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Drosophila prolongata is a member of the melanogaster species group and rhopaloa subgroup native to the subtropical highlands of Southeast Asia. This species exhibits an array of recently evolved male-specific morphological, physiological, and behavioral traits that distinguish it from its closest relatives, making it an attractive model for studying the evolution of sexual dimorphism and testing theories of sexual selection. The lack of genomic resources has impeded the dissection of the molecular basis of sex-specific development and behavior in this species. To address this, we assembled the genome of D. prolongata using long-read sequencing and Hi-C scaffolding, resulting in a highly complete and contiguous (scaffold N50 2.2 Mb) genome assembly of 220 Mb. The repetitive content of the genome is 24.6%, the plurality of which are long terminal repeats retrotransposons (33.2%). Annotations based on RNA-seq data and homology to related species revealed a total of 19,330 genes, of which 16,170 are protein-coding. The assembly includes 98.5% of Diptera BUSCO genes, including 93.8% present as a single copy. Despite some likely regional duplications, the completeness of this genome suggests that it can be readily used for gene expression, genome-wide association studies (GWAS), and other genomic analyses.

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer patient deaths due to extensive molecular heterogeneity, high recurrence rates, and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. Here, we performed a genome-wide CRISPR/Cas9 screen with PI3Kα and AKT inhibitors to find targetable synthetic lethalities in TNBC. Cholesterol homeostasis was identified as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro in mouse TNBC xenografts and in patient-derived estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single-agent or combination treatment with AKT inhibitor and pitavastatin, which was rescued by inhibition of the cholesterol-trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels, and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. These findings support combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC. Significance: Two FDA-approved compounds, AKT inhibitors and pitavastatin, synergize to induce cell death in triple-negative breast cancer, motivating evaluation of the efficacy of this combination in clinical trials.

Anti-predator behaviours in response to predator cues can be innate, or they can be learned through prior experience and remembered over time. The duration and strength of continued anti-predator behaviour after predator cues are no longer present, and the potential for an enhanced response when re-exposed to predator cues later is less known but could account for the observed variation in anti-predator responses. We measured the carryover effects of past predation exposure and the potential for anti-predator learning and memory in the marine snail Nucella canaliculata from six populations distributed over 1000 km of coastline. We exposed lab-reared snails to cues associated with a common crab predator or seawater control in two serial experiments separated by over seven months. Responses were population- and sex-dependent, with some populations retaining anti-predator behaviours while others showed a capacity for learning and memory. Male snails showed a strong carryover of risk aversion, while females were able to return to normal feeding rates and grow more quickly. These behavioural differences culminated in strong impacts on feeding and growth rates, demonstrating that this variation has implications for the strength of trait-mediated indirect interactions, which can impact entire ecosystems.

A catalogue of neuronal cell types has often been called a 'parts list' of the brain1, and regarded as a prerequisite for understanding brain function2,3. In the optic lobe of Drosophila, rules of connectivity between cell types have already proven to be essential for understanding fly vision4,5. Here we analyse the fly connectome to complete the list of cell types intrinsic to the optic lobe, as well as the rules governing their connectivity. Most new cell types contain 10 to 100 cells, and integrate information over medium distances in the visual field. Some existing type families (Tm, Li, and LPi)6-10 at least double in number of types. A new serpentine medulla (Sm) interneuron family contains more types than any other. Three families of cross-neuropil types are revealed. The consistency of types is demonstrated by analysing the distances in high-dimensional feature space, and is further validated by algorithms that select small subsets of discriminative features. We use connectivity to hypothesize about the functional roles of cell types in motion, object and colour vision. Connectivity with 'boundary types' that straddle the optic lobe and central brain is also quantified. We showcase the advantages of connectomic cell typing: complete and unbiased sampling, a rich array of features based on connectivity and reduction of the connectome to a substantially simpler wiring diagram of cell types, with immediate relevance for brain function and development.

Many animals use visual information to navigate1-4, but how such information is encoded and integrated by the navigation system remains incompletely understood. In Drosophila melanogaster, EPG neurons in the central complex compute the heading direction5 by integrating visual input from ER neurons6-12, which are part of the anterior visual pathway (AVP)10,13-16. Here we densely reconstruct all neurons in the AVP using electron-microscopy data17. The AVP comprises four neuropils, sequentially linked by three major classes of neurons: MeTu neurons10,14,15, which connect the medulla in the optic lobe to the small unit of the anterior optic tubercle (AOTUsu) in the central brain; TuBu neurons9,16, which connect the AOTUsu to the bulb neuropil; and ER neurons6-12, which connect the bulb to the EPG neurons. On the basis of morphologies, connectivity between neural classes and the locations of synapses, we identify distinct information channels that originate from four types of MeTu neurons, and we further divide these into ten subtypes according to the presynaptic connections in the medulla and the postsynaptic connections in the AOTUsu. Using the connectivity of the entire AVP and the dendritic fields of the MeTu neurons in the optic lobes, we infer potential visual features and the visual area from which any ER neuron receives input. We confirm some of these predictions physiologically. These results provide a strong foundation for understanding how distinct sensory features can be extracted and transformed across multiple processing stages to construct higher-order cognitive representations.

Excessive growth hormone (GH) has been shown to promote joint degeneration in both preclinical and clinical studies. Little is known about the effect of disrupted GH or GH receptor (GHR) on joint health. The goal of this study is to investigate joint pathology in mice with either germline (GHR-/-) or adult inducible (iGHR-/-) GHR deficiency. Knee joints from male and female GHR-/- and WT mice at 24 months of age were processed for histological analysis. Also, knee joints from male and female iGHR-/- and WT mice at 22 months of age were scanned by micro-CT (μCT) for subchondral bone changes and characterized via histology for cartilage degeneration. Joint sections were also stained for the chondrocyte hypertrophy marker, COLX, and the cartilage degeneration marker, ADAMTS-5, using immunohistochemistry. Compared to WT mice, GHR-/- mice had remarkably smooth articular joint surfaces and an even distribution of proteoglycan with no signs of degeneration. Quantitatively, GHR-/- mice had lower OARSI and Mankin scores compared to WT controls. By contrast, iGHR-/- mice were only moderately protected from developing aging-associated OA. iGHR-/- mice had a significantly lower Mankin score compared to WT. However, Mankin scores were not significantly different between iGHR-/- and WT when males and females were analyzed separately. OARSI scores did not differ significantly between WT and iGHR-/- in either individual or combined sex analyses. Both GHR-/- and iGHR-/- mice had fewer COLX+ hypertrophic chondrocytes compared to WT, while no significant difference was observed in ADAMTS-5 staining. Compared to WT, a significantly lower trabecular thickness in the subchondral bone was observed in the iGHR-/- male mice but not in the female mice. However, there were no significant differences between WT and iGHR-/- mice in the bone volume to total tissue volume (BV/TV), bone mineral density (BMD), and trabecular number in either sex. This study identified that both germline and adult-induced GHR deficiency protected mice from developing aging-associated OA with more effective protection in GHR-/- mice.

Genetically heterogeneous UM-HET3 mice born in 2020 were used to test possible lifespan effects of alpha-ketoglutarate (AKG), 2,4-dinitrophenol (DNP), hydralazine (HYD), nebivolol (NEBI), 16α-hydroxyestriol (OH_Est), and sodium thiosulfate (THIO), and to evaluate the effects of canagliflozin (Cana) when started at 16 months of age. OH_Est produced a 15% increase (p = 0.0001) in median lifespan in males but led to a significant (7%) decline in female lifespan. Cana, started at 16 months, also led to a significant increase (14%, p = 0.004) in males and a significant decline (6%, p = 0.03) in females. Cana given to mice at 6 months led, as in our previous study, to an increase in male lifespan without any change in female lifespan, suggesting that this agent may lead to female-specific late-life harm. We found that blood levels of Cana were approximately 20-fold higher in aged females than in young males, suggesting a possible mechanism for the sex-specific disparities in its effects. NEBI was also found to produce a female-specific decline (4%, p = 0.03) in lifespan. None of the other tested drugs provided a lifespan benefit in either sex. These data bring to 7 the list of ITP-tested drugs that induce at least a 10% lifespan increase in one or both sexes, add a fourth drug with demonstrated mid-life benefits on lifespan, and provide a testable hypothesis that might explain the sexual dimorphism in lifespan effects of the SGLT2 inhibitor Cana.

Salmonella translocate to the gut epithelium via microfold cells lining the follicle-associated epithelium (FAE). How Salmonella localize to the FAE is not well characterized. Here we use live imaging and competitive assays between wild-type and chemotaxis-deficient mutants to show that Salmonella enterica serotype Typhimurium (S. Typhimurium) localize to the FAE independently of chemotaxis in an ex vivo mouse caecum infection model. Electrical recordings revealed polarized FAE with sustained outward current and small transepithelial potential, while the surrounding villus is depolarized with inward current and large transepithelial potential. The distinct electrical potentials attracted S. Typhimurium to the FAE while Escherichia coli (E. coli) localized to the villi, through a process called galvanotaxis. Chloride flux involving the cystic fibrosis transmembrane conductance regulator (CFTR) generated the ionic currents around the FAE. Pharmacological inhibition of CFTR decreased S. Typhimurium FAE localization but increased E. coli recruitment. Altogether, our findings demonstrate that bioelectric cues contribute to S. Typhimurium targeting of specific gut epithelial locations, with potential implications for other enteric bacterial infections.

Copper plays a key role in host-pathogen interaction. We find that during Leishmania major infection, the parasite-harboring macrophage regulates its copper homeostasis pathway in a way to facilitate copper-mediated neutralization of the pathogen. Copper-ATPase ATP7A transports copper to amastigote-harboring phagolysosomes to induce stress on parasites. Leishmania in order to evade the copper stress, utilizes a variety of manipulative measures to lower the host-induced copper stress. It induces deglycosylation and degradation of host-ATP7A and downregulation of copper importer, CTR1 by cysteine oxidation. Additionally, Leishmania induces CTR1 endocytosis that arrests copper uptake. In mouse model of infection, we report an increase in systemic bioavailable copper in infected animals. Heart acts as the major organ for diverting its copper reserves to systemic circulation to fight-off infection by downregulating its CTR1. Our study explores reciprocal mechanism of manipulation of host copper homeostasis pathway by macrophage and Leishmania to gain respective advantages in host-pathogen interaction.

Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 - as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.

Wildlife tagging provides critical insights into animal movement ecology, physiology, and behavior amid global ecosystem changes. However, the stress induced by capture, handling, and tagging can impact post-release locomotion and activity and, consequently, the interpretation of study results. Here, we analyze post-tagging effects on 1585 individuals of 42 terrestrial mammal species using collar-collected GPS and accelerometer data. Species-specific displacements and overall dynamic body acceleration, as a proxy for activity, were assessed over 20 days post-release to quantify disturbance intensity, recovery duration, and speed. Differences were evaluated, considering species-specific traits and the human footprint of the study region. Over 70% of the analyzed species exhibited significant behavioral changes following collaring events. Herbivores traveled farther with variable activity reactions, while omnivores and carnivores were initially less active and mobile. Recovery duration proved brief, with alterations diminishing within 4-7 tracking days for most species. Herbivores, particularly males, showed quicker displacement recovery (4 days) but slower activity recovery (7 days). Individuals in high human footprint areas displayed faster recovery, indicating adaptation to human disturbance. Our findings emphasize the necessity of extending tracking periods beyond 1 week and particular caution in remote study areas or herbivore-focused research, specifically in smaller mammals.

Sterile inflammation after myocardial infarction is classically credited to myeloid cells interacting with dead cell debris in the infarct zone1,2. Here we show that cardiomyocytes are the dominant initiators of a previously undescribed type I interferon response in the infarct borderzone. Using spatial transcriptomics analysis in mice and humans, we find that myocardial infarction induces colonies of interferon-induced cells (IFNICs) expressing interferon-stimulated genes decorating the borderzone, where cardiomyocytes experience mechanical stress, nuclear rupture and escape of chromosomal DNA. Cardiomyocyte-selective deletion of Irf3 abrogated IFNIC colonies, whereas mice lacking Irf3 in fibroblasts, macrophages, neutrophils or endothelial cells, Ccr2-deficient mice or plasmacytoid-dendritic-cell-depleted mice did not. Interferons blunted the protective matricellular programs and contractile function of borderzone fibroblasts, and increased vulnerability to pathological remodelling. In mice that died after myocardial infarction, IFNIC colonies were immediately adjacent to sites of ventricular rupture, while mice lacking IFNICs were protected from rupture and exhibited improved survival3. Together, these results reveal a pathological borderzone niche characterized by a cardiomyocyte-initiated innate immune response. We suggest that selective inhibition of IRF3 activation in non-immune cells could limit ischaemic cardiomyopathy while avoiding broad immunosuppression.

We lack the fundamental information needed to understand how DNA damage in the brain is generated and how it is controlled over a lifetime in the absence of replication check points. To address these questions, here, we integrate cell-type and region-specific features of DNA repair activity in the normal brain. The brain has the same repair proteins as other tissues, but normal, canonical repair activity is unequal and is characterized by high base excision repair (BER) and low double strand break repair (DSBR). The natural imbalance creates conditions where single strand breaks (SSBs) can convert to double strand breaks (DSBs) and reversibly switch between states in response to oxidation both in vivo and in vitro. Our data suggest that, in a normal background of repair, SSBs and DSBs are in an equilibrium which is pushed or pulled by metabolic state. Interconversion of SSB to DSBs provides a physiological check point, which would allow the formation of unrepaired DSBs for productive functions, but would also restrict them from exceeding tolerable limits.

In frontotemporal lobar degeneration (FTLD), pathological protein aggregation in specific brain regions is associated with declines in human-specialized social-emotional and language functions. In most patients, disease protein aggregates contain either TDP-43 (FTLD-TDP) or tau (FTLD-tau). Here, we explored whether FTLD-associated regional degeneration patterns relate to regional gene expression of human accelerated regions (HARs), conserved sequences that have undergone positive selection during recent human evolution. To this end, we used structural neuroimaging from patients with FTLD and human brain regional transcriptomic data from controls to identify genes expressed in FTLD-targeted brain regions. We then integrated primate comparative genomic data to test our hypothesis that FTLD targets brain regions linked to expression levels of recently evolved genes. In addition, we asked whether genes whose expression correlates with FTLD atrophy are enriched for genes that undergo cryptic splicing when TDP-43 function is impaired. We found that FTLD-TDP and FTLD-tau subtypes target brain regions with overlapping and distinct gene expression correlates, highlighting many genes linked to neuromodulatory functions. FTLD atrophy-correlated genes were strongly enriched for HARs. Atrophy-correlated genes in FTLD-TDP showed greater overlap with TDP-43 cryptic splicing genes and genes with more numerous TDP-43 binding sites compared with atrophy-correlated genes in FTLD-tau. Cryptic splicing genes were enriched for HAR genes, and vice versa, but this effect was due to the confounding influence of gene length. Analyses performed at the individual-patient level revealed that the expression of HAR genes and cryptically spliced genes within putative regions of disease onset differed across FTLD-TDP subtypes. Overall, our findings suggest that FTLD targets brain regions that have undergone recent evolutionary specialization and provide intriguing potential leads regarding the transcriptomic basis for selective vulnerability in distinct FTLD molecular-anatomical subtypes.

BACKGROUND: Prenatal fish intake is a key source of omega-3 (ω-3) polyunsaturated fatty acids needed for brain development, yet intake is generally low, and studies addressing associations with autism spectrum disorder (ASD) and related traits are lacking. OBJECTIVE: This study aimed to examine associations of prenatal fish intake and ω-3 supplement use with both autism diagnosis and broader autism-related traits. METHODS: Participants were drawn from 32 cohorts in the Environmental influences on Child Health Outcomes Cohort Consortium. Children were born between 1999 and 2019 and part of ongoing follow-up with data available for analysis by August 2022. Exposures included self-reported maternal fish intake and ω-3/fish oil supplement use during pregnancy. Outcome measures included parent report of clinician-diagnosed ASD and parent-reported autism-related traits measured by the Social Responsiveness Scale (SRS)-second edition (n = 3939 and v3609 for fish intake analyses, respectively; n = 4537 and n = 3925 for supplement intake analyses, respectively). RESULTS: In adjusted regression models, relative to no fish intake, fish intake during pregnancy was associated with reduced odds of autism diagnosis (odds ratio: 0.84; 95% confidence interval [CI]: 0.77, 0.92), and a modest reduction in raw total SRS scores (β: -1.69; 95% CI: -3.3, -0.08). Estimates were similar across categories of fish consumption from any or less than once per week to more than twice per week. For ω-3 supplement use, relative to no use, no significant associations with autism diagnosis were identified, whereas a modest relation with SRS score was suggested (β: 1.98; 95% CI: 0.33, 3.64). CONCLUSIONS: These results extend previous work by suggesting that prenatal fish intake, but not ω-3 supplement use, may be associated with lower likelihood of both autism diagnosis and related traits. Given the low-fish intake in the United States general population and the rising autism prevalence, these findings suggest the need for better public health messaging regarding guidelines on fish intake for pregnant individuals.

PURPOSE: Population-based studies on the associations of plant-based foods, red meat or dairy with gut microbiome are scarce. We examined whether the consumption of plant-based foods (vegetables, potatoes, fruits, cereals), red and processed meat (RPM) or dairy (fermented milk, cheese, other dairy products) are related to gut microbiome in Finnish adults. METHODS: We utilized data from the National FINRISK/FINDIET 2002 Study (n = 1273, aged 25-64 years, 55% women). Diet was assessed with 48-hour dietary recalls. Gut microbiome was analyzed using shallow shotgun sequencing. We applied multivariate analyses with linear models and permutational ANOVAs adjusted for relevant confounders. RESULTS: Fruit consumption was positively (beta = 0.03, SE = 0.01, P = 0.04), while a dairy subgroup including milk, cream and ice-creams was inversely associated (beta=-0.03, SE 0.01, P = 0.02) with intra-individual gut microbiome diversity (alpha-diversity). Plant-based foods (R2 = 0.001, P = 0.03) and dairy (R2 = 0.002, P = 0.01) but not RPM (R2 = 0.001, P = 0.38) contributed to the compositional differences in gut microbiome (beta-diversity). Plant-based foods were associated with several butyrate producers/cellulolytic species including Roseburia hominis. RPM associations included an inverse association with R. hominis. Dairy was positively associated with several lactic producing/probiotic species including Lactobacillus delbrueckii and potentially opportunistic pathogens including Citrobacter freundii. Dairy, fermented milk, vegetables, and cereals were associated with specific microbial functions. CONCLUSION: Our results suggest a potential association between plant-based foods and dairy or their subgroups with microbial diversity measures. Furthermore, our findings indicated that all the food groups were associated with distinct overall microbial community compositions. Plant-based food consumption particularly was associated with a larger number of putative beneficial species.

Oxytocin (OXT) is a peptide hormone and a neuropeptide that regulates various peripheral physiological processes and modulates behavioral responses in the central nervous system. While the humoral release occurs from the axons arriving at the median eminence, the neuropeptide is also released from oxytocinergic cell axons in various brain structures that contain its receptor, and from their dendrites in hypothalamic nuclei and potentially into the cerebrospinal fluid (CSF). Understanding oxytocins complex functions requires the knowledge on patterns of oxytocinergic projections in relationship to its receptor (OXTR). This study provides the first comprehensive examination of the oxytocinergic system in the prairie vole (Microtus ochrogaster), an animal exhibiting social behaviors that mirror human social behaviors linked to oxytocinergic functioning. Using light and electron microscopy, we characterized the neuroanatomy of the oxytocinergic system in this species. OXT+ cell bodies were found primarily in the hypothalamus, and axons were densest in subcortical regions. Examination of the OXT+ fibers and their relationship to oxytocin receptor transcripts (Oxtr) revealed that except for some subcortical structures, the presence of axons was not correlated with the amount of Oxtr across the brain. Of particular interest, the cerebral cortex that had high expression of Oxtr transcripts contained little to no fibers. Electron microscopy is used to quantify dense cored vesicles (DCV) in OXT+ axons and to identify potential axonal release sites. The ependymal cells that line the ventricles were frequently permissive of DCV-containing OXT+ dendrites reaching the third ventricle. Our results highlight a mechanism in which oxytocin is released directly into the ventricles and circulates throughout the ventricular system, may serve as the primary source for oxytocin that binds to OXTR in the cerebral cortex.

Here we conducted wireless electrophysiological recording of hippocampal neurons from Egyptian fruit bats in the presence of human experimenters. In flying bats, many neurons modulated their activity depending on the identity of the human at the landing target. In stationary bats, many neurons carried significant spatial information about the position and identity of humans traversing the environment. Our results reveal that hippocampal activity is robustly modulated by the presence, movement and identity of human experimenters.

Meningiomas are associated with inactivation of NF2/Merlin, but approximately one-third of meningiomas with favorable clinical outcomes retain Merlin expression. Biochemical mechanisms underlying Merlin-intact meningioma growth are incompletely understood, and non-invasive biomarkers that may be used to guide treatment de-escalation or imaging surveillance are lacking. Here, we use single-cell RNA sequencing, proximity-labeling proteomic mass spectrometry, mechanistic and functional approaches, and magnetic resonance imaging (MRI) across meningioma xenografts and patients to define biochemical mechanisms and an imaging biomarker that underlie Merlin-intact meningiomas. We find Merlin serine 13 (S13) dephosphorylation drives meningioma Wnt signaling and tumor growth by attenuating inhibitory interactions with β-catenin and activating the Wnt pathway. MRI analyses show Merlin-intact meningiomas with S13 phosphorylation and favorable clinical outcomes are associated with high apparent diffusion coefficient (ADC). These results define mechanisms underlying a potential imaging biomarker that could be used to guide treatment de-escalation or imaging surveillance for patients with Merlin-intact meningiomas.

Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

BackgroundGastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival.MethodsTwo patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue.ResultsM5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800.ConclusionsHumanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection.

The insights into interactions between host genetics and gut microbiome (GM) in colorectal tumor susceptibility (CTS) remains lacking. We used Collaborative Cross mouse population model to identify genetic and microbial determinants of Azoxymethane-induced CTS. We identified 4417 CTS-associated single nucleotide polymorphisms (SNPs) containing 334 genes that were transcriptionally altered in human colorectal cancers (CRCs) and consistently clustered independent human CRC cohorts into two subgroups with different prognosis. We discovered a set of genera in early-life associated with CTS and defined a 16-genus signature that accurately predicted CTS, the majority of which were correlated with human CRCs. We identified 547 SNPs associated with abundances of these genera. Mediation analysis revealed GM as mediators partially exerting the effect of SNP UNC3869242 within Duox2 on CTS. Intestine cell-specific depletion of Duox2 altered GM composition and contribution of Duox2 depletion to CTS was significantly influenced by GM. Our findings provide potential novel targets for personalized CRC prevention and treatment.

Bile acids (BA) are among the most abundant metabolites produced by the gut microbiome. Primary BAs produced in the liver are converted by gut bacterial 7-α-dehydroxylation into secondary BAs, which can differentially regulate host health via signaling based on their varying affinity for BA receptors. Despite the importance of secondary BAs in host health, the regulation of 7-α-dehydroxylation and the role of diet in modulating this process is incompletely defined. Understanding this process could lead to dietary guidelines that beneficially shift BA metabolism. Dietary fiber regulates gut microbial composition and metabolite production. We tested the hypothesis that feeding mice a diet rich in a fermentable dietary fiber, resistant starch (RS), would alter gut bacterial BA metabolism. Male and female wild-type mice were fed a diet supplemented with RS or an isocaloric control diet (IC). Metabolic parameters were similar between groups. RS supplementation increased gut luminal deoxycholic acid (DCA) abundance. However, gut luminal cholic acid (CA) abundance, the substrate for 7-α-dehydroxylation in DCA production, was unaltered by RS. Further, RS supplementation did not change the mRNA expression of hepatic BA producing enzymes or ileal BA transporters. Metagenomic assessment of gut bacterial composition revealed no change in the relative abundance of bacteria known to perform 7-α-dehydroxylation. P. ginsenosidimutans and P. multiformis were positively correlated with gut luminal DCA abundance and increased in response to RS supplementation. These data demonstrate that RS supplementation enriches gut luminal DCA abundance without increasing the relative abundance of bacteria known to perform 7-α-dehydroxylation.

Adult horses are susceptible to equine coronavirus (ECoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), although, only ECoV has been linked to clinical disease. Little information is available regarding the seroprevalence against ECoV and SARS-CoV-2 in adult healthy horses. The goal of the present study was to determine the seroprevalence against two coronaviruses known to infect horses using convenience samples collected from horses recently imported from Europe to the United States from 2019 to 2023. A total of 385 banked serum samples were tested against ECoV and SARS-CoV-2 using previously validated ELISA assays. Prevalence factors including date of arrival in the United States, signalment and country of origin were available for the majority of the horses. A total of 9/385 (2.3%) and 4/385 (1.0%) horses tested seropositive for ECoV and SARS-CoV-2, respectively. The ECoV seropositive horses were all mares, ages 4 to 26 years (median 9 years) and originated from Germany, the Netherlands, Ireland, Belgium and Italy. These mares were predominantly imported during the summer and fall months. All SARS-CoV-2 seropositive horses were mares ages 5 to 10 years (median 7.5 years) imported from the Netherlands and the United Kingdom. The majority of the SARS-CoV-2 seropositive horses were imported during the colder months of the year. The study results support the presence of ECoV in Europe and report on the first SARS-CoV-2 seropositive healthy adult horses outside the United States. Commingling for movements by air and close contact to humans may predispose transmission with ECoV and SARS-CoV-2, respectively.

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

EWS fusion oncoproteins underlie several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer driven by EWS-WT1 fusion proteins. Here we combine chromatin occupancy and 3D profiles to identify EWS-WT1-dependent gene regulation networks and target genes. We show that EWS-WT1 is a powerful chromatin activator controlling an oncogenic gene expression program that characterizes primary tumors. Similar to wild type WT1, EWS-WT1 has two isoforms that differ in their DNA binding domain and we find that they have distinct DNA binding profiles and are both required to generate viable tumors that resemble primary DSRCT. Finally, we identify candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex oncogenic activity of EWS-WT1.

Autism spectrum disorders (ASD) can be caused by environmental factors. These factors act early in the development of the nervous system and induce stereotyped repetitive behaviors and diminished social interactions, among other outcomes. Little is known about how these behaviors are produced. In pregnant women, delivery of valproic acid (VPA) (to control seizure activity or stabilize mood) or immune activation by a virus increases the incidence of ASD in offspring. We found that either VPA or Poly Inosine:Cytosine (which mimics a viral infection), administered at mouse embryonic day 12.5, induced a neurotransmitter switch from GABA to glutamate in PV- and CCK-expressing interneurons in the medial prefrontal cortex by postnatal day 10. The switch was present for only a brief period during early postnatal development, observed in male and female mice at postnatal day 21 and reversed in both males and females by postnatal day 30. At postnatal day 90, male mice exhibited stereotyped repetitive behaviors and diminished social interaction while female mice exhibited only stereotyped repetitive behavior. Transfecting GAD1 in PV- and CCK-expressing interneurons at postnatal day 10, to reintroduce GABA expression, overrode the switch and prevented expression of autistic-like behavior. These findings point to an important role of neurotransmitter switching in mediating the environmental causes of autism.

TBI-223, a novel oxazolidinone for tuberculosis, is designed to provide improved efficacy and safety compared to linezolid in combination with bedaquiline and pretomanid (BPaL). We aim to optimize the dosing of TBI-223 within the BPaL regimen for enhanced therapeutic outcomes. TBI-223 is investigated in preclinical monotherapy, multidrug therapy, and lesion penetration experiments to describe its efficacy and safety versus linezolid. A translational platform incorporating linezolid and BPaL data from preclinical experiments and 4 clinical trials (NCT00396084, NCT02333799, NCT03086486, NCT00816426) is developed, enabling validation of the framework. TBI-223 preclinical and Phase 1 data (NCT03758612) are applied to the translational framework to predict clinical outcomes and optimize TBI-223 dosing in combination with bedaquiline and pretomanid. Results indicate that daily doses of 1200-2400 mg TBI-223 may achieve efficacy comparable to the BPaL regimen, with >90% of patients predicted to reach culture conversion by two months.

UNLABELLED: Gastrointestinal diseases are the most frequently reported clinical problems in captive common marmosets (Callithrix jacchus), often affecting the health and welfare of the animal and ultimately their use as a research subject. The microbiome has been shown to be intimately connected to diet and gastrointestinal health. Here, we use shotgun metagenomics and untargeted metabolomics in fecal samples of common marmosets collected before, during, and after a dietary transition from a biscuit to a gel diet. The overall health of marmosets, measured as weight recovery and reproductive outcome, improved after the diet transition. Moreover, each marmoset pair had significant shifts in the microbiome and metabolome after the diet transition. In general, we saw a decrease in Escherichia coli and Prevotella species and an increase in Bifidobacterium species. Untargeted metabolic profiles indicated that polyamine levels, specifically cadaverine and putrescine, were high after diet transition, suggesting either an increase in excretion or a decrease in intestinal reabsorption at the intestinal level. In conclusion, our data suggest that Bifidobacterium species could potentially be useful as probiotic supplements to the laboratory marmoset diet. Future studies with a larger sample size will be beneficial to show that this is consistent with the diet change. IMPORTANCE: Appropriate diet and health of the common marmoset in captivity are essential both for the welfare of the animal and to improve experimental outcomes. Our study shows that a gel diet compared to a biscuit diet improves the health of a marmoset colony, is linked to increases in Bifidobacterium species, and increases the removal of molecules associated with disease. The diet transition had an influence on the molecular changes at both the pair and time point group levels, but only at the pair level for the microbial changes. It appears to be more important which genes and functions present changed rather than specific microbes. Further studies are needed to identify specific components that should be considered when choosing an appropriate diet and additional supplementary foods, as well as to validate the benefits of providing probiotics. Probiotics containing Bifidobacterium species appear to be useful as probiotic supplements to the laboratory marmoset diet, but additional work is needed to validate these findings.

In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone loss1. Although oestrogen normally counteracts excessive bone resorption by promoting bone formation, this sex steroid drops precipitously during this postpartum period. Here we report that brain-derived cellular communication network factor 3 (CCN3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to build bone in lactating females. We began by showing that our previously reported female-specific, dense bone phenotype2 originates from a humoral factor that promotes bone mass and acts on skeletal stem cells to increase their frequency and osteochondrogenic potential. This circulatory factor was then identified as CCN3, a brain-derived hormone from ARCKISS1 neurons that is able to stimulate mouse and human skeletal stem cell activity, increase bone remodelling and accelerate fracture repair in young and old mice of both sexes. The role of CCN3 in normal female physiology was revealed after detecting a burst of CCN3 expression in ARCKISS1 neurons coincident with lactation. After reducing CCN3 in ARCKISS1 neurons, lactating mothers lost bone and failed to sustain their progeny when challenged with a low-calcium diet. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone for both sexes and define a new maternal brain hormone for ensuring species survival in mammals.

Accumulated evidence suggested that gut microbial dysbiosis interplayed with progressive chronic kidney disease (CKD). However, no available therapy is effective in suppressing progressive CKD. Here, using microbiomics in 480 participants including healthy controls and patients with stage 1-5 CKD, we identified an elongation taxonomic chain Bacilli-Lactobacillales-Lactobacillaceae-Lactobacillus-Lactobacillus johnsonii correlated with patients with CKD progression, whose abundance strongly correlated with clinical kidney markers. L. johnsonii abundance reduced with progressive CKD in rats with adenine-induced CKD. L. johnsonii supplementation ameliorated kidney lesion. Serum indole-3-aldehyde (IAld), whose level strongly negatively correlated with creatinine level in CKD rats, decreased in serum of rats induced using unilateral ureteral obstruction (UUO) and 5/6 nephrectomy (NX) as well as late CKD patients. Treatment with IAld dampened kidney lesion through suppressing aryl hydrocarbon receptor (AHR) signal in rats with CKD or UUO, and in cultured 1-hydroxypyrene-induced HK-2 cells. Renoprotective effect of IAld was partially diminished in AHR deficiency mice and HK-2 cells. Our further data showed that treatment with L. johnsonii attenuated kidney lesion by suppressing AHR signal via increasing serum IAld level. Taken together, targeting L. johnsonii might reverse patients with CKD. This study provides a deeper understanding of how microbial-produced tryptophan metabolism affects host disease and discovers potential pathways for prophylactic and therapeutic treatments for CKD patients.

BACKGROUND: Aedes albopictus is an important vector for pathogens such as dengue, Zika, and chikungunya viruses. While insecticides is the mainstay for mosquito control, their widespread and excessive use has led to the increased resistance in Ae. albopictus globally. Gut symbiotic bacteria are believed to play a potential role in insect physiology, potentially linking to mosquitoes metabolic resistance against insecticides. METHODS: We investigated the role of symbiotic bacteria in the development of resistance in Ae. albopictus by comparing gut symbiotic bacteria between deltamethrin-sensitive and deltamethrin-resistant populations. Adults were reared from field-collected larvae. Sensitive and resistant mosquitoes were screened using 0.03% and 0.09% deltamethrin, respectively, on the basis of the World Health Organization (WHO) tube bioassay. Sensitive and resistant field-collected larvae were screened using 5 × LC50 (lethal concentration at 50% mortality) and 20 × LC50 concentration of deltamethrin, respectively. Laboratory strain deltamethrin-sensitive adults and larvae were used as controls. The DNA of gut samples from these mosquitoes were extracted using the magnetic bead method. Bacterial 16S rDNA was sequenced using BGISEQ method. We isolated and cultured gut microorganisms from adult and larvae mosquitoes using four different media: Luria Bertani (LB), brain heart infusion (BHI), nutrient agar (NA), and salmonella shigella (SS). RESULTS: Sequencing revealed significantly higher gut microbial diversity in field-resistant larvae compared with field-sensitive and laboratory-sensitive larvae (P

Mapping neuronal networks is a central focus in neuroscience. While volume electron microscopy (vEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide molecular information to identify cell types or functions. We developed an approach that uses fluorescent single-chain variable fragments (scFvs) to perform multiplexed detergent-free immunolabeling and volumetric-correlated-light-and-electron-microscopy on the same sample. We generated eight fluorescent scFvs targeting brain markers. Six fluorescent probes were imaged in the cerebellum of a female mouse, using confocal microscopy with spectral unmixing, followed by vEM of the same sample. The results provide excellent ultrastructure superimposed with multiple fluorescence channels. Using this approach, we documented a poorly described cell type, two types of mossy fiber terminals, and the subcellular localization of one type of ion channel. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable molecular overlays for connectomic studies.

Protein-protein interactions (PPIs) are fundamental to understanding biological systems as protein complexes are the active molecular modules critical for carrying out cellular functions. Dysfunctional PPIs have been associated with various diseases including cancer. Systems-wide PPI analysis not only sheds light on pathological mechanisms, but also represents a paradigm in identifying potential therapeutic targets. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for defining endogenous PPIs of cellular networks. While proteome-wide studies have been performed in cell lysates, intact cells and tissues, applications of XL-MS in clinical samples have not been reported. In this study, we adopted a DSBSO-based in vivo XL-MS platform to map interaction landscapes from two breast cancer patient-derived xenograft (PDX) models. As a result, we have generated a PDX interaction network comprising 2,557 human proteins and identified interactions unique to breast cancer subtypes. Interestingly, most of the observed differences in PPIs correlated well with protein abundance changes determined by TMT-based proteome quantitation. Collectively, this work has demonstrated the feasibility of XL-MS analysis in clinical samples, and established an analytical workflow for tissue cross-linking that can be generalized for mapping PPIs from patient samples in the future to dissect disease-relevant cellular networks.

OBJECTIVES: Many cats do not see a veterinarian on an annual basis, and their caregivers face many barriers to accessing veterinary care. A potential solution to overcome some of these barriers is video telemedicine. Thus, the aim of this study was to understand companion cat caregivers perceptions of using veterinary video telemedicine with their cats. METHODS: An online quantitative questionnaire was used to survey US cat caregivers on their experiences of and attitudes to using video telemedicine with their cats. Participants were required to reside in the USA, be the primary caregiver of at least one cat and be aged 18 years or older. RESULTS: The majority (97.3%) of the 1254 respondents indicated they had never used a video telemedicine appointment with their cat(s) before; however, most (85.7%) indicated they were very or somewhat interested in using video telemedicine with their cat. Overall, caregivers perceived video telemedicine visits as less stressful for themselves (P

BackgroundPulmonary ischaemia-reperfusion injury (IRI) is a major contributor to poor lung transplant outcomes. We recently demonstrated a central role of airway-centred natural killer (NK) cells in mediating IRI; however, there are no existing effective therapies for directly targeting NK cells in humans.MethodsWe hypothesised that a depleting anti-CD94 monoclonal antibody (mAb) would provide therapeutic benefit in mouse and human models of IRI based on high levels of KLRD1 (CD94) transcripts in bronchoalveolar lavage samples from lung transplant patients.ResultsWe found that CD94 is highly expressed on mouse and human NK cells, with increased expression during IRI. Anti-mouse and anti-human mAbs against CD94 showed effective NK cell depletion in mouse and human models and blunted lung damage and airway epithelial killing, respectively. In two different allogeneic orthotopic lung transplant mouse models, anti-CD94 treatment during induction reduced early lung injury and chronic inflammation relative to control therapies. Anti-CD94 did not increase donor antigen-presenting cells that could alter long-term graft acceptance.ConclusionsLung transplant induction regimens incorporating anti-CD94 treatment may safely improve early clinical outcomes.

Delay discounting refers to the behavioral tendency to devalue rewards as a function of their delay in receipt. Heightened delay discounting has been associated with substance use disorders and multiple co-occurring psychopathologies. Human and animal genetic studies have established that delay discounting is heritable, but only a few associated genes have been identified. We aimed to identify novel genetic loci associated with delay discounting through a genome-wide association study (GWAS) using Heterogeneous Stock (HS) rats, a genetically diverse outbred population derived from eight inbred founder strains. We assessed delay discounting in 650 male and female HS rats using an adjusting amount procedure in which rats chose between smaller immediate sucrose rewards or a larger reward at various delays. Preference switch points were calculated and both exponential and hyperbolic functions were fitted to these indifference points. Area under the curve (AUC) and the discounting parameter k of both functions were used as delay discounting measures. GWAS for AUC, exponential k, and one indifference point identified significant loci on chromosomes 20 and 14. The gene Slc35f1, which encodes a member of the solute carrier family, was the sole gene within the chromosome 20 locus. That locus also contained an eQTL for Slc35f1, suggesting that heritable differences in the expression might be responsible for the association with behavior. Adgrl3, which encodes a latrophilin subfamily G-protein coupled receptor, was the sole gene within the chromosome 14 locus. These findings implicate novel genes in delay discounting and highlight the need for further exploration.

UNLABELLED: Antagonism of the PD-1/PD-L1 axis is a critical therapeutic strategy for patients with advanced bladder cancer. IFNγ functions as a key regulator of PD-L1 in both immune as well as cancer cells. Forkhead box P3 (FOXP3) is a transcription factor synonymous in T regulatory cell function but with increasingly described functions in cancer cells. Here, we investigated the relationship between FOXP3 and PD-L1 in bladder cancer. We showed that FOXP3 is critical in the ability for IFNγ to activate PD-L1 in bladder cancer cells. FOXP3 can bind to the PD-L1 promoter and induces a gene program that leads to regulation of multiple immune-related genes and genes involved in epithelial-to-mesenchymal transition (EMT). Using in vitro and in vivo human and murine models, we showed that FOXP3 can influence bladder cancer EMT as well as promote cancer metastases. Furthermore, FOXP3 may be a convergent factor for multiple activators of PD-L1, including the chemotherapeutic drug cisplatin. SIGNIFICANCE: Historically a key transcription factor driving T regulatory cell function, FOXP3 has an increasingly recognized role in cancer cells. In bladder cancer, we defined a novel mechanism whereby FOXP3 mediates the activation of the immune checkpoint PD-L1 by the cytokine IFNγ. We also showed that FOXP3 induces other immune checkpoints as well as genes involved in EMT, promoting immune resistance and cancer metastases.

ObjectivesChimpanzees (Pan troglodytes) are patrilocal, with males remaining in their natal community and females dispersing when they reach sexual maturity. However, the details of female chimpanzee dispersal, such as their possible origin, are difficult to assess, even in habituated communities. This study investigates the utility of 87Sr/86Sr analysis for (1) assessing Sr baseline differences between chimpanzee territories and (2) identifying the status (immigrant or natal) of females of unknown origin within the territories of five neighboring communities in Taï National Park (Côte d'Ivoire).Materials and methodsTo create a local Sr isoscape for the Taï Chimpanzee Project (TCP) study area, we sampled environmental samples from TCP-established territories (n = 35). To assess dispersal patterns, 34 tooth enamel samples (one per individual) were selected from the Taï chimpanzee skeletal collection. 87Sr/86Sr analysis was performed on all 69 samples at the W.M. Keck Lab. The theoretical density and overlap of chimpanzee communities as well as generalized linear mixed models (GLMMs) were used to test each question.Results87Sr/86Sr ratios for natal male chimpanzees ranged from 0.71662 to 0.72187, which is well within the corresponding environmental baseline range of 0.70774-0.73460. The local Sr isoscapes fit was estimated with the root-mean-square error value, which was 0.0048 (22% of the whole 87Sr/86Sr data range). GLMMs identified significant differences in 87Sr/86Sr ratios between natal and unknown North community origin groups, suggesting that after 1980, females of unknown origin could be immigrants to North community (n = 7, z-ratio = -4.08, p = 0.0001, power = 0.94).DiscussionThis study indicates that 87Sr/86This study indicates that 87Sr/86Sr analysis can successfully identify immigrant females in skeletal collections obtained from wild chimpanzee communities, enabling the tracking of female dispersal patterns historically. There are, however, significant limitations within the scope of this study, such as (1) the absence of reliable maps for the TCP study area, (2) limited capacity for environmental sampling, (3) small sample sizes, and (4) tooth formation in wild chimpanzees.

Cognitive skills, such as innovative problem-solving, are hypothesized to aid animals in urban environments. However, the significance of innovation in wild populations, and its expression across individuals and socio-ecological conditions, is poorly understood. To identify how and when innovation arises in urban-dwelling species, we used advanced technologies and new testing and analytical methods to evaluate innovative problem-solving abilities of wild raccoons (Procyon lotor). We deployed multi-compartment puzzle boxes with either one or multiple solution types and identified raccoons using radio frequency identification. Raccoons solved these novel extractive foraging tasks, and their success was influenced by age and exploratory diversity. Successful raccoons always discovered multiple different solution types, highlighting flexible problem-solving. Using a unique, comparative sequence analysis approach, we found that variation in raccoon solving techniques was greater between individuals than within individuals, and this self-similarity intensified during times of competition. Finally, the inclusion of an easier solution in the multi-solution trials enabled previously unsuccessful raccoons to bootstrap their learning and successfully open multiple difficult solutions. Our study suggests that innovative problem-solving is probably influenced by many factors and has provided novel field and analytical methods, as well as new insights on the socio-ecological dynamics of urban populations.

Central nervous system (CNS) lesions become surrounded by neuroprotective borders of newly proliferated reactive astrocytes; however, fundamental features of these cells are poorly understood. Here we show that following spinal cord injury or stroke, 90% and 10% of border-forming astrocytes derive, respectively, from proliferating local astrocytes and oligodendrocyte progenitor cells in adult mice of both sexes. Temporal transcriptome analysis, single-nucleus RNA sequencing and immunohistochemistry show that after focal CNS injury, local mature astrocytes dedifferentiate, proliferate and become transcriptionally reprogrammed to permanently altered new states, with persisting downregulation of molecules associated with astrocyte-neuron interactions and upregulation of molecules associated with wound healing, microbial defense and interactions with stromal and immune cells. These wound repair astrocytes share morphologic and transcriptional features with perimeningeal limitans astrocytes and are the predominant source of neuroprotective borders that re-establish CNS integrity around lesions by separating neural parenchyma from stromal and immune cells as occurs throughout the healthy CNS.

Glucagon plays a central role in amino acid (AA) homeostasis. The dog is an established model of glucagon biology, and recently, metabolomic changes in people associated with glucagon infusions have been reported. Glucagon also has effects on the kidney; however, changes in urinary AA concentrations associated with glucagon remain under investigation. Therefore, we aimed to fill these gaps in the canine model by determining the effects of glucagon on the canine plasma metabolome and measuring urine AA concentrations. Employing two constant rate glucagon infusions (CRI) - low-dose (CRI-LO: 3 ng/kg/min) and high-dose (CRI-HI: 50 ng/kg/min) on five research beagles, we monitored interstitial glucose and conducted untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) on plasma samples and urine AA concentrations collected pre- and post-infusion. The CRI-HI induced a transient glucose peak (90-120 min), returning near baseline by infusion end, while only the CRI-LO resulted in 372 significantly altered plasma metabolites, primarily reductions (333). Similarly, CRI-HI affected 414 metabolites, with 369 reductions, evidenced by distinct clustering post-infusion via data reduction (PCA and sPLS-DA). CRI-HI notably decreased circulating AA levels, impacting various AA-related and energy-generating metabolic pathways. Urine analysis revealed increased 3-methyl-l-histidine and glutamine, and decreased alanine concentrations post-infusion. These findings demonstrate glucagons glucose-independent modulation of the canine plasma metabolome and highlight the dogs relevance as a translational model for glucagon biology. Understanding these effects contributes to managing dysregulated glucagon conditions and informs treatments impacting glucagon homeostasis.

BACKGROUND: Deeper insights into ERBB2-driven cancers are essential to develop new treatment approaches for ERBB2+ breast cancers (BCs). We employed the Collaborative Cross (CC) mouse model to unearth genetic factors underpinning Erbb2-driven mammary tumour development and metastasis. METHODS: 732 F1 hybrid female mice between FVB/N MMTV-Erbb2 and 30 CC strains were monitored for mammary tumour phenotypes. GWAS pinpointed SNPs that influence various tumour phenotypes. Multivariate analyses and models were used to construct the polygenic score and to develop a mouse tumour susceptibility gene signature (mTSGS), where the corresponding human ortholog was identified and designated as hTSGS. The importance and clinical value of hTSGS in human BC was evaluated using public datasets, encompassing TCGA, METABRIC, GSE96058, and I-SPY2 cohorts. The predictive power of mTSGS for response to chemotherapy was validated in vivo using genetically diverse MMTV-Erbb2 mice. FINDINGS: Distinct variances in tumour onset, multiplicity, and metastatic patterns were observed in F1-hybrid female mice between FVB/N MMTV-Erbb2 and 30 CC strains. Besides lung metastasis, liver and kidney metastases emerged in specific CC strains. GWAS identified specific SNPs significantly associated with tumour onset, multiplicity, lung metastasis, and liver metastasis. Multivariate analyses flagged SNPs in 20 genes (Stx6, Ramp1, Traf3ip1, Nckap5, Pfkfb2, Trmt1l, Rprd1b, Rer1, Sepsecs, Rhobtb1, Tsen15, Abcc3, Arid5b, Tnr, Dock2, Tti1, Fam81a, Oxr1, Plxna2, and Tbc1d31) independently tied to various tumour characteristics, designated as a mTSGS. hTSGS scores (hTSGSS) based on their transcriptional level showed prognostic values, superseding clinical factors and PAM50 subtype across multiple human BC cohorts, and predicted pathological complete response independent of and superior to MammaPrint score in I-SPY2 study. The power of mTSGS score for predicting chemotherapy response was further validated in an in vivo mouse MMTV-Erbb2 model, showing that, like findings in human patients, mouse tumours with low mTSGS scores were most likely to respond to treatment. INTERPRETATION: Our investigation has unveiled many new genes predisposing individuals to ERBB2-driven cancer. Translational findings indicate that hTSGS holds promise as a biomarker for refining treatment strategies for patients with BC. FUNDING: The U.S. Department of Defense (DoD) Breast Cancer Research Program (BCRP) (BC190820), United States; MCIN/AEI/10.13039/501100011039 (PID2020-118527RB-I00, PDC2021-121735-I00), the European Union Next Generation EU/PRTR, the Regional Government of Castile and León (CSI144P20), European Union.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 base pair region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals in whom it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologues. Using RNA sequencing, we show how 5 splice-site use is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 base pair region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

Hematopoietic stem cells and multipotential progenitors emerge in multiple, overlapping waves of fetal development. Some of these populations seed the bone marrow and sustain adult B- and T-cell development long-term after birth. However, others are present transiently, but whether they are vestigial or generate B and T cells that contribute to the adult immune system is not well understood. We now report that transient fetal progenitors distinguished by expression of low levels of the PU.1 transcription factor generated activated and memory T and B cells that colonized and were maintained in secondary lymphoid tissues. These included the small and large intestines, where they may contribute to the maintenance of gut homeostasis through at least middle age. At least some of the activated/memory cells may have been the progeny of B-1 and marginal zone B cells, as transient PU.1low fetal progenitors efficiently generated those populations. Taken together, our data demonstrate the potential of B- and T-cell progeny of transient PU.1low fetal progenitors to make an early and long-term contribution to the adult immune system.

Cerebellar injury in preterm infants with central nervous system (CNS) hemorrhage results in lasting neurological deficits and an increased risk of autism. The impact of blood-induced pathways on cerebellar development remains largely unknown, so no specific treatments have been developed to counteract the harmful effects of blood after neurovascular damage in preterm infants. Here, we show that fibrinogen, a blood-clotting protein, plays a central role in impairing neonatal cerebellar development. Longitudinal MRI of preterm infants revealed that cerebellar bleeds were the most critical factor associated with poor cerebellar growth. Using inflammatory and hemorrhagic mouse models of neonatal cerebellar injury, we found that fibrinogen increased innate immune activation and impeded neurogenesis in the developing cerebellum. Fibrinogen inhibited sonic hedgehog (SHH) signaling, the main mitogenic pathway in cerebellar granule neuron progenitors (CGNPs), and was sufficient to disrupt cerebellar growth. Genetic fibrinogen depletion attenuated neuroinflammation, promoted CGNP proliferation, and preserved normal cerebellar development after neurovascular damage. Our findings suggest that fibrinogen alters the balance of SHH signaling in the neurovascular niche and may serve as a therapeutic target to mitigate developmental brain injury after CNS hemorrhage.

Recent emerging zoonotic disease outbreaks, such as that of SARS-CoV-2, have demonstrated the need for wider companion animal disease surveillance. We tested 1000 dogs and cats belonging to employees of a US veterinary hospital network that were exposed to human COVID-19 cases in the household between 1 January 2020 and 10 March 2022 for SARS-CoV-2 and surveyed their owners about clinical signs and risk factors. The seropositivity was 33% for 747 dogs and 27% for 253 cats. Pet seropositivity correlated with the US human case rates over time, exhibiting peaks corresponding with the major COVID-19 surges. Antibodies persisted longer than previously documented (828 days in dogs; 650 days in cats). Increasing age and duration of proximity to infected people were associated with increased seropositivity in dogs but not cats. Cats were more likely to have clinical signs, but an association between seropositivity and the presence of clinical signs was not found in either species.

Exaggerated airway constriction triggered by repeated exposure to allergen, also called hyperreactivity, is a hallmark of asthma. Whereas vagal sensory neurons are known to function in allergen-induced hyperreactivity1-3, the identity of downstream nodes remains poorly understood. Here we mapped a full allergen circuit from the lung to the brainstem and back to the lung. Repeated exposure of mice to inhaled allergen activated the nuclei of solitary tract (nTS) neurons in a mast cell-, interleukin-4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA sequencing, followed by RNAscope assay at baseline and allergen challenges, showed that a Dbh+ nTS population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted hyperreactivity whereas chemogenetic activation promoted it. Viral tracing indicated that Dbh+ nTS neurons project to the nucleus ambiguus (NA) and that NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that directly drive airway constriction. Delivery of noradrenaline antagonists to the NA blunted hyperreactivity, suggesting noradrenaline as the transmitter between Dbh+ nTS and NA. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. This knowledge informs how neural modulation could be used to control allergen-induced airway hyperreactivity.

UNLABELLED: Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

The influence of light spectral properties on circadian rhythms is of substantial interest to laboratory-based investigation of the circadian system and to field-based understanding of the effects of artificial light at night. The trade-offs between intensity and spectrum regarding masking behaviors are largely unknown, even for well-studied organisms. We used a custom LED illumination system to document the response of wild-type house mice (Mus musculus) to 1-h nocturnal exposure of all combinations of four intensity levels (0.01, 0.5, 5 and 50 lx) and three correlated color temperatures (CCT; 1750, 1950 and 3000 K). Higher intensities of light (50 lx) suppressed cage activity substantially, and consistently more for the higher CCT light (91% for 3000 K, 53% for 1750 K). At the lowest intensity (0.01 lx), mean activity was increased, with the greatest increases for the lowest CCT (12.3% increase at 1750 K, 3% increase at 3000 K). Multiple linear regression confirmed the influence of both CCT and intensity on changes in activity, with the scaled effect size of intensity 3.6 times greater than that of CCT. Activity suppression was significantly lower for male than for female mice. Assessment of light-evoked cFos expression in the suprachiasmatic nucleus at 50 lx showed no significant difference between high and low CCT exposure. The significant differences by spectral composition illustrate a need to account for light spectrum in circadian studies of behavior, and confirm that spectral controls can mitigate some, but certainly not all, of the effects of light pollution on species in the wild.