e-SpRY achieved up to 70% correction, reaching efficiencies greater than three-and-one-half times higher than previous CRISPR nuclease and donor template approaches. We profiled potential off-target DNA edits, transcriptome-wide RNA edits, and chromosomal perturbations in base-edited HSPCs, which together revealed minimal off-target or bystander edits. Edited alleles persisted after transplantation of the base-edited HSPCs into immunodeficient mice. Together, these investigational new drug-enabling studies demonstrated efficient and precise correction of an X-CGD mutation with PAMless BEs, supporting a first-in-human clinical trial (NCT06325709) and providing a potential blueprint for treatment of other IEI mutations.

Human genetic studies of critical COVID-19 pneumonia have revealed the essential role of type I interferon-dependent innate immunity to SARS-CoV-2 infection. Conversely, an association between the HLA-B∗15:01 allele and asymptomatic SARS-CoV-2 infection in unvaccinated individuals was recently reported, suggesting a contribution of pre-existing T cell-dependent adaptive immunity. We report a lack of association of classical HLA alleles, including HLA-B∗15:01, with pre-omicron asymptomatic SARS-CoV-2 infection in unvaccinated participants in a prospective population-based study in the United States (191 asymptomatic vs. 945 symptomatic COVID-19 cases). Moreover, we found no such association in the international COVID Human Genetic Effort cohort (206 asymptomatic vs. 574 mild or moderate COVID-19 cases and 1,625 severe or critical COVID-19 cases). Finally, in the Human Challenge Characterisation study, the three HLA-B∗15:01 individuals infected with SARS-CoV-2 developed symptoms. As with other acute primary infections studied, no classical HLA alleles favoring an asymptomatic course of SARS-CoV-2 infection were identified., Competing Interests: Declaration of interests E.T.C., K.M.S.B., and A.B. are employees of Helix., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inflammatory monocytes (iMO) are recruited from the bone marrow to the brain during viral encephalitis. C-C motif chemokine receptor (CCR) 2 deficiency substantially reduces iMO recruitment for most, but not all encephalitic viruses. Here we show CCR7 acts synergistically with CCR2 to control this process. Following Herpes simplex virus type-1 (HSV-1), or La Crosse virus (LACV) infection, we find iMO proportions are reduced by approximately half in either Ccr2 or Ccr7 knockout mice compared to control mice. However, Ccr2/Ccr7 double knockouts eliminate iMO recruitment following infection with either virus, indicating these receptors together control iMO recruitment. We also find that LACV induces a more robust iMO recruitment than HSV-1. However, unlike iMOs in HSV-1 infection, LACV-recruited iMOs do not influence neurological disease development. LACV-induced iMOs have higher expression of proinflammatory and proapoptotic but reduced mitotic, phagocytic and phagolysosomal transcripts compared to HSV-1-induced iMOs. Thus, virus-specific activation of iMOs affects their recruitment, activation, and function., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Regulators of G protein signaling (RGS) proteins constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase-activating protein activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and airway smooth muscle contraction, whereas RGS4 KO mice unexpectedly have decreased AHR because of increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here, we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GTPase-activating protein activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared with either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3K and inhibited PI3K-dependent PGE2 secretion elicited by transforming growth factor beta in airway epithelial cells. Together, these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Clarkson disease, or monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome (ISCLS), is a rare, relapsing-remitting disorder featuring the abrupt extravasation of fluids and proteins into peripheral tissues, which in turn leads to hypotensive shock, severe hemoconcentration, and hypoalbuminemia. The specific leakage factor(s) and pathways in ISCLS are unknown, and there is no effective treatment for acute flares. Here, we characterize an autonomous vascular endothelial defect in ISCLS that was recapitulated in patient-derived endothelial cells (ECs) in culture and in a mouse model of disease. ISCLS-derived ECs were functionally hyperresponsive to permeability-inducing factors like VEGF and histamine, in part due to increased endothelial nitric oxide synthase (eNOS) activity. eNOS blockade by administration of N(γ)-nitro-l-arginine methyl ester (l-NAME) ameliorated vascular leakage in an SJL/J mouse model of ISCLS induced by histamine or VEGF challenge. eNOS mislocalization and decreased protein phosphatase 2A (PP2A) expression may contribute to eNOS hyperactivation in ISCLS-derived ECs. Our findings provide mechanistic insights into microvascular barrier dysfunction in ISCLS and highlight a potential therapeutic approach.

Human genetic studies of critical COVID-19 pneumonia have revealed the essential role of type I interferon-dependent innate immunity to SARS-CoV-2 infection. Conversely, an association between the HLA-B*15:01 allele and asymptomatic SARS-CoV-2 infection in unvaccinated individuals was recently reported, suggesting a contribution of pre-existing T cell-dependent adaptive immunity. We report a lack of association of classical HLA alleles, including HLA-B*15:01, with pre-omicron asymptomatic SARS-CoV-2 infection in unvaccinated participants in a prospective population-based study in the US (191 asymptomatic vs. 945 symptomatic COVID-19 cases). Moreover, we found no such association in the international COVID Human Genetic Effort cohort (206 asymptomatic vs. 574 mild or moderate COVID-19 cases and 1,625 severe or critical COVID-19 cases). Finally, in the Human Challenge Characterisation study, the three HLA-B*15:01 individuals infected with SARS-CoV-2 developed symptoms. As with other acute primary infections, no classical HLA alleles favoring an asymptomatic course of SARS-CoV-2 infection were identified. These findings suggest that memory T-cell immunity to seasonal coronaviruses does not strongly influence the outcome of SARS-CoV-2 infection in unvaccinated individuals.

Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at -80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws.

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18- hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.

Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the southwestern United States and Mexico. Illness occurs in approximately 30% of those infected, less than 1% of whom develop disseminated disease. To address why some individuals allow dissemination, we enrolled patients with DCM and performed whole-exome sequencing. In an exploratory set of 67 patients with DCM, 2 had haploinsufficient STAT3 mutations, and defects in β-glucan sensing and response were seen in 34 of 67 cases. Damaging CLEC7A and PLCG2 variants were associated with impaired production of β-glucan-stimulated TNF-α from PBMCs compared with healthy controls. Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were overrepresented in DCM, including CLEC7A Y238* and PLCG2 R268W. A validation cohort of 111 patients with DCM confirmed the PLCG2 R268W, CLEC7A I223S, and CLEC7A Y238* variants. Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived hydrogen peroxide [H2O2] in transfected cells. Heterozygous DUOX1 or DUOXA1 variants that impaired H2O2 production were overrepresented in discovery and validation cohorts. Patients with DCM have impaired β-glucan sensing or response affecting TNF-α and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.

Background: Clarkson disease (monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome, ISCLS) is a rare idiopathic condition marked by transient, relapsing-remitting episodes of systemic microvascular hyper-permeability, which liberates plasma fluid and macromolecules into the peripheral tissues. This pathology manifests clinically as the abrupt onset of hypotensive shock, hemoconcentration, and hypoalbuminemia., Methods: We analysed endothelial glycocalyx (eGCX)-related markers in plasma from patients with ISCLS during acute disease flares and convalescence by ELISA and comprehensive proteomic profiling. We evaluated eGCX-related components and gene expression in cultured endothelial cells using RNA-sequencing, real-time PCR, and fluorescence staining., Results: Serum levels of eGCX-related core components including hyaluronic acid (HA) and the core proteoglycan soluble syndecan-1 (sCD138) were elevated at baseline and during acute ISCLS flares. Serial measurements demonstrated that sCD138 levels peaked during the recovery (post-leak) phase of the illness. Proteomic analysis of matched acute and convalescent ISCLS plasma revealed increased abundance of eGCX-related proteins, including glypicans, thrombospondin-1 (TSP-1), and eGCX-degrading enzymes in acute compared to remission plasma. Abundance of endothelial cell damage markers did not differ in acute and baseline plasma. Expression of several eGCX-related genes and surface carbohydrate content in endothelial cells from patients with ISCLS did not differ significantly from that observed in healthy control cells., Conclusions: eGCX dysfunction, but not endothelial injury, may contribute to clinical symptoms of acute ISCLS. Serum levels of of eGCX components including sCD138 may be measured during acute episodes of ISCLS to monitor clinical status and therapeutic responses., (© 2022. The Author(s).)

The relationships between adaptive evolution, phenotypic plasticity, and canalization remain incompletely understood. Theoretical and empirical studies have made conflicting arguments on whether adaptive evolution may enhance or oppose the plastic response. Gene regulatory traits offer excellent potential to study the relationship between plasticity and adaptation, and they can now be studied at the transcriptomic level. Here, we take advantage of three closely related pairs of natural populations of Drosophila melanogaster from contrasting thermal environments that reflect three separate instances of cold tolerance evolution. We measure the transcriptome-wide plasticity in gene expression levels and alternative splicing (intron usage) between warm and cold laboratory environments. We find that suspected adaptive changes in both gene expression and alternative splicing tend to neutralize the ancestral plastic response. Further, we investigate the hypothesis that adaptive evolution can lead to decanalization of selected gene regulatory traits. We find strong evidence that suspected adaptive gene expression (but not splicing) changes in cold-adapted populations are more vulnerable to the genetic perturbation of inbreeding than putatively neutral changes. We find some evidence that these patterns may reflect a loss of genetic canalization accompanying adaptation, although other processes including hitchhiking recessive deleterious variants may contribute as well. Our findings augment our understanding of genetic and environmental effects on gene regulation in the context of adaptive evolution., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Important uncertainties persist regarding the genetic architecture of adaptive trait evolution in natural populations, including the number of genetic variants involved, whether they are drawn from standing genetic variation, and whether directional selection drives them to complete fixation. Here, we take advantage of a unique natural population of Drosophila melanogaster from the Ethiopian highlands, which has evolved larger body size than any other known population of this species. We apply a bulk segregant quantitative trait locus mapping approach to 4 unique crosses between highland Ethiopian and lowland Zambian populations for both thorax length and wing length. Results indicated a persistently variable genetic basis for these evolved traits (with largely distinct sets of quantitative trait loci for each cross), and at least a moderately polygenic architecture with relatively strong effects present. We complemented these mapping experiments with population genetic analyses of quantitative trait locus regions and gene ontology enrichment analysis, generating strong hypotheses for specific genes and functional processes that may have contributed to these adaptive trait changes. Finally, we find that the genetic architectures indicated by our quantitative trait locus mapping results for size traits mirror those from similar experiments on other recently evolved traits in this species. Collectively, these studies suggest a recurring pattern of polygenic adaptation in this species, in which causative variants do not approach fixation and moderately strong effect loci are present., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Population genetics seeks to illuminate the forces shaping genetic variation, often based on a single snapshot of genomic variation. However, utilizing multiple sampling times to study changes in allele frequencies can help clarify the relative roles of neutral and non-neutral forces on short time scales. This study compares whole-genome sequence variation of recently collected natural population samples of Drosophila melanogaster against a collection made approximately 35 years prior from the same locality-encompassing roughly 500 generations of evolution. The allele frequency changes between these time points would suggest a relatively small local effective population size on the order of 10,000, significantly smaller than the global effective population size of the species. Some loci display stronger allele frequency changes than would be expected anywhere in the genome under neutrality-most notably the tandem paralogs Cyp6a17 and Cyp6a23, which are impacted by structural variation associated with resistance to pyrethroid insecticides. We find a genome-wide excess of outliers for high genetic differentiation between old and new samples, but a larger number of adaptation targets may have affected SNP-level differentiation versus window differentiation. We also find evidence for strengthening latitudinal allele frequency clines: northern-associated alleles have increased in frequency by an average of nearly 2.5% at SNPs previously identified as clinal outliers, but no such pattern is observed at random SNPs. This project underscores the scientific potential of using multiple sampling time points to investigate how evolution operates in natural populations, by quantifying how genetic variation has changed over ecologically relevant timescales., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Acute respiratory virus infections can have profound and long-term effects on lung function that persist even after the acute responses have fully resolved. In this study, we examined gene expression by RNA sequencing in the lung tissue of wild-type BALB/c mice that were recovering from a sublethal infection with the pneumonia virus of mice (PVM), a natural rodent pathogen of the same virus family and genus as the human respiratory syncytial virus. We compared these responses to gene expression in PVM-infected mice treated with Lactobacillus plantarum , an immunobiotic agent that limits inflammation and averts the negative clinical sequelae typically observed in response to acute infection with this pathogen. Our findings revealed prominent differential expression of inflammation-associated genes as well as numerous genes and gene families implicated in mitosis and cell-cycle regulation, including cyclins, cyclin-dependent kinases, cell division cycle genes, E2F transcription factors, kinesins, centromere proteins, and aurora kinases, among others. Of particular note was the differential expression of the cell division cycle gene Cdc20b , which was previously identified as critical for the ex vivo differentiation of multi-ciliated cells. Collectively, these findings provided us with substantial insight into post-viral repair processes and broadened our understanding of the mechanisms underlying Lactobacillus -mediated protection.

No longer regarded simply as end-stage cytotoxic effectors, eosinophils are now recognized as complex cells with unique phenotypes that develop in response stimuli in the local microenvironment. In our previous study, we documented eosinophil infiltration in damaged muscle characteristic of dystrophin-deficient (mdx) mice that model Duchenne muscular dystrophy. Specifically, we found that eosinophils did not promote the generation of muscle lesions, as these persisted in eosinophil-deficient mdx.PHIL mice. To obtain additional insight into these findings, we performed RNA sequencing of eosinophils isolated from muscle tissue of mdx, IL5tg, and mdx.IL5tg mice. We observed profound up-regulation of classical effector proteins (major basic protein-1, eosinophil peroxidase, and eosinophil-associated ribonucleases) in eosinophils isolated from lesion-free muscle from IL5tg mice. By contrast, we observed significant up-regulation of tissue remodeling genes, including proteases, extracellular matrix components, collagen, and skeletal muscle precursors, as well as the immunomodulatory receptor, Trem2, in eosinophils isolated from skeletal muscle tissue from the dystrophin-deficient mdx mice. Although the anti-inflammatory properties of Trem2 have been described in the monocyte/macrophage lineage, no previous studies have documented its expression in eosinophils. We found that Trem2 was critical for full growth and differentiation of bone marrow-derived eosinophil cultures and full expression of TLR4. Immunoreactive Trem2 was also detected on human peripheral blood eosinophils at levels that correlated with donor body mass index and total leukocyte count. Taken together, our findings provide important insight into the immunomodulatory and remodeling capacity of mouse eosinophils and the flexibility of their gene expression profiles in vivo., (©2021 Society for Leukocyte Biology.)

Changes in gene regulation at multiple levels may comprise an important share of the molecular changes underlying adaptive evolution in nature. However, few studies have assayed within- and between-population variation in gene regulatory traits at a transcriptomic scale, and therefore inferences about the characteristics of adaptive regulatory changes have been elusive. Here, we assess quantitative trait differentiation in gene expression levels and alternative splicing (intron usage) between three closely related pairs of natural populations of Drosophila melanogaster from contrasting thermal environments that reflect three separate instances of cold tolerance evolution. The cold-adapted populations were known to show population genetic evidence for parallel evolution at the SNP level, and here we find evidence for parallel expression evolution between them, with stronger parallelism at larval and adult stages than for pupae. We also implement a flexible method to estimate cis- vs trans-encoded contributions to expression or splicing differences at the adult stage. The apparent contributions of cis- vs trans-regulation to adaptive evolution vary substantially among population pairs. While two of three population pairs show a greater enrichment of cis-regulatory differences among adaptation candidates, trans-regulatory differences are more likely to be implicated in parallel expression changes between population pairs. Genes with significant cis-effects are enriched for signals of elevated genetic differentiation between cold- and warm-adapted populations, suggesting that they are potential targets of local adaptation. These findings expand our knowledge of adaptive gene regulatory evolution and our ability to make inferences about this important and widespread process., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. The importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes has become clear in recent years. However, the control of opportunistic pathogens-and especially intracellular bacteria-by type I IFNs remains less appreciated. In this study, we use the opportunistic, Gram-negative bacterial pathogen Burkholderia cenocepacia (Bc) to show that type I IFNs are capable of limiting bacterial replication in macrophages, preventing illness in immunocompetent mice. Sustained type I IFN signaling through cytosolic receptors allows for increased expression of autophagy and linear ubiquitination mediators, which slows bacterial replication. Transcriptomic analyses and in vivo studies also show that LPS stimulation does not replicate the conditions of intracellular Gram-negative bacterial infection as it pertains to type I IFN stimulation or signaling. This study highlights the importance of type I IFNs in protection against opportunistic pathogens through innate immunity, without the need for damaging inflammatory responses., Competing Interests: The authors have declared that no competing interests exist.

Tumors frequently subvert major histocompatibility complex class I (MHC-I) peptide presentation to evade CD8 + T cell immunosurveillance, though how this is accomplished is not always well defined. To identify the global regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of genes that positively and negatively modulate MHC-I cell surface expression. Validated genes clustered in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Genes can exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors displayed genetic alterations in multiple regulators. We established SUGT1 as a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhanced DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity., Competing Interests: Declaration of Interests N.P.R. and R.J.K. are now employees of Lyell Immunopharma and hold equity., (Published by Elsevier Inc.)

One major foundation of cancer etiology is the process of clonal expansion. The mechanisms underlying the complex process of a single cell leading to a clonal dominant tumor, are poorly understood. Our study aims to analyze mitochondrial DNA (mtDNA) for somatic single nucleotide polymorphisms (SNPs) variants, to determine if they are conserved throughout clonal expansion in mammary tissues and tumors. To test this hypothesis, we took advantage of a mouse mammary tumor virus (MMTV)-infected mouse model (CzechII). CzechII mouse mtDNA was extracted, from snap-frozen normal, hyperplastic, and tumor mammary epithelial outgrowth fragments. Next generation deep sequencing was used to determine if mtDNA " de novo " SNP variants are conserved during serial transplantation of both normal and neoplastic mammary clones. Our results support the conclusion that mtDNA " de novo " SNP variants are selected for and maintained during serial passaging of clonal phenotypically heterogeneous normal cellular populations; neoplastic cellular populations; metastatic clonal cellular populations and in individual tumor transplants, grown from the original metastatic tumor. In one case, a mammary tumor arising from a single cell, within a clonal hyperplastic outgrowth, contained only mtDNA copies, harboring a deleterious " de novo " SNP variant, suggesting that only one mtDNA template may act as a template for all mtDNA copies regardless of cell phenotype. This process has been attributed to "heteroplasmic-shifting". A process that is thought to result from selective pressure and may be responsible for pathogenic mutated mtDNA copies becoming homogeneous in clonal dominant oncogenic tissues., Competing Interests: Conflicts of Interest The authors declare no potential conflicts of interest.

Resting eggs banks are unique windows that allow us to directly observe shifts in population genetics, and phenotypes over time as natural populations evolve. Though a variety of planktonic organisms also produce resting stages, the keystone freshwater consumer, Daphnia, is a well-known model for paleogenetics and resurrection ecology. Nevertheless, paleogenomic investigations are limited largely because resting eggs do not contain enough DNA for genomic sequencing. In fact, genomic studies even on extant populations include a laborious preparatory phase of batch culturing dozens of individuals to generate sufficient genomic DNA. Here, we furnish a protocol to generate whole genomes of single ephippial (resting) eggs and single daphniids. Whole genomes of single ephippial eggs and single adults were amplified using Qiagen REPLI-g Single Cell kit reaction, followed by NEBNext Ultra DNA Library Prep Kit for library construction and Illumina sequencing. We compared the quality of the single-egg and single-individual amplified genomes to the standard batch genomic DNA extraction in the absence of genome amplification. At mean 20× depth, coverage was essentially identical for the amplified single individual relative to the unamplified batch extracted genome (>90% of the genome was covered and callable). Finally, while amplification resulted in the slight loss of heterozygosity for the amplified genomes, estimates were largely comparable and illustrate the utility and limitations of this approach in estimating population genetic parameters over long periods of time in natural populations of Daphnia and also other small species known to produce resting stages., (© 2017 John Wiley & Sons Ltd.)

Drosophila melanogaster originated in tropical Africa before expanding into strikingly different temperate climates in Eurasia and beyond. Here, we find elevated cold tolerance in three distinct geographic regions: beyond the well-studied non-African case, we show that populations from the highlands of Ethiopia and South Africa have significantly increased cold tolerance as well. We observe greater cold tolerance in outbred versus inbred flies, but only in populations with higher inversion frequencies. Each cold-adapted population shows lower inversion frequencies than a closely-related warm-adapted population, suggesting that inversion frequencies may decrease with altitude in addition to latitude. Using the FST-based "Population Branch Excess" statistic (PBE), we found only limited evidence for parallel genetic differentiation at the scale of ∼4 kb windows, specifically between Ethiopian and South African cold-adapted populations. And yet, when we looked for single nucleotide polymorphisms (SNPs) with codirectional frequency change in two or three cold-adapted populations, strong genomic enrichments were observed from all comparisons. These findings could reflect an important role for selection on standing genetic variation leading to "soft sweeps". One SNP showed sufficient codirectional frequency change in all cold-adapted populations to achieve experiment-wide significance: an intronic variant in the synaptic gene Prosap. Another codirectional outlier SNP, at senseless-2, had a strong association with our cold trait measurements, but in the opposite direction as predicted. More generally, proteins involved in neurotransmission were enriched as potential targets of parallel adaptation. The ability to study cold tolerance evolution in a parallel framework will enhance this classic study system for climate adaptation., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Unraveling the genetic architecture of adaptive phenotypic divergence is a fundamental quest in evolutionary biology. In Drosophila melanogaster, high-altitude melanism has evolved in separate mountain ranges in sub-Saharan Africa, potentially as an adaptation to UV intensity. We investigated the genetic basis of this melanism in three populations using a new bulk segregant analysis mapping method. We identified 19 distinct QTL regions from nine mapping crosses, with several QTL peaks overlapping between two or all populations, and yet different crosses involving the same melanic population commonly yielded distinct QTL. The strongest QTL often overlapped well-known pigmentation genes, but we typically did not find wide signals of genetic differentiation (F ST ) between lightly and darkly pigmented populations at these genes. Instead, we found small numbers of highly differentiated SNPs at the probable causative genes. A simulation analysis showed that these patterns of polymorphism were consistent with selection on standing genetic variation. Overall, our results suggest that, even for potentially simpler traits like pigmentation, the complexity of adaptive trait evolution poses important challenges for QTL mapping and population genetic analysis., (Copyright © 2016 Bastide et al.)

Sex-limited polymorphisms are an intriguing form of sexual dimorphism that offer unique opportunities to reconstruct the evolutionary changes that decouple male and female traits encoded by a shared genome. We investigated the genetic basis of a Mendelian female-limited color dimorphism (FLCD) that segregates in natural populations of more than 20 species of the Drosophila montium subgroup. In these species, females have alternative abdominal color morphs, light and dark, whereas males have only one color morph in each species. A comprehensive molecular phylogeny of the montium subgroup supports multiple origins of FLCD. Despite this, we mapped FLCD to the same locus in four distantly related species-the transcription factor POU domain motif 3 (pdm3), which acts as a repressor of abdominal pigmentation in D. melanogaster. In D. serrata, FLCD maps to a structural variant in the first intron of pdm3; however, this variant is not found in the three other species-D. kikkawai, D. leontia, and D. burlai-and sequence analysis strongly suggests the pdm3 alleles responsible for FLCD originated independently at least three times. We propose that cis-regulatory changes in pdm3 form sexually dimorphic and monomorphic alleles that segregate within species and are preserved, at least in one species, by structural variation. Surprisingly, pdm3 has not been implicated in the evolution of sex-specific pigmentation outside the montium subgroup, suggesting that the genetic paths to sexual dimorphism may be constrained within a clade but variable across clades., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Understanding the physiological and genetic basis of growth and body size variation has wide-ranging implications, from cancer and metabolic disease to the genetics of complex traits. We examined the evolution of body and wing size in high-altitude Drosophila melanogaster from Ethiopia, flies with larger size than any previously known population. Specifically, we sought to identify life history characteristics and cellular mechanisms that may have facilitated size evolution. We found that the large-bodied Ethiopian flies laid significantly fewer but larger eggs relative to lowland, smaller-bodied Zambian flies. The highland flies were found to achieve larger size in a similar developmental period, potentially aided by a reproductive strategy favoring greater provisioning of fewer offspring. At the cellular level, cell proliferation was a strong contributor to wing size evolution, but both thorax and wing size increases involved important changes in cell size. Nuclear size measurements were consistent with elevated somatic ploidy as an important mechanism of body size evolution. We discuss the significance of these results for the genetic basis of evolutionary changes in body and wing size in Ethiopian D. melanogaster.

In higher organisms, the phenotypic impacts of potentially harmful or beneficial mutations are often modulated by complex developmental networks. Stabilizing selection may favor the evolution of developmental canalization--that is, robustness despite perturbation--to insulate development against environmental and genetic variability. In contrast, directional selection acts to alter the developmental process, possibly undermining the molecular mechanisms that buffer a trait's development, but this scenario has not been shown in nature. Here, we examined the developmental consequences of size increase in highland Ethiopian Drosophila melanogaster. Ethiopian inbred strains exhibited much higher frequencies of wing abnormalities than lowland populations, consistent with an elevated susceptibility to the genetic perturbation of inbreeding. We then used mutagenesis to test whether Ethiopian wing development is, indeed, decanalized. Ethiopian strains were far more susceptible to this genetic disruption of development, yielding 26 times more novel wing abnormalities than lowland strains in F2 males. Wing size and developmental perturbability cosegregated in the offspring of between-population crosses, suggesting that genes conferring size differences had undermined developmental buffering mechanisms. Our findings represent the first observation, to our knowledge, of morphological evolution associated with decanalization in the same tissue, underscoring the sensitivity of development to adaptive change.

Clines in life history traits, presumably driven by spatially varying selection, are widespread. Major latitudinal clines have been observed, for example, in Drosophila melanogaster, an ancestrally tropical insect from Africa that has colonized temperate habitats on multiple continents. Yet, how geographic factors other than latitude, such as altitude or longitude, affect life history in this species remains poorly understood. Moreover, most previous work has been performed on derived European, American and Australian populations, but whether life history also varies predictably with geography in the ancestral Afro-tropical range has not been investigated systematically. Here, we have examined life history variation among populations of D. melanogaster from sub-Saharan Africa. Viability and reproductive diapause did not vary with geography, but body size increased with altitude, latitude and longitude. Early fecundity covaried positively with altitude and latitude, whereas lifespan showed the opposite trend. Examination of genetic variance-covariance matrices revealed geographic differentiation also in trade-off structure, and QST -FST analysis showed that life history differentiation among populations is likely shaped by selection. Together, our results suggest that geographic and/or climatic factors drive adaptive phenotypic differentiation among ancestral African populations and confirm the widely held notion that latitude and altitude represent parallel gradients., (© 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.)

Hundreds of wild-derived Drosophila melanogaster genomes have been published, but rigorous comparisons across data sets are precluded by differences in alignment methodology. The most common approach to reference-based genome assembly is a single round of alignment followed by quality filtering and variant detection. We evaluated variations and extensions of this approach and settled on an assembly strategy that utilizes two alignment programs and incorporates both substitutions and short indels to construct an updated reference for a second round of mapping prior to final variant detection. Utilizing this approach, we reassembled published D. melanogaster population genomic data sets and added unpublished genomes from several sub-Saharan populations. Most notably, we present aligned data from phase 3 of the Drosophila Population Genomics Project (DPGP3), which provides 197 genomes from a single ancestral range population of D. melanogaster (from Zambia). The large sample size, high genetic diversity, and potentially simpler demographic history of the DPGP3 sample will make this a highly valuable resource for fundamental population genetic research. The complete set of assemblies described here, termed the Drosophila Genome Nexus, presently comprises 623 consistently aligned genomes and is publicly available in multiple formats with supporting documentation and bioinformatic tools. This resource will greatly facilitate population genomic analysis in this model species by reducing the methodological differences between data sets., (Copyright © 2015 by the Genetics Society of America.)

Experimental and comparative studies suggest that a major determinant of increased ossification of the mandibular symphysis is elevated masticatory stress related to a mechanically challenging diet. However, the morphology of this joint tracks variation in dietary properties in only some mammalian clades. Extant anthropoid primates are a notable exception: synostosis is ubiquitous in this speciose group, despite its great age and diverse array of feeding adaptations. One possible explanation for this pattern is that, once synostosis evolves, reversion to a lesser degree of fusion is unlikely or even constrained. If correct, this has important implications for functional and phylogenetic analyses of the mammalian feeding apparatus. To test this hypothesis, we generated a molecular tree for 76 vespertilionoid and noctilionoid chiropterans using Bayesian phylogenetic analysis and examined character evolution using parsimony and likelihood ancestral-state reconstructions along with the binary state speciation and extinction (BiSSE) model. Results indicate that reversals have occurred within Vespertilionoidea. In contrast, noctilionoids exhibit an anthropoid-like pattern, which suggests that more detailed comparisons of the functional and developmental bases for fusion in these bat clades may provide insight into why fusion is maintained in some lineages but not in others. Potential functional and developmental explanations for the lack of reversal are discussed., (© 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.)

The role of rodents in the epidemiology of zoonotic hepatitis E virus (HEV) infection has been a subject of considerable debate. Seroprevalence studies suggest widespread HEV infection in commensal Rattus spp. rats, but experimental transmission has been largely unsuccessful and recovery of zoonotic genotype 3 HEV RNA from wild Rattus spp. rats has never been confirmed. We surveyed R. rattus and R. norvegicus rats from across the United States and several international populations by using a hemi-nested reverse transcription PCR approach. We isolated HEV RNA in liver tissues from 35 of 446 rats examined. All but 1 of these isolates was relegated to the zoonotic HEV genotype 3, and the remaining sequence represented the recently discovered rat genotype from the United States and Germany. HEV-positive rats were detected in urban and remote localities. Genetic analyses suggest all HEV genotype 3 isolates obtained from wild Rattus spp. rats were closely related.

Biological invasions result in novel species interactions, which can have significant evolutionary impacts on both native and invading taxa. One evolutionary concern with invasions is hybridization among lineages that were previously isolated, but make secondary contact in their invaded range(s). Black rats, consisting of several morphologically very similar but genetically distinct taxa that collectively have invaded six continents, are arguably the most successful mammalian invaders on the planet. We used mitochondrial cytochrome b sequences, two nuclear gene sequences (Atp5a1 and DHFR) and nine microsatellite loci to examine the distribution of three invasive black rat lineages (Rattus tanezumi, Rattus rattus I and R. rattus IV) in the United States and Asia and to determine the extent of hybridization among these taxa. Our analyses revealed two mitochondrial lineages that have spread to multiple continents, including a previously undiscovered population of R. tanezumi in the south-eastern United States, whereas the third lineage (R. rattus IV) appears to be confined to Southeast Asia. Analyses of nuclear DNA (both sequences and microsatellites) suggested significant hybridization is occurring among R. tanezumi and R. rattus I in the United States and also suggest hybridization between R. tanezumi and R. rattus IV in Asia, although further sampling of the latter species pair in Asia is required. Furthermore, microsatellite analyses suggest unidirectional introgression from both R. rattus I and R. rattus IV into R. tanezumi. Within the United States, introgression appears to be occurring to such a pronounced extent that we were unable to detect any nuclear genetic signal for R. tanezumi, and a similar pattern was detected in Asia., (© 2012 Blackwell Publishing Ltd.)

The order Piroplasmida consists of several genera of tick-borne parasites that infect mammals, and to a lesser extent birds, and are therefore of medical and economic importance. Despite their importance, considerable confusion exists concerning the relationship among piroplasmid species, specifically concerning the number of genera and the intergeneric relationships. To examine evolutionary relationships among piroplasmids, we conducted phylogenetic analyses of 192 18S rDNA sequences from the genera Theileria, Babesia and Cytauxzoon. Our analyses revealed eight clades potentially representing distinct genera, and we distinguish the Duncani Group and Microti Group as genetically distinct groups of species requiring detailed analysis of morphology and life-history to allow formal generic description. The piroplasmid phylogeny revealed considerable host diversity and limited host specificity, suggesting piroplasmids have undergone frequent host switches during their evolution. Our analyses provide the first reported evolutionary timescale for piroplasmids independent of the assumption of parasite-host cospeciation, which is invalid for piroplasmids. Evolutionary rate analyses revealed considerable substitution rate heterogeneity, which we attribute to host switching and diversification. Finally, we call for a comprehensive phylogenetic, morphological and life-history analysis for these medically relevant taxa to resolve relationships and understand host specificity., (Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The bat fly (Trichobius major) is a blood-feeding ectoparasite of the cave myotis (Myotis velifer). A recent mitochondrial DNA (mtDNA) study examining population structure of T. major in the South Central United States detected a single haplotype from all individuals examined (N = 48 from 12 different caves), representing one of only a few known examples of such widespread mtDNA uniformity. We examined nuclear genetic diversity using amplified fragment length polymorphism and detected high levels of nuclear genetic diversity in all populations sampled. Amplified fragment length polymorphism analyses indicated significant levels of gene flow among caves >700 km apart, suggesting the absence of mtDNA diversity in T. major is the result of a selective sweep, not a demographic event (i.e., a recent bottleneck). One mechanism by which mtDNA sweeps occur in arthropods is through bacterial parasites that manipulate host reproduction and mtDNA inheritance. We used PCR to test for the presence of all known reproductive parasites and detected a widespread infection (91.33% infection rate) of T. major with a novel Arsenophonus bacterium, as well as the infection of 2 individuals (1.16% infection rate) with a novel strain of Rickettsia. We discuss the implications for T. major phylogeography and the necessity of a bigenomic approach in arthropod population genetics.