Remarkably High Repeat Content in the Genomes of Sparrows: The Importance of Genome Assembly Completeness for Transposable Element Discovery.

Transposable elements (TE) play critical roles in shaping genome evolution. Highly repetitive TE sequences are also a major source of assembly gaps making it difficult to fully understand the impact of these elements on host genomes. The increased capacity of long-read sequencing technologies to span highly repetitive regions promises to provide new insights into patterns of TE activity across diverse taxa. Here we report the generation of highly contiguous reference genomes using PacBio long-read and Omni-C technologies for three species of Passerellidae sparrow. We compared these assemblies to three chromosome-level sparrow assemblies and nine other sparrow assemblies generated using a variety of short- and long-read technologies. All long-read based assemblies were longer (range: 1.12 to 1.41 Gb) than short-read assemblies (0.91 to 1.08 Gb) and assembly length was strongly correlated with the amount of repeat content. Repeat content for Bell's sparrow (31.2% of genome) was the highest level ever reported within the order Passeriformes, which comprises over half of avian diversity. The highest levels of repeat content (79.2% to 93.7%) were found on the W chromosome relative to other regions of the genome. Finally, we show that proliferation of different TE classes varied even among species with similar levels of repeat content. These patterns support a dynamic model of TE expansion and contraction even in a clade where TEs were once thought to be fairly depauperate and static. Our work highlights how the resolution of difficult-to-assemble regions of the genome with new sequencing technologies promises to transform our understanding of avian genome evolution.

Color-Flu Fluorescent Reporter Influenza A Viruses Allow for In Vivo Studies of Innate Immune Function in Zebrafish.

Influenza virus infection can cause severe respiratory disease and is estimated to cause millions of illnesses annually. Studies on the contribution of the innate immune response to influenza A virus (IAV) to viral pathogenesis may yield new antiviral strategies. Zebrafish larvae are useful models for studying the innate immune response to pathogens, including IAV, in vivo. Here, we demonstrate how Color-flu, four fluorescent IAV strains originally developed for mice, can be used to study the host response to infection by simultaneously monitoring infected cells, neutrophils, and macrophages in vivo. Using this model, we show how the angiotensin-converting enzyme inhibitor, ramipril, and mitophagy inhibitor, MDIVI-1, improved survival, decreased viral burden, and improved the respiratory burst response to IAV infection. The Color-flu zebrafish larvae model of IAV infection is complementary to other models where the dynamics of infection and the response of innate immune cells can be visualized in a transparent host in vivo.

Multi-spectral Fluorescent Reporter Influenza A Viruses Allow for in vivo Studies of Innate Immune Function in Zebrafish.

Influenza virus infection can cause severe respiratory disease and is estimated to cause millions of illnesses annually. Studies of the contribution of the innate immune response to influenza A virus (IAV) to viral pathogenesis may yield new antiviral strategies. Zebrafish larvae are useful models to study the innate immune response to pathogens, including IAV, in vivo. Here, we demonstrate how Color-flu, four fluorescent IAV strains originally developed for mice, can be used to study host-virus interactions by simultaneously monitoring virus particles, neutrophils, and macrophages in vivo. Using this model, we show how the angiotensin-converting enzyme inhibitor, ramipril, and mitophagy inhibitor, MDIVI-1, improved survival, decreased viral burden, and improved the respiratory burst response to IAV infection. The Color-flu zebrafish model of IAV infection is complementary to other models as it is the only model where interactions between virus particles and host cells in an intact vertebrate can be visualized in vivo.

A New Phenotype in Candida-Epithelial Cell Interaction Distinguishes Colonization- versus Vulvovaginal Candidiasis-Associated Strains.

Vulvovaginal candidiasis (VVC) affects nearly 3/4 of women during their lifetime, and its symptoms seriously reduce quality of life. Although Candida albicans is a common commensal, it is unknown if VVC results from a switch from a commensal to pathogenic state, if only some strains can cause VVC, and/or if there is displacement of commensal strains with more pathogenic strains. We studied a set of VVC and colonizing C. albicans strains to identify consistent in vitro phenotypes associated with one group or the other. We find that the strains do not differ in overall genetic profile or behavior in culture media (i.e., multilocus sequence type [MLST] profile, rate of growth, and filamentation), but they show strikingly different behaviors during their interactions with vaginal epithelial cells. Epithelial infections with VVC-derived strains yielded stronger fungal proliferation and shedding of fungi and epithelial cells. Transcriptome sequencing (RNA-seq) analysis of representative epithelial cell infections with selected pathogenic or commensal isolates identified several differentially activated epithelial signaling pathways, including the integrin, ferroptosis, and type I interferon pathways; the latter has been implicated in damage protection. Strikingly, inhibition of type I interferon signaling selectively increases fungal shedding of strains in the colonizing cohort, suggesting that increased shedding correlates with lower interferon pathway activation. These data suggest that VVC strains may intrinsically have enhanced pathogenic potential via differential elicitation of epithelial responses, including the type I interferon pathway. Therefore, it may eventually be possible to evaluate pathogenic potential in vitro to refine VVC diagnosis. IMPORTANCE Despite a high incidence of VVC, we still have a poor understanding of this female-specific disease whose negative impact on women's quality of life has become a public health issue. It is not yet possible to determine by genotype or laboratory phenotype if a given Candida albicans strain is more or less likely to cause VVC. Here, we show that Candida strains causing VVC induce more fungal shedding from epithelial cells than strains from healthy women. This effect is also accompanied by increased epithelial cell detachment and differential activation of the type I interferon pathway. These distinguishing phenotypes suggest it may be possible to evaluate the VVC pathogenic potential of fungal isolates. This would permit more targeted antifungal treatments to spare commensals and could allow for displacement of pathogenic strains with nonpathogenic colonizers. We expect these new assays to provide a more targeted tool for identifying fungal virulence factors and epithelial responses that control fungal vaginitis.

Retraction Notice to: Modulation of TNFα Activity by the microRNA Let-7 Coordinates Zebrafish Heart Regeneration.

[This retracts the article DOI: 10.1016/j.isci.2019.06.017.].

Retraction Notice to: Modulation of TNFa Activity by the microRNA Let-7 Coordinates Zebrafish Heart Regeneration.

[This retracts the article DOI: 10.1016/j.isci.2019.04.009.].

Rearrangement in the Hypervariable Region of JC Polyomavirus Genomes Isolated from Patient Samples and Impact on Transcription Factor-Binding Sites and Disease Outcomes.

JC polyomavirus (JCPyV) is the causative agent of the fatal, incurable, neurological disease, progressive multifocal leukoencephalopathy (PML). The virus is present in most of the adult population as a persistent, asymptotic infection in the kidneys. During immunosuppression, JCPyV reactivates and invades the central nervous system. A main predictor of disease outcome is determined by mutations within the hypervariable region of the viral genome. In patients with PML, JCPyV undergoes genetic rearrangements in the noncoding control region (NCCR). The outcome of these rearrangements influences transcription factor binding to the NCCR, orchestrating viral gene transcription. This study examines 989 NCCR sequences from patient isolates deposited in GenBank to determine the frequency of mutations based on patient isolation site and disease status. The transcription factor binding sites (TFBS) were also analyzed to understand how these rearrangements could influence viral transcription. It was determined that the number of TFBS was significantly higher in PML samples compared to non-PML samples. Additionally, TFBS that could promote JCPyV infection were more prevalent in samples isolated from the cerebrospinal fluid compared to other locations. Collectively, this research describes the extent of mutations in the NCCR that alter TFBS and how they correlate with disease outcome.

Beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in the zebrafish model of Duchenne muscular dystrophy.

Neuromuscular electrical stimulation (NMES) allows activation of muscle fibers in the absence of voluntary force generation. NMES could have the potential to promote muscle homeostasis in the context of muscle disease, but the impacts of NMES on diseased muscle are not well understood. We used the zebrafish Duchenne muscular dystrophy (dmd) mutant and a longitudinal design to elucidate the consequences of NMES on muscle health. We designed four neuromuscular stimulation paradigms loosely based on weightlifting regimens. Each paradigm differentially affected neuromuscular structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES improves muscle and neuromuscular junction morphology, swimming, and survival. Heme oxygenase and integrin alpha7 are required for eNMES-mediated improvement. Our data indicate that neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function.

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.1 µM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

PI3K/AKT/mTOR Signaling Pathway Is Required for JCPyV Infection in Primary Astrocytes.

Astrocytes are a main target of JC polyomavirus (JCPyV) in the central nervous system (CNS), where the destruction of these cells, along with oligodendrocytes, leads to the fatal disease progressive multifocal leukoencephalopathy (PML). There is no cure currently available for PML, so it is essential to discover antivirals for this aggressive disease. Additionally, the lack of a tractable in vivo models for studying JCPyV infection makes primary cells an accurate alternative for elucidating mechanisms of viral infection in the CNS. This research to better understand the signaling pathways activated in response to JCPyV infection reveals and establishes the importance of the PI3K/AKT/mTOR signaling pathway in JCPyV infection in primary human astrocytes compared to transformed cell lines. Using RNA sequencing and chemical inhibitors to target PI3K, AKT, and mTOR, we have demonstrated the importance of this signaling pathway in JCPyV infection of primary astrocytes not observed in transformed cells. Collectively, these findings illuminate the potential for repurposing drugs that are involved with inhibition of the PI3K/AKT/mTOR signaling pathway and cancer treatment as potential therapeutics for PML, caused by this neuroinvasive virus.

Meta-analysis: Congruence of genomic and phenotypic differentiation across diverse natural study systems.

Linking genotype to phenotype is a primary goal for understanding the genomic underpinnings of evolution. However, little work has explored whether patterns of linked genomic and phenotypic differentiation are congruent across natural study systems and traits. Here, we investigate such patterns with a meta-analysis of studies examining population-level differentiation at subsets of loci and traits putatively responding to divergent selection. We show that across the 31 studies (88 natural population-level comparisons) we examined, there was a moderate (R 2 = 0.39) relationship between genomic differentiation (F ST ) and phenotypic differentiation (P ST ) for loci and traits putatively under selection. This quantitative relationship between P ST and F ST for loci under selection in diverse taxa provides broad context and cross-system predictions for genomic and phenotypic adaptation by natural selection in natural populations. This context may eventually allow for more precise ideas of what constitutes "strong" differentiation, predictions about the effect size of loci, comparisons of taxa evolving in nonparallel ways, and more. On the other hand, links between P ST and F ST within studies were very weak, suggesting that much work remains in linking genomic differentiation to phenotypic differentiation at specific phenotypes. We suggest that linking genotypes to specific phenotypes can be improved by correlating genomic and phenotypic differentiation across a spectrum of diverging populations within a taxon and including wide coverage of both genomes and phenomes.

The MAPK/ERK Pathway and the Role of DUSP1 in JCPyV Infection of Primary Astrocytes.

JC polyomavirus (JCPyV) is a neuroinvasive pathogen causing a fatal, demyelinating disease of the central nervous system (CNS) known as progressive multifocal leukoencephalopathy (PML). Within the CNS, JCPyV predominately targets two cell types: oligodendrocytes and astrocytes. The underlying mechanisms of astrocytic infection are poorly understood, yet recent findings suggest critical differences in JCPyV infection of primary astrocytes compared to a widely studied immortalized cell model. RNA sequencing was performed in primary normal human astrocytes (NHAs) to analyze the transcriptomic profile that emerges during JCPyV infection. Through a comparative analysis, it was validated that JCPyV requires the mitogen-activated protein kinase, extracellular signal-regulated kinase (MAPK/ERK) pathway, and additionally requires the expression of dual-specificity phosphatases (DUSPs). Specifically, the expression of DUSP1 is needed to establish a successful infection in NHAs, yet this was not observed in an immortalized cell model of JCPyV infection. Additional analyses demonstrated immune activation uniquely observed in NHAs. These results support the hypothesis that DUSPs within the MAPK/ERK pathway impact viral infection and influence potential downstream targets and cellular pathways. Collectively, this research implicates DUSP1 in JCPyV infection of primary human astrocytes, and most importantly, further resolves the signaling events that lead to successful JCPyV infection in the CNS.

Modeling Virus-Induced Inflammation in Zebrafish: A Balance Between Infection Control and Excessive Inflammation.

The inflammatory response to viral infection in humans is a dynamic process with complex cell interactions that are governed by the immune system and influenced by both host and viral factors. Due to this complexity, the relative contributions of the virus and host factors are best studied in vivo using animal models. In this review, we describe how the zebrafish (Danio rerio) has been used as a powerful model to study host-virus interactions and inflammation by combining robust forward and reverse genetic tools with in vivo imaging of transparent embryos and larvae. The innate immune system has an essential role in the initial inflammatory response to viral infection. Focused studies of the innate immune response to viral infection are possible using the zebrafish model as there is a 4-6 week timeframe during development where they have a functional innate immune system dominated by neutrophils and macrophages. During this timeframe, zebrafish lack a functional adaptive immune system, so it is possible to study the innate immune response in isolation. Sequencing of the zebrafish genome has revealed significant genetic conservation with the human genome, and multiple studies have revealed both functional conservation of genes, including those critical to host cell infection and host cell inflammatory response. In addition to studying several fish viruses, zebrafish infection models have been developed for several human viruses, including influenza A, noroviruses, chikungunya, Zika, dengue, herpes simplex virus type 1, Sindbis, and hepatitis C virus. The development of these diverse viral infection models, coupled with the inherent strengths of the zebrafish model, particularly as it relates to our understanding of macrophage and neutrophil biology, offers opportunities for far more intensive studies aimed at understanding conserved host responses to viral infection. In this context, we review aspects relating to the evolution of innate immunity, including the evolution of viral pattern recognition receptors, interferons and interferon receptors, and non-coding RNAs.

Chronic cortisol exposure in early development leads to neuroendocrine dysregulation in adulthood.

OBJECTIVE: Chronic early life stress can affect development of the neuroendocrine stress system, leading to its persistent dysregulation and consequently increased disease risk in adulthood. One contributing factor is thought to be epigenetic programming in response to chronic cortisol exposure during early development. We have previously shown that zebrafish embryos treated chronically with cortisol develop into adults with constitutively elevated whole-body cortisol and aberrant immune gene expression. Here we further characterize that phenotype by assessing persistent effects of the treatment on cortisol tissue distribution and dynamics, chromatin accessibility, and activities of glucocorticoid-responsive regulatory genes klf9 and fkbp5. To that end cortisol levels in different tissues of fed and fasted adults were measured using ELISA, open chromatin in adult blood cells was mapped using ATAC-seq, and gene activity in adult blood and brain cells was measured using qRT-PCR. RESULTS: Adults derived from cortisol-treated embryos have elevated whole-body cortisol with aberrantly regulated tissue distribution and dynamics that correlate with differential activity of klf9 and fkbp5 in blood and brain.

Quantitative Trait Locus and Integrative Genomics Revealed Candidate Modifier Genes for Ectopic Mineralization in Mouse Models of Pseudoxanthoma Elasticum.

Pseudoxanthoma elasticum, a prototype of heritable multisystem ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter, ABCC6. The phenotypic spectrum of pseudoxanthoma elasticum varies, and the correlation between genotype and phenotype has not been established. To identify genetic modifiers, we performed quantitative trait locus analysis in inbred mouse strains that carry the same hypomorphic allele in Abcc6 yet with highly variable ectopic mineralization phenotypes of pseudoxanthoma elasticum. Abcc6 was confirmed as a major determinant for ectopic mineralization in multiple tissues. Integrative analysis using functional genomics tools that included GeneWeaver, String, and Mouse Genome Informatics identified a total of nine additional candidate modifier genes that could influence the organ-specific ectopic mineralization phenotypes. Integration of the candidate genes into the existing ectopic mineralization gene network expands the current knowledge on the complexity of the network that, as a whole, governs ectopic mineralization in soft connective tissues.

Modulation of TNFα Activity by the microRNA Let-7 Coordinates Zebrafish Heart Regeneration.

The adult zebrafish is capable of regenerating heart muscle, resolving collagen tissue, and fully restoring heart function throughout its life. In this study, we show that the highly upregulated, epicardium-enriched microRNA let-7i functions in wound closure and cardiomyocyte proliferation. RNA sequencing experiments identified upregulated expression of members of the tumor necrosis factor (TNF) signaling pathway in the absence of let-7. Importantly, co-suppression of TNF and let-7 activity rescued epicardium migration and cardiomyocyte proliferation defects induced by depletion of let-7 alone. Sensitizing animals to low levels of TNF activity before injury culminated in repressed cardiomyocyte proliferation and wound closure defects, suggesting that levels of inflammation at the onset of injury are critical for heart regeneration. Our studies indicate that injury-induced reduction in TNF signaling by let-7 in the epicardium creates a pro-regenerative environment for cardiomyocyte proliferation during adult heart regeneration.

Profiling microRNA expression in Atlantic killifish (Fundulus heteroclitus) gill and responses to arsenic and hyperosmotic stress.

The Atlantic killifish (Fundulus heteroclitus), native to estuarine areas of the Atlantic coast of the United States, has become a valuable ecotoxicological model as a result of its ability to acclimate to rapid environmental changes and adapt to polluted habitats. MicroRNAs (miRNAs) are highly conserved small RNAs that regulate gene expression and play critical roles in stress responses in a variety of organisms. Global miRNA expression in killifish and the potential roles miRNA have in environmental acclimation have yet to be characterized. Accordingly, we profiled miRNA expression in killifish gill for the first time and identified a small group of highly expressed, well-conserved miRNAs as well as 16 novel miRNAs not yet identified in other organisms. Killifish respond to large fluctuations in salinity with rapid changes in gene expression and protein trafficking to maintain osmotic balance, followed by a secondary phase of gene and protein expression changes that enable remodeling of the gills. Arsenic, a major environmental toxicant, was previously shown to inhibit gene expression responses in killifish gill, as well the ability of killifish to acclimate to a rapid increase in salinity. Thus, we examined the individual and combined effects of salinity and arsenic on miRNA expression in killifish gill. Using small RNA sequencing, we identified 270 miRNAs expressed in killifish, and found that miR-135b was differentially expressed in response to arsenic and at 24 h following transfer to salt water. Predicted targets of miR-135b are involved in ion transport, cell motility and migration, GTPase mediated signal transduction and organelle assembly. Consistent with previous studies of these two environmental stressors, we found a significant interaction (i.e., arsenic dependent salinity effect), whereby killifish exposed to arsenic exhibited an opposite response in miR-135b expression at 24 h post hyperosmotic challenge compared to controls. By examining mRNA expression of predicted miRNA targets during salinity acclimation and arsenic exposure, we found that miR-135b targets were significantly more likely to decrease during salinity acclimation than non-targets. Our identification of a significant interaction effect of arsenic and salinity on miR-135b expression supports the hypothesis that arsenic alters upstream regulators of stress response networks, which may adversely affect the killifish response to osmotic stress. The characterization of miRNAs in this ecotoxicological model will be a valuable resource for future studies investigating the role of miRNAs in response to environmental stress.

RegenDbase: a comparative database of noncoding RNA regulation of tissue regeneration circuits across multiple taxa.

Regeneration is an endogenous process of tissue repair that culminates in complete restoration of tissue and organ function. While regenerative capacity in mammals is limited to select tissues, lower vertebrates like zebrafish and salamanders are endowed with the capacity to regenerate entire limbs and most adult tissues, including heart muscle. Numerous profiling studies have been conducted using these research models in an effort to identify the genetic circuits that accompany tissue regeneration. Most of these studies, however, are confined to an individual injury model and/or research organism and focused primarily on protein encoding transcripts. Here we describe RegenDbase, a new database with the functionality to compare and contrast gene regulatory pathways within and across tissues and research models. RegenDbase combines pipelines that integrate analysis of noncoding RNAs in combination with protein encoding transcripts. We created RegenDbase with a newly generated comprehensive dataset for adult zebrafish heart regeneration combined with existing microarray and RNA-sequencing studies on multiple injured tissues. In this current release, we detail microRNA-mRNA regulatory circuits and the biological processes these interactions control during the early stages of heart regeneration. Moreover, we identify known and putative novel lncRNAs and identify their potential target genes based on proximity searches. We postulate that these candidate factors underscore robust regenerative capacity in lower vertebrates. RegenDbase provides a systems-level analysis of tissue regeneration genetic circuits across injury and animal models and addresses the growing need to understand how noncoding RNAs influence these changes in gene expression.