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Connexin hemichannels were identified as the first members of the eukaryotic large-pore channel family that mediate permeation of both atomic ions and small molecules between the intracellular and extracellular environments. The conventional view is that their pore is a large passive conduit through which both ions and molecules diffuse in a similar manner. In stark contrast to this notion, we demonstrate that the permeation of ions and of molecules in connexin hemichannels can be uncoupled and differentially regulated. We find that human connexin mutations that produce pathologies and were previously thought to be loss-of-function mutations due to the lack of ionic currents are still capable of mediating the passive transport of molecules with kinetics close to those of wild-type channels. This molecular transport displays saturability in the micromolar range, selectivity, and competitive inhibition, properties that are tuned by specific interactions between the permeating molecules and the N-terminal domain that lies within the pore-a general feature of large-pore channels. We propose that connexin hemichannels and, likely, other large-pore channels, are hybrid channel/transporter-like proteins that might switch between these two modes to promote selective ion conduction or autocrine/paracrine molecular signaling in health and disease processes.

Multiple sclerosis (MS) is a chronic and debilitating neurological disease that results in inflammatory demyelination. While endogenous remyelination helps to recover function, this restorative process tends to become less efficient over time. Currently, intense efforts aimed at the mechanisms that promote remyelination are being considered promising therapeutic approaches. The M1 muscarinic acetylcholine receptor (M1R) was previously identified as a negative regulator of oligodendrocyte differentiation and myelination. Here, we validate M1R as a target for remyelination by characterizing expression in human and rodent oligodendroglial cells (including those in human MS tissue) using a highly selective M1R probe. As a breakthrough to conventional methodology, we conjugated a fluorophore to a highly M1R selective peptide (MT7) which targets the M1R in the subnanomolar range. This allows for exceptional detection of M1R protein expression in the human CNS. More importantly, we introduce PIPE-307, a brain-penetrant, small-molecule antagonist with favorable drug-like properties that selectively targets M1R. We evaluate PIPE-307 in a series of in vitro and in vivo studies to characterize potency and selectivity for M1R over M2-5R and confirm the sufficiency of blocking this receptor to promote differentiation and remyelination. Further, PIPE-307 displays significant efficacy in the mouse experimental autoimmune encephalomyelitis model of MS as evaluated by quantifying disability, histology, electron microscopy, and visual evoked potentials. Together, these findings support targeting M1R for remyelination and support further development of PIPE-307 for clinical studies.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early β-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of β-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in β-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any β-catenin transcriptional activity as measured by β-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in β-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl.

Controlling the principal African malaria vector, the mosquito Anopheles gambiae, is considered essential to curtail malaria transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass releases of nonbiting, nondriving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here, we introduce a vector control technology termed precision-guided sterile insect technique (pgSIT), in A. gambiae for inducible, programmed male sterilization and female elimination for wide-scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male sterility and >99.9% female lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce sustained population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, and safe suppression in the species.

Legacy effects describe the persistent, long-term impacts on an ecosystem following the removal of an abiotic or biotic feature. Redlining, a policy that codified racial segregation and disinvestment in minoritized neighborhoods, has produced legacy effects with profound impacts on urban ecosystem structure and health. These legacies have detrimentally impacted public health outcomes, socioeconomic stability, and environmental health. However, the collateral impacts of redlining on wildlife communities are uncertain. Here, we investigated whether faunal biodiversity was associated with redlining. We used home-owner loan corporation (HOLC) maps [grades A (i.e., "best" and "greenlined"), B, C, and D (i.e., "hazardous" and "redlined")] across four cities in California and contributory science data (iNaturalist) to estimate alpha and beta diversity across six clades (mammals, birds, insects, arachnids, reptiles, and amphibians) as a function of HOLC grade. We found that in greenlined neighborhoods, unique species were detected with less sampling effort, with redlined neighborhoods needing over 8,000 observations to detect the same number of unique species. Historically redlined neighborhoods had lower native and nonnative species richness compared to greenlined neighborhoods across each city, with disparities remaining at the clade level. Further, community composition (i.e., beta diversity) consistently differed among HOLC grades for all cities, including large differences in species assemblage observed between green and redlined neighborhoods. Our work spotlights the lasting effects of social injustices on the community ecology of cities, emphasizing that urban conservation and management efforts must incorporate an antiracist, justice-informed lens to improve biodiversity in urban environments.

The heart beats approximately 100,000 times per day in humans, imposing substantial energetic demands on cardiac muscle. Adenosine triphosphate (ATP) is an essential energy source for normal function of cardiac muscle during each beat, as it powers ion transport, intracellular Ca2+ handling, and actin-myosin cross-bridge cycling. Despite this, the impact of excitation-contraction coupling on the intracellular ATP concentration ([ATP]i) in myocytes is poorly understood. Here, we conducted real-time measurements of [ATP]i in ventricular myocytes using a genetically encoded ATP fluorescent reporter. Our data reveal rapid beat-to-beat variations in [ATP]i. Notably, diastolic [ATP]i was

In many organisms, most notably Drosophila, homologous chromosomes associate in somatic cells, a phenomenon known as somatic pairing, which takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, mediated by different proteins that bind to these different regions. Here, we use computational modeling to evaluate an alternative "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. In this model, buttons are nonuniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a nonhomolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. By simulating randomly generated nonuniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. We conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.

Infanticide and adoption have been attributed to sexual selection, where an individual later reproduces with the parent whose offspring it killed or adopted. While sexually selected infanticide is well known, evidence for sexually selected adoption is anecdotal. We report on both behaviors at 346 nests over 27 y in green-rumped parrotlets (Forpus passerinus) in Venezuela. Parrotlets are monogamous with long-term pair bonds, exhibit a strongly male-biased adult sex ratio, and nest in cavities that are in short supply, creating intense competition for nest sites and mates. Infanticide attacks occurred at 256 nests in two distinct contexts: 1) Attacks were primarily committed by nonbreeding pairs (69%) attempting to evict parents from the cavity. Infanticide attacks per nest were positively correlated with population size and evicting pairs never adopted abandoned offspring. Competition for limited nest sites was a primary cause of eviction-driven infanticide, and 2) attacks occurred less frequently at nests where one mate died (31%), was perpetrated primarily by stepparents of both sexes, and was independent of population size. Thus, within a single species and mating system, infanticide occurred in multiple contexts due to multiple drivers. Nevertheless, 48% of stepparents of both sexes adopted offspring, and another 23% of stepfathers exhibited both infanticide and long-term care. Stepfathers were often young males who subsequently nested with widows, reaching earlier ages of first breeding than competitors and demonstrating sexually selected adoption. Adoption and infanticide conferred similar fitness benefits to stepfathers and appeared to be equivalent strategies driven by limited breeding opportunities, male-biased sex ratios, and long-term monogamy.

Affective touch-a slow, gentle, and pleasant form of touch-activates a different neural network than which is activated during discriminative touch in humans. Affective touch perception is enabled by specialized low-threshold mechanoreceptors in the skin with unmyelinated fibers called C tactile (CT) afferents. These CT afferents are conserved across mammalian species, including macaque monkeys. However, it is unknown whether the neural representation of affective touch is the same across species and whether affective touch's capacity to activate the hubs of the brain that compute socioaffective information requires conscious perception. Here, we used functional MRI to assess the preferential activation of neural hubs by slow (affective) vs. fast (discriminative) touch in anesthetized rhesus monkeys (Macaca mulatta). The insula, anterior cingulate cortex (ACC), amygdala, and secondary somatosensory cortex were all significantly more active during slow touch relative to fast touch, suggesting homologous activation of the interoceptive-allostatic network across primate species during affective touch. Further, we found that neural responses to affective vs. discriminative touch in the insula and ACC (the primary cortical hubs for interoceptive processing) changed significantly with age. Insula and ACC in younger animals differentiated between slow and fast touch, while activity was comparable between conditions for aged monkeys (equivalent to >70 y in humans). These results, together with prior studies establishing conserved peripheral nervous system mechanisms of affective touch transduction, suggest that neural responses to affective touch are evolutionarily conserved in monkeys, significantly impacted in old age, and do not necessitate conscious experience of touch.

Global control of infectious diseases depends on the continuous development and deployment of diverse vaccination strategies. Currently available live-attenuated and killed virus vaccines typically take a week or longer to activate specific protection by the adaptive immunity. The mosquito-transmitted Nodamura virus (NoV) is attenuated in mice by mutations that prevent expression of the B2 viral suppressor of RNA interference (VSR) and consequently, drastically enhance in vivo production of the virus-targeting small-interfering RNAs. We reported recently that 2 d after immunization with live-attenuated VSR-disabled NoV (NoVΔB2), neonatal mice become fully protected against lethal NoV challenge and develop no detectable infection. Using Rag1-/- mice that produce no mature B and T lymphocytes as a model, here we examined the hypothesis that adaptive immunity is dispensable for the RNAi-based protective immunity activated by NoVΔB2 immunization. We show that immunization of both neonatal and adult Rag1-/- mice with live but not killed NoVΔB2 induces full protection against NoV challenge at 2 or 14 d postimmunization. Moreover, NoVΔB2-induced protective antiviral immunity is virus-specific and remains effective in adult Rag1-/- mice 42 and 90 d after a single-shot immunization. We conclude that immunization with the live-attenuated VSR-disabled RNA virus vaccine activates rapid and long-lasting protective immunity against lethal challenges by a distinct mechanism independent of the adaptive immunity mediated by B and T cells. Future studies are warranted to determine whether additional animal and human viruses attenuated by VSR inactivation induce similar protective immunity in healthy and adaptive immunity-compromised individuals.

Listeria monocytogenes is a bacterial pathogen that can cause life-threatening central nervous system (CNS) infections. While mechanisms by which L. monocytogenes and other pathogens traffic to the brain have been studied, a quantitative understanding of the underlying dynamics of colonization and replication within the brain is still lacking. In this study, we used barcoded L. monocytogenes to quantify the bottlenecks and dissemination patterns that lead to cerebral infection. Following intravenous (IV) inoculation, multiple independent invasion events seeded all parts of the CNS from the blood, however, only one clone usually became dominant in the brain. Sequential IV inoculations and intracranial inoculations suggested that clones that had a temporal advantage (i.e., seeded the CNS first), rather than a spatial advantage (i.e., invaded a particular brain region), were the main drivers of clonal dominance. In a foodborne model of cerebral infection with immunocompromised mice, rare invasion events instead led to a highly infected yet monoclonal CNS. This restrictive bottleneck likely arose from pathogen transit into the blood, rather than directly from the blood to the brain. Collectively, our findings provide a detailed quantitative understanding of the L. monocytogenes population dynamics that lead to CNS infection and a framework for studying the dynamics of other cerebral infections.

The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during cell division by monitoring kinetochore-microtubule attachment. Plants produce both sequence-conserved and diverged SAC components, and it has been largely unknown how SAC activation leads to the assembly of these proteins at unattached kinetochores to prevent cells from entering anaphase. In Arabidopsis thaliana, the noncanonical BUB3.3 protein was detected at kinetochores throughout mitosis, unlike MAD1 and the plant-specific BUB1/MAD3 family protein BMF3 that associated with unattached chromosomes only. When BUB3.3 was lost by a genetic mutation, mitotic cells often entered anaphase with misaligned chromosomes and presented lagging chromosomes after they were challenged by low doses of the microtubule depolymerizing agent oryzalin, resulting in the formation of micronuclei. Surprisingly, BUB3.3 was not required for the kinetochore localization of other SAC proteins or vice versa. Instead, BUB3.3 specifically bound to BMF3 through two internal repeat motifs that were not required for BMF3 kinetochore localization. This interaction enabled BMF3 to recruit CDC20, a downstream SAC target, to unattached kinetochores. Taken together, our findings demonstrate that plant SAC utilizes unconventional protein interactions for arresting mitosis, with BUB3.3 directing BMF3's role in CDC20 recruitment, rather than the recruitment of BUB1/MAD3 proteins observed in fungi and animals. This distinct mechanism highlights how plants adapted divergent versions of conserved cell cycle machinery to achieve specialized SAC control.

Genomic evidence supports an important role for selection in shaping patterns of introgression along the genome, but frameworks for understanding the evolutionary dynamics within hybrid populations that underlie these patterns have been lacking. Due to the clock-like effect of recombination in hybrids breaking up parental haplotypes, drift and selection produce predictable patterns of ancestry variation at varying spatial genomic scales through time. Here, we develop methods based on the Discrete Wavelet Transform to study the genomic scale of local ancestry variation and its association with recombination rates and show that these methods capture temporal dynamics of drift and genome-wide selection after hybridization. We apply these methods to published datasets from hybrid populations of swordtail fish (Xiphophorus) and baboons (Papio) and to inferred Neanderthal introgression in modern humans. Across systems, upward of 20% of variation in local ancestry at the broadest genomic scales can be attributed to systematic selection against introgressed alleles, consistent with strong selection acting on early-generation hybrids. Signatures of selection at fine genomic scales suggest selection over longer time scales; however, we suggest that our ability to confidently infer selection at fine scales is likely limited by inherent biases in current methods for estimating local ancestry from contiguous segments of genomic similarity. Wavelet approaches will become widely applicable as genomic data from systems with introgression become increasingly available and can help shed light on generalities of the genomic consequences of interspecific hybridization.

Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.

Cancer therapy, including immunotherapy, is inherently limited by chronic inflammation-induced tumorigenesis and toxicity within the tumor microenvironment. Thus, stimulating the resolution of inflammation may enhance immunotherapy and improve the toxicity of immune checkpoint inhibition (ICI). As epoxy-fatty acids (EpFAs) are degraded by the enzyme soluble epoxide hydrolase (sEH), the inhibition of sEH increases endogenous EpFA levels to promote the resolution of cancer-associated inflammation. Here, we demonstrate that systemic treatment with ICI induces sEH expression in multiple murine cancer models. Dietary omega-3 polyunsaturated fatty acid supplementation and pharmacologic sEH inhibition, both alone and in combination, significantly enhance anti-tumor activity of ICI in these models. Notably, pharmacological abrogation of the sEH pathway alone or in combination with ICI counter-regulates an ICI-induced pro-inflammatory and pro-tumorigenic cytokine storm. Thus, modulating endogenous EpFA levels through dietary supplementation or sEH inhibition may represent a unique strategy to enhance the anti-tumor activity of paradigm cancer therapies.

Kelp forests are highly productive and economically important ecosystems worldwide, especially in the North Pacific Ocean. However, current hypotheses for their evolutionary origins are reliant on a scant fossil record. Here, we report fossil hapteral kelp holdfasts from western Washington State, USA, indicating that kelp has existed in the northeastern Pacific Ocean since the earliest Oligocene. This is consistent with the proposed North Pacific origin of kelp associated with global cooling around the Eocene-Oligocene transition. These fossils also support the hypotheses that a hapteral holdfast, rather than a discoid holdfast, is the ancestral state in complex kelps and suggest that early kelps likely had a flexible rather than a stiff stipe. Early kelps were possibly grazed upon by mammals like desmostylians, but fossil evidence of the complex ecological interactions known from extant kelp forests is lacking. The fossil record further indicates that the present-day, multi-story kelp forest had developed at latest after the mid-Miocene climate optimum. In summary, the fossils signify a stepwise evolution of the kelp ecosystem in the North Pacific, likely enabled by changes in the ocean-climate system.

Across internally fertilising species, males transfer ejaculate proteins that trigger wide-ranging changes in female behaviour and physiology. Much theory has been developed to explore the drivers of ejaculate protein evolution. The accelerating availability of high-quality genomes now allows us to test how these proteins are evolving at fine taxonomic scales. Here, we use genomes from 264 species to chart the evolutionary history of Sex Peptide (SP), a potent regulator of female post-mating responses in Drosophila melanogaster. We infer that SP first evolved in the Drosophilinae subfamily and has since followed markedly different evolutionary trajectories in different lineages. Outside of the Sophophora-Lordiphosa, SP exists largely as a single-copy gene with independent losses in several lineages. Within the Sophophora-Lordiphosa, the SP gene family has repeatedly and independently expanded. Up to seven copies, collectively displaying extensive sequence variation, are present in some species. Despite these changes, SP expression remains restricted to the male reproductive tract. Alongside, we document considerable interspecific variation in the presence and morphology of seminal microcarriers that, despite the critical role SP plays in microcarrier assembly in D. melanogaster, appears to be independent of changes in the presence/absence or sequence of SP. We end by providing evidence that SP's evolution is decoupled from that of its receptor, Sex Peptide Receptor, in which we detect no evidence of correlated diversifying selection. Collectively, our work describes the divergent evolutionary trajectories that a novel gene has taken following its origin and finds a surprisingly weak coevolutionary signal between a supposedly sexually antagonistic protein and its receptor.

Cross-ecosystem subsidies are critical to ecosystem structure and function, especially in recipient ecosystems where they are the primary source of organic matter to the food web. Subsidies are indicative of processes connecting ecosystems and can couple ecological dynamics across system boundaries. However, the degree to which such flows can induce cross-ecosystem cascades of spatial synchrony, the tendency for system fluctuations to be correlated across locations, is not well understood. Synchrony has destabilizing effects on ecosystems, adding to the importance of understanding spatiotemporal patterns of synchrony transmission. In order to understand whether and how spatial synchrony cascades across the marine-terrestrial boundary via resource subsidies, we studied the relationship between giant kelp forests on rocky nearshore reefs and sandy beach ecosystems that receive resource subsidies in the form of kelp wrack (detritus). We found that synchrony cascades from rocky reefs to sandy beaches, with spatiotemporal patterns mediated by fluctuations in live kelp biomass, wave action, and beach width. Moreover, wrack deposition synchronized local abundances of shorebirds that move among beaches seeking to forage on wrack-associated invertebrates, demonstrating that synchrony due to subsidies propagates across trophic levels in the recipient ecosystem. Synchronizing resource subsidies likely play an underappreciated role in the spatiotemporal structure, functioning, and stability of ecosystems.

The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2E62K also causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2E62K stimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2+/E62K bone-marrow-derived macrophages also cannibalize primary Rac2+/E62K T cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2+/E62K macrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2E62K also enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2+/E62K human immunodeficiency and enhance CAR-M cancer immunotherapy.

Seasonal influenza results in 3 to 5 million cases of severe disease and 250,000 to 500,000 deaths annually. Macrophages have been implicated in both the resolution and progression of the disease, but the drivers of these outcomes are poorly understood. We probed mouse lung transcriptomic datasets using the Digital Cell Quantifier algorithm to predict immune cell subsets that correlated with mild or severe influenza A virus (IAV) infection outcomes. We identified a unique lung macrophage population that transcriptionally resembled small serosal cavity macrophages and whose presence correlated with mild disease. Until now, the study of serosal macrophage translocation in the context of viral infections has been neglected. Here, we show that pleural macrophages (PMs) migrate from the pleural cavity to the lung after infection with IAV. We found that the depletion of PMs increased morbidity and pulmonary inflammation. There were increased proinflammatory cytokines in the pleural cavity and an influx of neutrophils within the lung. Our results show that PMs are recruited to the lung during IAV infection and contribute to recovery from influenza. This study expands our knowledge of PM plasticity and identifies a source of lung macrophages independent of monocyte recruitment and local proliferation.

To mediate critical host-microbe interactions in the human small intestine, Paneth cells constitutively produce abundant levels of α-defensins and other antimicrobials. We report that the expression profile of these antimicrobials is dramatically askew in human small intestinal organoids (enteroids) as compared to that in paired tissue from which they are derived, with a reduction of α-defensins to nearly undetectable levels. Murine enteroids, however, recapitulate the expression profile of Paneth cell α-defensins seen in tissue. WNT/TCF signaling has been found to be instrumental in the regulation of α-defensins, yet in human enteroids exogenous stimulation of WNT signaling appears insufficient to rescue α-defensin expression. By stark contrast, forkhead box O (FOXO) inhibitor AS1842856 induced the expression of α-defensin mRNA in enteroids by >100,000-fold, restoring DEFA5 and DEFA6 to levels comparable to those found in primary human tissue. These results newly identify FOXO signaling as a pathway of biological and potentially therapeutic relevance for the regulation of human Paneth cell α-defensins in health and disease.

Analyses of genome sequence data have revealed pervasive interspecific gene flow and enriched our understanding of the role of gene flow in speciation and adaptation. Inference of gene flow using genomic data requires powerful statistical methods. Yet current likelihood-based methods involve heavy computation and are feasible for small datasets only. Here, we implement the multispecies-coalescent-with-migration model in the Bayesian program bpp, which can be used to test for gene flow and estimate migration rates, as well as species divergence times and population sizes. We develop Markov chain Monte Carlo algorithms for efficient sampling from the posterior, enabling the analysis of genome-scale datasets with thousands of loci. Implementation of both introgression and migration models in the same program allows us to test whether gene flow occurred continuously over time or in pulses. Analyses of genomic data from Anopheles mosquitoes demonstrate rich information in typical genomic datasets about the mode and rate of gene flow.

Although robustly expressed in the disease-free (DF) breast stroma, CD36 is consistently absent from the stroma surrounding invasive breast cancers (IBCs). In this study, we primarily observed CD36 expression in adipocytes and intralobular capillaries within the DF breast. Larger vessels concentrated in interlobular regions lacked CD36 and were instead marked by the expression of CD31. When evaluated in perilesional capillaries surrounding ductal carcinoma in situ, a nonobligate IBC precursor, CD36 loss was more commonly observed in lesions associated with subsequent IBC. Peroxisome proliferator-activated receptor γ (PPARγ) governs the expression of CD36 and genes involved in differentiation, metabolism, angiogenesis, and inflammation. Coincident with CD36 loss, we observed a dramatic suppression of PPARγ and its target genes in capillary endothelial cells (ECs) and pericytes, which typically surround and support the stability of the capillary endothelium. Factors present in conditioned media from malignant cells repressed PPARγ and its target genes not only in cultured ECs and pericytes but also in adipocytes, which require PPARγ for proper differentiation. In addition, we identified a role for PPARγ in opposing the transition of pericytes toward a tumor-supportive myofibroblast phenotype. In mouse xenograft models, early intervention with rosiglitazone, a PPARγ agonist, demonstrated significant antitumor effects; however, following the development of a palpable tumor, the antitumor effects of rosiglitazone were negated by the repression of PPARγ in the mouse stroma. In summary, PPARγ activity in healthy tissues places several stromal cell types in an antitumorigenic state, directly inhibiting EC proliferation, maintaining adipocyte differentiation, and suppressing the transition of pericytes into tumor-supportive myofibroblasts.

Women remain underrepresented in most math-intensive fields. [Breda and Napp, Proc. Natl. Acad. Sci. U.S.A. 116, 15435 (2019)] reported that girls' comparative advantage in reading over math (i.e., the intraindividual differences between girls' reading vs. math performance, compared to such differences for boys) could explain up to 80% of the gender gap in students' intentions to pursue math-intensive studies and careers, in conflict with findings from previous research. We conducted a conceptual replication and expanded upon Breda and Napp's study by using new global data (PISA2018, N = 466,165) and a recent US nationally representative longitudinal study (High School Longitudinal Study of 2009, N = 6,560). We coded students' intended majors and careers and their actual college majors. The difference between a student's math vs. reading performance explained only small proportions of the gender gap in students' intentions to pursue math-intensive fields (0.4 to 10.2%) and in their enrollment in math-intensive college majors (12.3%). Consistent with previous studies, our findings suggest girls' comparative advantage in reading explains a minority of the gender gap in math-related majors and occupational intentions and choices. Potential reasons for differences in the estimated effect sizes include differences in the operationalization of math-related choices, the operationalization of math and reading performance, and possibly the timing of measuring intentions and choices. Therefore, it seems premature to conclude that girls' comparative advantage in reading, rather than the cumulative effects of other structural and/or psychological factors, can largely explain the persistent gender gap in math-intensive educational and career choices.

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances, treatment options for OS have remained unchanged since the advent of standard chemotherapeutics and offer less than a 25% 5-y survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and necessitates the study of this disease in an innovative system. Here, we adapt a previously described engineered bone marrow (eBM) construct for use as a three-dimensional platform to study how microenvironmental and immune factors affect OS tumor progression. We form eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs after 8 wk for study. We interrogate the influence of the anatomical implantation site on eBM tissue quality, test ex vivo stability under normoxic (5% O2) and standard (21% O2) culture conditions, culture OS cells within these constructs, and compare them to human OS samples. We show that eBM stably recapitulates the composition of native bone marrow. OS cells exhibit differential behavior dependent on metastatic potential when cultured in eBM, thus mimicking in vivo conditions. Furthermore, we highlight the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment and show that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that is useful for mechanistic bone cancer research and drug screening.

Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) in brain capillary endothelial cells, leading to the loss of inwardly rectifying K+ (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP2 by converting it to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD.

The acquisition of novel sexually dimorphic traits poses an evolutionary puzzle: How do new traits arise and become sex-limited? Recently acquired color vision, sexually dimorphic in animals like primates and butterflies, presents a compelling model for understanding how traits become sex-biased. For example, some Heliconius butterflies uniquely possess UV (ultraviolet) color vision, which correlates with the expression of two differentially tuned UV-sensitive rhodopsins, UVRh1 and UVRh2. To discover how such traits become sexually dimorphic, we studied Heliconius charithonia, which exhibits female-specific UVRh1 expression. We demonstrate that females, but not males, discriminate different UV wavelengths. Through whole-genome shotgun sequencing and assembly of the H. charithonia genome, we discovered that UVRh1 is present on the W chromosome, making it obligately female-specific. By knocking out UVRh1, we show that UVRh1 protein expression is absent in mutant female eye tissue, as in wild-type male eyes. A PCR survey of UVRh1 sex-linkage across the genus shows that species with female-specific UVRh1 expression lack UVRh1 gDNA in males. Thus, acquisition of sex linkage is sufficient to achieve female-specific expression of UVRh1, though this does not preclude other mechanisms, like cis-regulatory evolution from also contributing. Moreover, both this event, and mutations leading to differential UV opsin sensitivity, occurred early in the history of Heliconius. These results suggest a path for acquiring sexual dimorphism distinct from existing mechanistic models. We propose a model where gene traffic to heterosomes (the W or the Y) genetically partitions a trait by sex before a phenotype shifts (spectral tuning of UV sensitivity).

Plants produce various pigments that not only appear as attractive colors but also provide valuable resources in applications in daily life and scientific research. Biosynthesis pathways for these natural plant pigments are well studied, and most have multiple enzymes that vary among plant species. However, adapting these pathways to animals remains a challenge. Here, we describe successful biosynthesis of betalains, water-soluble pigments found only in a single plant order, Caryophyllales, in transgenic silkworms by coexpressing three betalain synthesis genes, cytochrome P450 enzyme CYP76AD1, DOPA 4,5-dioxygenase, and betanidin 5-O-glucosyltransferase. Betalains can be synthesized in various tissues under the control of the ubiquitous IE1 promoter but accumulate mainly in the hemolymph with yields as high as 274 μg/ml. Additionally, transformed larvae and pupae show a strong red color easily distinguishable from wild-type animals. In experiments in which expression is controlled by the promoter of silk gland-specific gene, fibroin heavy-chain, betalains are found predominantly in the silk glands and can be secreted into cocoons through spinning. Betalains in transformed cocoons are easily recovered from cocoon shells in water with average yields reaching 14.4 μg/mg. These data provide evidence that insects can synthesize natural plant pigments through a complex, multiple enzyme-mediated synthesis pathway. Such pigments also can serve as dominant visible markers in insect transgenesis applications. This study provides an approach to producing valuable plant-derived compounds by using genetically engineered silkworms as a bioreactor.

Aquaporin-4 (AQP4)-specific Th17 cells are thought to have a central role in neuromyelitis optica (NMO) pathogenesis. When modeling NMO, only AQP4-reactive Th17 cells from AQP4-deficient (AQP4-/-), but not wild-type (WT) mice, caused CNS autoimmunity in recipient WT mice, indicating that a tightly regulated mechanism normally ensures tolerance to AQP4. Here, we found that pathogenic AQP4 T cell epitopes bind MHC II with exceptionally high affinity. Examination of T cell receptor (TCR) α/β usage revealed that AQP4-specific T cells from AQP4-/- mice employed a distinct TCR repertoire and exhibited clonal expansion. Selective thymic AQP4 deficiency did not fully restore AQP4-reactive T cells, demonstrating that thymic negative selection alone did not account for AQP4-specific tolerance in WT mice. Indeed, AQP4-specific Th17 cells caused paralysis in recipient WT or B cell-deficient mice, which was followed by complete recovery that was associated with apoptosis of donor T cells. However, donor AQP4-reactive T cells survived and caused persistent paralysis in recipient mice deficient in both T and B cells or mice lacking T cells only. Thus, AQP4 CNS autoimmunity was limited by T cell-dependent deletion of AQP4-reactive T cells. In contrast, myelin oligodendrocyte glycoprotein (MOG)-specific T cells survived and caused sustained disease in WT mice. These findings underscore the importance of peripheral T cell deletional tolerance to AQP4, which may be relevant to understanding the balance of AQP4-reactive T cells in health and in NMO. T cell tolerance to AQP4, expressed in multiple tissues, is distinct from tolerance to MOG, an autoantigen restricted in its expression.

Proposed genetic approaches for reducing human malaria include population modification, which introduces genes into vector mosquitoes to reduce or prevent parasite transmission. We demonstrate the potential of Cas9/guide RNA (gRNA)-based gene-drive systems linked to dual antiparasite effector genes to spread rapidly through mosquito populations. Two strains have an autonomous gene-drive system coupled to dual anti-Plasmodium falciparum effector genes comprising single-chain variable fragment monoclonal antibodies targeting parasite ookinetes and sporozoites in the African malaria mosquitoes Anopheles gambiae (AgTP13) and Anopheles coluzzii (AcTP13). The gene-drive systems achieved full introduction within 3 to 6 mo after release in small cage trials. Life-table analyses revealed no fitness loads affecting AcTP13 gene-drive dynamics but AgTP13 males were less competitive than wild types. The effector molecules reduced significantly both parasite prevalence and infection intensities. These data supported transmission modeling of conceptual field releases in an island setting that shows meaningful epidemiological impacts at different sporozoite threshold levels (2.5 to 10 k) for human infection by reducing malaria incidence in optimal simulations by 50 to 90% within as few as 1 to 2 mo after a series of releases, and by ≥90% within 3 mo. Modeling outcomes for low sporozoite thresholds are sensitive to gene-drive system fitness loads, gametocytemia infection intensities during parasite challenges, and the formation of potentially drive-resistant genome target sites, extending the predicted times to achieve reduced incidence. TP13-based strains could be effective for malaria control strategies following validation of sporozoite transmission threshold numbers and testing field-derived parasite strains. These or similar strains are viable candidates for future field trials in a malaria-endemic region.

Changes in phenology in response to ongoing climate change have been observed in numerous taxa around the world. Differing rates of phenological shifts across trophic levels have led to concerns that ecological interactions may become increasingly decoupled in time, with potential negative consequences for populations. Despite widespread evidence of phenological change and a broad body of supporting theory, large-scale multitaxa evidence for demographic consequences of phenological asynchrony remains elusive. Using data from a continental-scale bird-banding program, we assess the impact of phenological dynamics on avian breeding productivity in 41 species of migratory and resident North American birds breeding in and around forested areas. We find strong evidence for a phenological optimum where breeding productivity decreases in years with both particularly early or late phenology and when breeding occurs early or late relative to local vegetation phenology. Moreover, we demonstrate that landbird breeding phenology did not keep pace with shifts in the timing of vegetation green-up over a recent 18-y period, even though avian breeding phenology has tracked green-up with greater sensitivity than arrival for migratory species. Species whose breeding phenology more closely tracked green-up tend to migrate shorter distances (or are resident over the entire year) and breed earlier in the season. These results showcase the broadest-scale evidence yet of the demographic impacts of phenological change. Future climate change-associated phenological shifts will likely result in a decrease in breeding productivity for most species, given that bird breeding phenology is failing to keep pace with climate change.

Traumatic brain injury (TBI) is a pervasive problem worldwide for which no effective treatment is currently available. Although most studies have focused on the pathology of the injured brain, we have noted that the liver plays an important role in TBI. Using two mouse models of TBI, we found that the enzymatic activity of hepatic soluble epoxide hydrolase (sEH) was rapidly decreased and then returned to normal levels following TBI, whereas such changes were not observed in the kidney, heart, spleen, or lung. Interestingly, genetic downregulation of hepatic Ephx2 (which encodes sEH) ameliorates TBI-induced neurological deficits and promotes neurological function recovery, whereas overexpression of hepatic sEH exacerbates TBI-associated neurological impairments. Furthermore, hepatic sEH ablation was found to promote the generation of A2 phenotype astrocytes and facilitate the production of various neuroprotective factors associated with astrocytes following TBI. We also observed an inverted V-shaped alteration in the plasma levels of four EET (epoxyeicosatrienoic acid) isoforms (5,6-, 8,9-,11,12-, and 14,15-EET) following TBI which were negatively correlated with hepatic sEH activity. However, hepatic sEH manipulation bidirectionally regulates the plasma levels of 14,15-EET, which rapidly crosses the blood-brain barrier. Additionally, we found that the application of 14,15-EET mimicked the neuroprotective effect of hepatic sEH ablation, while 14,15-epoxyeicosa-5(Z)-enoic acid blocked this effect, indicating that the increased plasma levels of 14,15-EET mediated the neuroprotective effect observed after hepatic sEH ablation. These results highlight the neuroprotective role of the liver in TBI and suggest that targeting hepatic EET signaling could represent a promising therapeutic strategy for treating TBI.

When evolution leads to differences in body size, organs generally scale along. A well-known example of the tight relationship between organ and body size is the scaling of mammalian molar teeth. To investigate how teeth scale during development and evolution, we compared molar development from initiation through final size in the mouse and the rat. Whereas the linear dimensions of the rat molars are twice that of the mouse molars, their shapes are largely the same. Here, we focus on the first lower molars that are considered the most reliable dental proxy for size-related patterns due to their low within-species variability. We found that scaling of the molars starts early, and that the rat molar is patterned equally as fast but in a larger size than the mouse molar. Using transcriptomics, we discovered that a known regulator of body size, insulin-like growth factor 1 (Igf1), is more highly expressed in the rat molars compared to the mouse molars. Ex vivo and in vivo mouse models demonstrated that modulation of the IGF pathway reproduces several aspects of the observed scaling process. Furthermore, analysis of IGF1-treated mouse molars and computational modeling indicate that IGF signaling scales teeth by simultaneously enhancing growth and by inhibiting the cusp-patterning program, thereby providing a relatively simple mechanism for scaling teeth during development and evolution. Finally, comparative data from shrews to elephants suggest that this scaling mechanism regulates the minimum tooth size possible, as well as the patterning potential of large teeth.

T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCβ1 is coexpressed. Resting peripheral hM3Dq+PLCβ1 (hM3Dq/β1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCβ1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/β1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/β1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/β1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

The balance between neural stem cell proliferation and neuronal differentiation is paramount for the appropriate development of the nervous system. Sonic hedgehog (Shh) is known to sequentially promote cell proliferation and specification of neuronal phenotypes, but the signaling mechanisms responsible for the developmental switch from mitogenic to neurogenic have remained unclear. Here, we show that Shh enhances Ca2+ activity at the neural cell primary cilium of developing Xenopus laevis embryos through Ca2+ influx via transient receptor potential cation channel subfamily C member 3 (TRPC3) and release from intracellular stores in a developmental stage-dependent manner. This ciliary Ca2+ activity in turn antagonizes canonical, proliferative Shh signaling in neural stem cells by down-regulating Sox2 expression and up-regulating expression of neurogenic genes, enabling neuronal differentiation. These discoveries indicate that the Shh-Ca2+-dependent switch in neural cell ciliary signaling triggers the switch in Shh action from canonical-mitogenic to neurogenic. The molecular mechanisms identified in this neurogenic signaling axis are potential targets for the treatment of brain tumors and neurodevelopmental disorders.

To address claims of human exceptionalism, we determine where humans fit within the greater mammalian distribution of reproductive inequality. We show that humans exhibit lower reproductive skew (i.e., inequality in the number of surviving offspring) among males and smaller sex differences in reproductive skew than most other mammals, while nevertheless falling within the mammalian range. Additionally, female reproductive skew is higher in polygynous human populations than in polygynous nonhumans mammals on average. This patterning of skew can be attributed in part to the prevalence of monogamy in humans compared to the predominance of polygyny in nonhuman mammals, to the limited degree of polygyny in the human societies that practice it, and to the importance of unequally held rival resources to women's fitness. The muted reproductive inequality observed in humans appears to be linked to several unusual characteristics of our species-including high levels of cooperation among males, high dependence on unequally held rival resources, complementarities between maternal and paternal investment, as well as social and legal institutions that enforce monogamous norms.

T cell activation stimulates substantially increased protein synthesis activity to accumulate sufficient biomass for cell proliferation. The protein synthesis is fueled by the amino acids transported from the environment. Steroid nuclear receptor coactivator 2 (SRC2) is a member of a family of transcription coactivators. Here, we show that SRC2 recruited by c-Myc enhances CD4+ T cell activation to stimulate immune responses via upregulation of amino acid transporter Slc7a5. Mice deficient of SRC2 in T cells (SRC2fl/fl/CD4Cre) are resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and susceptible to Citrobacter rodentium (C. rodentium) infection. Adoptive transfer of naive CD4+ T cells from SRC2fl/fl/CD4Cre mice fails to elicit EAE and colitis in Rag1/ recipients. Further, CD4+ T cells from SRC2fl/fl/CD4Cre mice display defective T cell proliferation, cytokine production, and differentiation both in vitro and in vivo. Mechanically, SRC2 functions as a coactivator to work together with c-Myc to stimulate the expression of amino acid transporter Slc7a5 required for T cell activation. Slc7a5 fails to be up-regulated in CD4+ T cells from SRC2fl/fl/CD4Cre mice, and forced expression of Slc7a5 rescues proliferation, cytokine production, and the ability of SRC2fl/fl/CD4Cre CD4+ T cells to induce EAE. Therefore, SRC2 is essential for CD4+ T cell activation and, thus, a potential drug target for controlling CD4+ T cell-mediated autoimmunity.

Many chemosensory cues evoke responses of the same valence under widely varying physiological conditions. It remains unclear whether similar or distinct neural mechanisms are involved in the detection and processing of such chemosensory cues across contexts. We show that in Caenorhabditis elegans, a chemosensory cue is processed by distinct neural mechanisms at two different life stages that share the same valence state. Both starved adults and dauer larvae are attracted to carbon dioxide (CO2), but CO2 evokes different patterns of neural activity and different motor outputs at the two life stages. Moreover, the same interneuron within the CO2 microcircuit plays a different role in driving CO2-evoked motor output at the two life stages. The dauer-specific patterns of CO2-evoked activity in this interneuron require a dauer-specific gap junction complex and insulin signaling. Our results demonstrate that functionally distinct microcircuits are engaged in response to a chemosensory cue that triggers the same valence state at different life stages, revealing an unexpected complexity to chemosensory processing.

The vertebrate eye was described by Charles Darwin as one of the greatest potential challenges to a theory of natural selection by stepwise evolutionary processes. While numerous evolutionary transitions that led to the vertebrate eye have been explained, some aspects appear to be vertebrate specific with no obvious metazoan precursor. One critical difference between vertebrate and invertebrate vision hinges on interphotoreceptor retinoid-binding protein (IRBP, also known as retinol-binding protein, RBP3), which enables the physical separation and specialization of cells in the vertebrate visual cycle by promoting retinoid shuttling between cell types. While IRBP has been functionally described, its evolutionary origin has remained elusive. Here, we show that IRBP arose via acquisition of novel genetic material from bacteria by interdomain horizontal gene transfer (iHGT). We demonstrate that a gene encoding a bacterial peptidase was acquired prior to the radiation of extant vertebrates >500 Mya and underwent subsequent domain duplication and neofunctionalization to give rise to vertebrate IRBP. Our phylogenomic analyses on >900 high-quality genomes across the tree of life provided the resolution to distinguish contamination in genome assemblies from true instances of horizontal acquisition of IRBP and led us to discover additional independent transfers of the same bacterial peptidase gene family into distinct eukaryotic lineages. Importantly, this work illustrates the evolutionary basis of a key transition that led to the vertebrate visual cycle and highlights the striking impact that acquisition of bacterial genes has had on vertebrate evolution.

Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. Here, we show how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. We discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, we focused on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. We found that cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and we observed that D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were more susceptible to parasitoids than the wild type. We conclude that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.

Hepatic CD4 tissue-resident memory T cells (TRM) are required for robust protection against Salmonella infection; however, the generation of this T cell population is poorly understood. To interrogate the contribution of inflammation, we developed a simple Salmonella-specific T cell transfer system that allowed direct visualization of hepatic TRM formation. Salmonella-specific (SM1) T cell receptor (TCR) transgenic CD4 T cells were activated in vitro and adoptively transferred into C57BL/6 mice while hepatic inflammation was induced by acetaminophen overdose or L. monocytogenes infection. In both model systems, hepatic CD4 TRM formation was accentuated by local tissue responses. Liver inflammation also enhanced the suboptimal protection provided by a subunit Salmonella vaccine which typically induces circulating memory CD4 T cells. To further elucidate the mechanism of CD4 TRM formation in response to liver inflammation, various cytokines were examined by RNAseq, bone marrow chimeras, and in vivo neutralization. Surprisingly, IL-2 and IL-1 were found to enhance CD4 TRM formation. Thus, local inflammatory mediators enhance CD4 TRM populations and can boost the protective immunity provided by a suboptimal vaccine. This knowledge will be foundational for the development of a more effective vaccine against invasive nontyphoidal salmonellosis (iNTS).

Sleep is essential for our well-being, and chronic sleep deprivation has unfavorable health consequences. We recently demonstrated that two familial natural short sleep (FNSS) mutations, DEC2-P384R and Npsr1-Y206H, are strong genetic modifiers of tauopathy in PS19 mice, a model of tauopathy. To gain more insight into how FNSS variants modify the tau phenotype, we tested the effect of another FNSS gene variant, Adrb1-A187V, by crossing mice with this mutation onto the PS19 background. We found that the Adrb1-A187V mutation helped restore rapid eye movement (REM) sleep and alleviated tau aggregation in a sleep-wake center, the locus coeruleus (LC), in PS19 mice. We found that ADRB1+ neurons in the central amygdala (CeA) sent projections to the LC, and stimulating CeAADRB1+ neuron activity increased REM sleep. Furthermore, the mutant Adrb1 attenuated tau spreading from the CeA to the LC. Our findings suggest that the Adrb1-A187V mutation protects against tauopathy by both mitigating tau accumulation and attenuating tau spreading.

In neurodegenerative diseases, proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. However, there is a need to rapidly identify amyloid conformations in situ. Here, we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid-binding dyes to identify six distinct different conformational strains in vitro, as well as amyloid-β (Aβ) deposits in different transgenic mouse models. Our EMBER (excitation multiplexed bright emission recording) imaging method rapidly identifies conformational differences in Aβ and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. EMBER has in situ identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. In future studies, EMBER should enable high-throughput measurements of the fidelity of strain transmission in cellular and animal neurodegenerative diseases models, time course of amyloid strain propagation, and identification of pathogenic versus benign strains.

DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed Xenopus egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.

Sea sponges are the largest marine source of small-molecule natural products described to date. Sponge-derived molecules, such as the chemotherapeutic eribulin, the calcium-channel blocker manoalide, and antimalarial compound kalihinol A, are renowned for their impressive medicinal, chemical, and biological properties. Sponges contain microbiomes that control the production of many natural products isolated from these marine invertebrates. In fact, all genomic studies to date investigating the metabolic origins of sponge-derived small molecules concluded that microbes-not the sponge animal host-are the biosynthetic producers. However, early cell-sorting studies suggested the sponge animal host may play a role particularly in the production of terpenoid molecules. To investigate the genetic underpinnings of sponge terpenoid biosynthesis, we sequenced the metagenome and transcriptome of an isonitrile sesquiterpenoid-containing sponge of the order Bubarida. Using bioinformatic searches and biochemical validation, we identified a group of type I terpene synthases (TSs) from this sponge and multiple other species, the first of this enzyme class characterized from the sponge holobiome. The Bubarida TS-associated contigs consist of intron-containing genes homologous to sponge genes and feature GC percentage and coverage consistent with other eukaryotic sequences. We identified and characterized TS homologs from five different sponge species isolated from geographically distant locations, thereby suggesting a broad distribution amongst sponges. This work sheds light on the role of sponges in secondary metabolite production and speaks to the possibility that other sponge-specific molecules originate from the animal host.

Heavy metal contamination due to industrial and agricultural waste represents a growing threat to water supplies. Frequent and widespread monitoring for toxic metals in drinking and agricultural water sources is necessary to prevent their accumulation in humans, plants, and animals, which results in disease and environmental damage. Here, the metabolic stress response of bacteria is used to report the presence of heavy metal ions in water by transducing ions into chemical signals that can be fingerprinted using machine learning analysis of vibrational spectra. Surface-enhanced Raman scattering surfaces amplify chemical signals from bacterial lysate and rapidly generate large, reproducible datasets needed for machine learning algorithms to decode the complex spectral data. Classification and regression algorithms achieve limits of detection of 0.5 pM for As3+ and 6.8 pM for Cr6+, 100,000 times lower than the World Health Organization recommended limits, and accurately quantify concentrations of analytes across six orders of magnitude, enabling early warning of rising contaminant levels. Trained algorithms are generalizable across water samples with different impurities; water quality of tap water and wastewater was evaluated with 92% accuracy.

The amyloid aggregation of alpha-synuclein within the brain is associated with the pathogenesis of Parkinson's disease (PD) and other related synucleinopathies, including multiple system atrophy (MSA). Alpha-synuclein aggregates are a major therapeutic target for treatment of these diseases. We identify two small molecules capable of disassembling preformed alpha-synuclein fibrils. The compounds, termed CNS-11 and CNS-11g, disaggregate recombinant alpha-synuclein fibrils in vitro, prevent the intracellular seeded aggregation of alpha-synuclein fibrils, and mitigate alpha-synuclein fibril cytotoxicity in neuronal cells. Furthermore, we demonstrate that both compounds disassemble fibrils extracted from MSA patient brains and prevent their intracellular seeding. They also reduce in vivo alpha-synuclein aggregates in C. elegans. Both compounds also penetrate brain tissue in mice. A molecular dynamics-based computational model suggests the compounds may exert their disaggregating effects on the N terminus of the fibril core. These compounds appear to be promising therapeutic leads for targeting alpha-synuclein for the treatment of synucleinopathies.

Gould syndrome is a rare multisystem disorder resulting from autosomal dominant mutations in the collagen-encoding genes COL4A1 and COL4A2. Human patients and Col4a1 mutant mice display brain pathology that typifies cerebral small vessel diseases (cSVDs), including white matter hyperintensities, dilated perivascular spaces, lacunar infarcts, microbleeds, and spontaneous intracerebral hemorrhage. The underlying pathogenic mechanisms are unknown. Using the Col4a1+/G394V mouse model, we found that vasoconstriction in response to internal pressure-the vascular myogenic response-is blunted in cerebral arteries from middle-aged (12 mo old) but not young adult (3 mo old) animals, revealing age-dependent cerebral vascular dysfunction. The defect in the myogenic response was associated with a significant decrease in depolarizing cation currents conducted by TRPM4 (transient receptor potential melastatin 4) channels in native cerebral artery smooth muscle cells (SMCs) isolated from mutant mice. The minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is necessary for TRPM4 activity. Dialyzing SMCs with PIP2 and selective blockade of phosphoinositide 3-kinase (PI3K), an enzyme that converts PIP2 to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored TRPM4 currents. Acute inhibition of PI3K activity and blockade of transforming growth factor-beta (TGF-β) receptors also rescued the myogenic response, suggesting that hyperactivity of TGF-β signaling pathways stimulates PI3K to deplete PIP2 and impair TRPM4 channels. We conclude that age-related cerebral vascular dysfunction in Col4a1+/G394V mice is caused by the loss of depolarizing TRPM4 currents due to PIP2 depletion, revealing an age-dependent mechanism of cSVD.

TMEM161B encodes an evolutionarily conserved widely expressed novel 8-pass transmembrane protein of unknown function in human. Here we identify TMEM161B homozygous hypomorphic missense variants in our recessive polymicrogyria (PMG) cohort. Patients carrying TMEM161B mutations exhibit striking neocortical PMG and intellectual disability. Tmem161b knockout mice fail to develop midline hemispheric cleavage, whereas knock-in of patient mutations and patient-derived brain organoids show defects in apical cell polarity and radial glial scaffolding. We found that TMEM161B modulates actin filopodia, functioning upstream of the Rho-GTPase CDC42. Our data link TMEM161B with human PMG, likely regulating radial glia apical polarity during neocortical development.