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Great apes and humans develop many of the same health conditions, including cardiac disease as a leading cause of death. In humans, lipid markers are strong predictors of morbidity and mortality risk. To determine if they similarly predict risk in gorillas, we measured five serum lipid markers and calculated three lipoprotein ratios from zoo-housed western lowland gorillas (aged 6–52 years, n=61, subset with routine immobilizations only: n=47): total cholesterol (TC), triglycerides (TGs), high-density lipoprotein (HDL), low-density lipoprotein (LDL), apolipoprotein A1 (apoA1), TC∕HDL, LDL∕HDL, and TG∕HDL. We examined each in relation to age and sex, then analyzed whether they predicted all-cause morbidity, cardiac disease, and mortality using generalized linear models (GLMs). Older age was significantly associated with higher TG, TC∕HDL, LDL∕HDL, and TG∕HDL, and lower HDL and apoA1. With all ages combined, compared to females, males had significantly lower TG, TC∕HDL, LDL∕HDL, and TG∕HDL, and higher HDL. Using GLMs, age, sex, and lower LDL∕HDL were significant predictors of all-cause morbidity; this is consistent with research demonstrating lower LDL in humans with arthritis, which was the second most prevalent condition in this sample. In contrast to humans, lipid markers were not better predictors of cardiac disease and mortality risk in gorillas, with cardiac disease best predicted by age and sex alone, and mortality risk only by age. Similar results were observed when multimodel inference was used as an alternative analysis strategy, suggesting it can be used in place of or in addition to traditional methods for predicting risk.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare subtype of cutaneous T-cell lymphoma. There may be a significant histologic overlap with traumatic panniculitis and lupus profundus. We describe a 54-year-old woman who had received a diagnosis of SPTCL based upon a left parietal scalp biopsy 5 years earlier. This diagnosis was supported by immunohistochemistry (IHC) demonstrating a CD8+ predominant lymphocyte population in the subcutis. T-cell gene rearrangement studies were not performed at that time. The patient was treated and showed significant clinical improvement. When several tender erythematous subcutaneous nodules appeared on the upper back, left plantar surface and pretibial region, repeat biopsy was performed. Histology revealed a lobular and septal panniculitis with no vasculitis. The infiltrate contained abundant eosinophils and histiocytes not seen in the original biopsy specimen. IHC demonstrated a mixture of CD4+, CD8+ and CD7+ lymphocytes with abundant CD68+ histiocytes. T-cell gene rearrangement studies performed on one of the lesions failed to demonstrate clonality. It is important to recognize that patients with SPTCL are not exempt from other types of panniculitis, and complete histologic, IHC and molecular workups are essential to properly classify all cutaneous lesions in these patients.

Abstract Background The Caribbean offers a unique opportunity to study evolutionary dynamics in insular mammals. However, the recent extinction of most Caribbean non-volant mammals has obstructed evolutionary studies, and poor DNA preservation associated with tropical environments means that very few ancient DNA sequences are available for extinct vertebrates known from the region’s Holocene subfossil record. The endemic Caribbean eulipotyphlan family Nesophontidae (“island-shrews”) became extinct ~ 500 years ago, and the taxonomic validity of many Nesophontes species and their wider evolutionary dynamics remain unclear. Here we use both morphometric and palaeogenomic methods to clarify the status and evolutionary history of Nesophontes species from Hispaniola, the second-largest Caribbean island. Results Principal component analysis of 65 Nesophontes mandibles from late Quaternary fossil sites across Hispaniola identified three non-overlapping morphometric clusters, providing statistical support for the existence of three size-differentiated Hispaniolan Nesophontes species. We were also able to extract and sequence ancient DNA from a ~ 750-year-old specimen of Nesophontes zamicrus, the smallest non-volant Caribbean mammal, including a whole-mitochondrial genome and partial nuclear genes. Nesophontes paramicrus (39-47 g) and N. zamicrus (~ 10 g) diverged recently during the Middle Pleistocene (mean estimated divergence = 0.699 Ma), comparable to the youngest species splits in Eulipotyphla and other mammal groups. Pairwise genetic distance values for N. paramicrus and N. zamicrus based on mitochondrial and nuclear genes are low, but fall within the range of comparative pairwise data for extant eulipotyphlan species-pairs. Conclusions Our combined morphometric and palaeogenomic analyses provide evidence for multiple co-occurring species and rapid body size evolution in Hispaniolan Nesophontes, in contrast to patterns of genetic and morphometric differentiation seen in Hispaniola’s extant non-volant land mammals. Different components of Hispaniola’s mammal fauna have therefore exhibited drastically different rates of morphological evolution. Morphological evolution in Nesophontes is also rapid compared to patterns across the Eulipotyphla, and our study provides an important new example of rapid body size change in a small-bodied insular vertebrate lineage. The Caribbean was a hotspot for evolutionary diversification as well as preserving ancient biodiversity, and studying the surviving representatives of its mammal fauna is insufficient to reveal the evolutionary patterns and processes that generated regional diversity.

Bile acids (BAs) serve multiple biological functions, ranging from the absorption of lipids and fat-soluble vitamins to serving as signaling molecules through the direct activation of dedicated cellular receptors. Synthesized by both host and microbial pathways, BAs are increasingly understood as participating in the regulation of numerous pathways relevant to metabolic diseases, including lipid and glucose metabolism, energy expenditure, and inflammation. Quantitative analyses of BAs in biological matrices can be problematic due to their unusual and diverse physicochemical properties, making optimization of a method that shows good accuracy, precision, efficiency of extraction, and minimized matrix effects across structurally distinct human and murine BAs challenging. Herein we develop and clinically validate a stable-isotope-dilution LC/MS/MS method for the quantitative analysis of numerous primary and secondary BAs in both human and mouse biological matrices. We also utilize this tool to investigate gut microbiota participation in the generation of structurally specific BAs in both humans and mice. We examine circulating levels of specific BAs and in a clinical case-control study of age- and gender-matched type 2 diabetes mellitus (T2DM) versus nondiabetics. BAs whose circulating levels are associated with T2DM include numerous 12α-hydroxyl BAs (taurocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, deoxycholic acid, and 3-ketodeoxycholic acid), while taurohyodeoxycholic acid was negatively associated with diabetes. The LC/MS/MS-based platform described should serve as a robust, high-throughput investigative tool for studying the potential involvement of structurally specific BAs and the gut microbiome on both physiological and disease processes.

Fat contributes most of the energy for migratory flight of birds, whereas lean body tissues (muscles and organs) contribute amino acids and water to maintain metabolic and osmotic homeostasis. During refueling at stopover sites, both fat and lean mass are recovered, but the dynamics of this recovery are poorly understood. We used non-invasive quantitative magnetic resonance (QMR) analysis to measure fat and lean mass of > 3,500 individuals of 25 songbird species during six spring and three autumn migration seasons between 2009 and 2019 at Long Point, ON, Canada. We used allometric scaling analysis and linear mixed-effects modeling of body composition data at both the population level (single capture) and the individual level (recapture). In the population-level analysis, lean mass scaled hypoallometrically with body mass, such that for every 20% increase in body mass, lean mass was predicted to increase by 12.1% in spring and 12.8% in autumn. Fat scaled hyperallometrically with body mass, such that for every 20% increase in body mass, fat mass was predicted to increase by 144% in spring and 136% in autumn. At the individual level, these allometric relationships were more extreme. As a result of this differential allometry, at low body masses, lean and fat mass contributes nearly equally to changes in mass, but at high body mass fat deposition becomes progressively more dominant. Spring migrants deposited relatively more fat than autumn migrants, and in autumn juvenile birds tended to have greater lean mass than adults. Our findings show that lean mass deposition during refueling by songbirds is substantial, and in line with the losses of protein expected in flight. The process of fat and lean mass deposition is characterized by non-linear dynamics which are influenced by the current body composition, season, and, to a lesser extent, age. The patterns suggest that the need for dietary protein to rebuild lean mass will be greater when body mass is low, during autumn migration, and in juvenile birds.

Background Improved detection of hepatocellular carcinoma (HCC) is needed, as current detection methods, such as alpha fetoprotein (AFP) and ultrasound, suffer from poor sensitivity. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate many cellular functions and impact cancer development and progression. Notably, miRNAs are detectable in saliva and have shown potential as non-invasive biomarkers for a number of cancers including breast, oral, and lung cancers. Here, we present, to our knowledge, the first report of salivary miRNAs in HCC and compare these findings to patients with cirrhosis, a high-risk cohort for HCC. Methods We performed small RNA sequencing in 20 patients with HCC and 19 with cirrhosis. Eleven patients with HCC had chronic liver disease, and analyses were performed with these samples combined and stratified by the presence of chronic liver disease. P values were adjusted for multiple comparisons using a false discovery rate (FDR) approach and miRNA with FDR P < 0.05 were considered statistically significant. Differential expression of salivary miRNAs was compared to a previously published report of miRNAs in liver tissue of patients with HCC vs cirrhosis. Support vector machines and leave-one-out cross-validation were performed to determine if salivary miRNAs have predictive potential for detecting HCC. Results A total of 4,565 precursor and mature miRNAs were detected in saliva and 365 were significantly different between those with HCC compared to cirrhosis (FDR P < 0.05). Interestingly, 283 of these miRNAs were significantly downregulated in patients with HCC. Machine-learning identified a combination of 10 miRNAs and covariates that accurately classified patients with HCC (AUC = 0.87). In addition, we identified three miRNAs that were differentially expressed in HCC saliva samples and in a previously published study of miRNAs in HCC tissue compared to cirrhotic liver tissue. Conclusions This study demonstrates, for the first time, that miRNAs relevant to HCC are detectable in saliva, that salivary miRNA signatures show potential to be highly sensitive and specific non-invasive biomarkers of HCC, and that additional studies utilizing larger cohorts are needed.

The purpose of this study was to investigate within- and between-person associations between sleep and subsequent goal setting in adolescents. We conducted an intensive repeated measures longitudinal study to assess intra- and interindividual associations between sleep and goal setting and potential moderators of such associations. Thirty-nine seventh through 12th graders reported on their sleep quality and propensity to set goals in their daily lives several times per week for approximately four months. We used a combination of multilevel modeling with time-varying covariates and centering techniques to partition within- and between-person variance. We found significant and positive associations between sleep and goal setting within individuals, but no such associations between individuals. That is, students were more likely to set goals for their work after getting a good night’s sleep relative to their own average sleep quality, but getting good sleep on average relative to other individuals showed no association with average goal setting. These relationships were not moderated by participant age, gender, or sociodemographic status as indexed by maternal education. Differences in average sleep between adolescents matters less for their propensity to set goals than whether they experienced better- or worse-than-usual sleep the previous night given their own average. This finding represents the first evidence documenting effects of sleep on goal setting, which is an important psychological precursor to many youth behavioral and achievement outcomes. Our findings highlight the individuality of sleep needs and point to new directions for sleep-related practice and policy aimed at youth.

While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation.

Summary: Cellular cholesterol is regulated by at least two transcriptional mechanisms involving sterol-regulatory-element-binding proteins (SREBPs) and liver X receptors (LXRs). Although SREBP and LXR pathways are the predominant mechanisms that sense cholesterol in the endoplasmic reticulum and nucleus to alter sterol-regulated gene expression, evidence suggests cholesterol in plasma membrane can be sensed by proteins in the Hedgehog (Hh) pathway which regulate organ self-renewal and are a morphogenic driver during embryonic development. Cholesterol interacts with the G-protein-coupled receptor Smoothened (Smo), which impacts downstream Hh signaling. Although evidence suggests cholesterol influences Hh signaling, it is not known whether Smo-dependent sterol sensing impacts cholesterol homeostasis in vivo. We examined dietary-cholesterol-induced reorganization of whole-body sterol and bile acid (BA) homeostasis in adult mice with inducible hepatocyte-specific Smo deletion. These studies demonstrate Smo in hepatocytes plays a regulatory role in sensing and feedback regulation of cholesterol balance driven by excess dietary cholesterol.

The retinoid X receptor agonist bexarotene promotes remyelination in patients with multiple sclerosis. Murine studies have also demonstrated that RXR agonists have anti-inflammatory effects by enhancing the ability of all-trans-retinoic acid (atRA) to promote T-regulatory cell (Treg) induction and reduce Th17 differentiation in vitro. By stimulating human naïve CD4 T-cells in the presence of Treg or Th17 skewing cytokines, we show that bexarotene also tips the human Treg/Th17 axis in favor of Treg induction, but unlike murine cells this occurs independently of atRA and retinoic acid receptor signaling. Tregs induced in the presence of bexarotene express canonical markers of T-regulation and are functionally suppressive in vitro. Circulating Treg numbers did not increase in the blood of trial patients receiving bexarotene; we believe this is because Treg induction is likely to occur within tissues. These findings lend support to developing RXR agonists as treatments of autoimmune diseases, in particular multiple sclerosis.

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

ABSTRACT The marine lithospheric subsurface is one of the largest biospheres on Earth; however, little is known about the identity and ecological function of microorganisms found in low abundance in this habitat, though these organisms impact global-scale biogeochemical cycling. Here, we describe the diversity and metabolic potential of sediment and endolithic (within rock) microbial communities found in ultrasmall amounts (101 to 104 cells cm−3) in the subsurface of the Atlantis Massif, an oceanic core complex on the Mid-Atlantic Ridge that was sampled on International Ocean Discovery Program (IODP) Expedition 357. This study used fluorescence-activated cell sorting (FACS) to enable the first amplicon, metagenomic, and single-cell genomic study of the shallow (

Abstract Background Understanding the mechanisms by which organisms adapt to unfavourable conditions is a fundamental question in ecology and evolutionary biology. One such mechanism is diapause, a period of dormancy typically found in nematodes, fish, crustaceans and insects. This state is a key life-history event characterised by arrested development, suppressed metabolism and increased stress tolerance and allows an organism to avoid prolonged periods of harsh and inhospitable environmental conditions. For some species, diapause is preceded by mating which can have a profound effect on female behaviour, physiology and key biological processes, including immunity. However, our understanding of how mating impacts long-term immunity and whether these effects persist throughout diapause is currently limited. To address this, we explored molecular changes in the haemolymph of the ecologically important pollinator, the buff-tailed bumblebee Bombus terrestris. B. terrestris queens mate prior to entering diapause, a non-feeding period of arrested development that can last 6–9 months. Using mass-spectrometry-based proteomics, we quantified changes in the pre-diapause queen haemolymph after mating, as well as the subsequent protein expression of mated queens during and post-diapause. Results Our analysis identified distinct proteome profiles associated with diapause preparation, maintenance and termination. More specifically, mating pre-diapause was followed by an increase in the abundance of antimicrobial peptides, key effectors of the immune system. Furthermore, we identified the elevated abundance of these proteins to be maintained throughout diapause. This finding was in contrast to the general reduction observed in immune proteins during diapause suggestive of selective immune priming and expression during diapause. Diapause also affected the expression of proteins involved in cuticular maintenance, olfaction, as well as proteins of unknown function, which may have roles in diapause regulation. Conclusions Our results provide clear molecular evidence for the consequences and benefits of mating at the immune level as it precedes the selective increased abundance of antimicrobial peptides that are sustained throughout diapause. In addition, our results provide novel insights into the molecular mechanisms by which bumblebees prepare for, survive, and recover from diapause, insights that may have implications for our general understanding of these processes in other insect groups.

The authors show that the fungal pathogen Candida albicans exploits diverse host-associated signals, including specific nutrients and stresses, to promote immune evasion by masking cell wall β-glucan, a major pathogen-associated molecular pattern.

Abstract Background Evidence is mounting that patient-reported experience can provide a valuable indicator of the quality of healthcare services. However, little is known about the relationship between the experiences of people with severe mental illness and the quality of care they receive. We conducted a study to examine the relationship between patient-reported experience and the quality of care provided to people with schizophrenia. Methods We calculated a composite global rating of quality of care for people with schizophrenia using data from an audit of 64 mental health providers. We then examined associations between these ratings and mean patient satisfaction and patient-rated outcome using data from a survey of 5608 schizophrenic patients treated in these services. Results Global rating of quality of care was positively correlated with patient-rated outcome (r = 0.33; p = 0.01) but not with patient satisfaction (r = 0.21, p = 0.10). Patient-rated outcome was also positively correlated with patient involvement (r = 0.26, p = 0.04) and the quality of prescribing practice (r = 0.31, p = 0.02). High patient satisfaction scores were significantly associated with the extent of use of care plans within each organisation (r = 0.27, p = 0.03). Conclusions Among people with schizophrenia, patient-rated outcome provides a better guide to the quality of care than patient-rated satisfaction. Greater use of patient-reported outcome measures should be made when assessing the quality of care provided to people with psychosis.

Aspergillus fumigatus is the leading cause of the fungal invasive disease called aspergillosis, which is associated with a high mortality rate that can reach 50% in some groups of immunocompromised individuals. The increasing prevalence of azole-resistant A. fumigatus isolates, both in clinical settings and the environment, highlights the importance of discovering new fungal virulence factors that can potentially become targets for novel antifungals. Nitronate monooxygenases (Nmos) represent potential targets for antifungal compounds as no orthologs of those enzymes are present in humans. Nmos catalyse the denitrification of nitroalkanes, thereby detoxifying these mediators of nitro-oxidative stress, and therefore we tested whether Nmos provide protection for A. fumigatus against host-imposed stresses at sites of infection. The results of inhibition zone assays indicated that Nmo2 and Nmo5 are not essential for the oxidative stress resistance of A. fumigatus in vitro. In addition, the resazurin-based metabolic activity assay revealed that the growth of mutants lacking the nmo2 or nmo5 genes was only slightly reduced in the presence of 0.05 mM peroxynitrite. Nevertheless, both Nmo2 and Nmo5 were shown to contribute to defense against murine bone marrow-derived macrophages, and this was no longer observed when NADPH oxidase, the main generator of reactive oxygen species during infection, was inhibited in macrophages. Furthermore, we revealed that Nnmos promote the virulence of the fungus in the Galleria mellonella model of infection. Both nmo2 and nmo5 knock-out strains were less virulent than the wild-type control as recorded 72 h post-infection. Our results indicate that Nmos play a role in the virulence of A. fumigatus.

Improved blood tests assessing the functional status of rare gluten-specific CD4+ T cells are needed to effectively monitor experimental therapies for coeliac disease (CD). Our aim was to develop a simple, but highly sensitive cytokine release assay (CRA) for gluten-specific CD4+ T cells that did not require patients to undergo a prior gluten challenge, and would be practical in large, multi-centre clinical trials. We developed an enhanced CRA and used it in a phase 2 clinical trial (“RESET CeD”) of Nexvax2, a peptide-based immunotherapy for CD. Two participants with treated CD were assessed in a pilot study prior to and six days after a 3-day gluten challenge. Dye-dilution proliferation in peripheral blood mononuclear cells (PBMC) was assessed, and IL-2, IFN-γ and IL-10 were measured by multiplex electrochemiluminescence immunoassay (ECL) after 24-hour gluten-peptide stimulation of whole blood or matched PBMC. Subsequently, gluten-specific CD4+ T cells in blood were assessed in a subgroup of the RESET CeD Study participants who received Nexvax2 (maintenance dose 900 μg, n = 12) or placebo (n = 9). The pilot study showed that gluten peptides induced IL-2, IFN-γ and IL-10 release from PBMCs attributable to CD4+ T cells, but the PBMC CRA was substantially less sensitive than whole blood CRA. Only modest gluten peptide-stimulated IL-2 release could be detected without prior gluten challenge using PBMC. In contrast, whole blood CRA enabled detection of IL-2 and IFN-γ before and after gluten challenge. IL-2 and IFN-γ release in whole blood required more than 6 hours incubation. Delay in whole blood incubation of more than three hours from collection substantially reduced antigen-stimulated IL-2 and IFN-γ secretion. Nexvax2, but not placebo treatment in the RESET CeD Study was associated with significant reductions in gluten peptide-stimulated whole blood IL-2 and IFN-γ release, and CD4+ T cell proliferation. We conclude that using fresh whole blood instead of PBMC substantially enhances cytokine secretion stimulated by gluten peptides, and enables assessment of rare gluten-specific CD4+ T cells without requiring CD patients to undertake a gluten challenge. Whole blood assessment coupled with ultra-sensitive cytokine detection shows promise in the monitoring of rare antigen-specific T cells in clinical studies.

The chemical and isotopic compositions of volcanic arc lavas often show evidence for involvement of a sedimentary component during magma genesis. Determining where this sedimentary component is added to arc magmas is of vital importance for constraining the extent to which sediments and volatiles are recycled at subduction zones. Lavas from Martinique in the Lesser Antilles arc have wide ranging isotopic compositions extending to highly radiogenic values (e.g. 87/Sr/86Sr up to ∼0.710) that could, in principle, be explained by sediment addition to the mantle source or by crustal assimilation in the upper plate. We use Sr isotopic compositions of plagioclase from Martinique plutonic xenoliths to provide evidence supporting the crustal assimilation hypothesis. Plagioclase from plutonic xenoliths formed in the mid-crust (∼12 km) show a restricted range of unradiogenic Sr isotope ratios (87Sr/86Sr = 0.7041–0.7042) whereas plagioclase from upper crustal plutonic xenoliths (∼6 km) show greater intra-sample variation and more radiogenic Sr isotopic compositions up to 87Sr/86Sr = 0.7047. This trend is also observed in plutonic xenolith whole rock 87Sr/86Sr. Combined, these results indicate that the range of Sr isotope compositions becomes larger and more radiogenic in Martinique magmas as a result of sediment assimilation at shallow crustal levels. This is supported by Assimilation-Fractional Crystallization modeling, which shows that assimilation of chemically and isotopically heterogenous crustal sediments can produce the isotopic variation in Martinique plutonic xenoliths and lavas. Our results highlight the importance of constraining crustal contributions from the upper plate before using arc lava geochemistry to quantify sediment and volatile recycling at subduction zones and assessing potential heterogeneity of arc mantle sources.

Gut microbe–derived metabolites influence human physiology and disease. However, establishing mechanistic links between gut microbial metabolites and disease pathogenesis in animal models remains challenging. The major route of absorption for microbe-derived small molecules is venous drainage via the portal vein to the liver. In the event of presystemic hepatic metabolism, the route of metabolite administration becomes critical. To our knowledge, we describe here a novel portal vein cannulation technique using a s.c. implanted osmotic pump to achieve continuous portal vein infusion in mice. We first administered the microbial metabolite trimethylamine (TMA) over 4 weeks, during which increased peripheral plasma levels of TMA and its host liver-derived cometabolite, trimethylamine-N-oxide, were observed when compared with a vehicle control. Next, 4-hydroxyphenylacetic acid (4-HPAA), a microbial metabolite that undergoes extensive presystemic hepatic metabolism, was administered intraportally to examine effects on hepatic gene expression. As expected, hepatic levels of 4-HPAA were elevated when compared with the control group while peripheral plasma 4-HPAA levels remained the same. Moreover, significant changes in the hepatic transcriptome were revealed by an unbiased RNA-Seq approach. Collectively, to our knowledge this work describes a novel method for administering gut microbe–derived metabolites via the portal vein, mimicking their physiologic delivery in vivo.

Reactive Oxygen Species (ROS) are highly reactive molecules that can induce oxidative stress. For instance, the oxidative burst of immune cells is well known for its ability to inhibit the growth of invading pathogens. However, ROS also mediate redox signalling, which is important for the regulation of antimicrobial immunity. Here, we report a crucial role of mitochondrial ROS (mitoROS) in antifungal responses of macrophages. We show that mitoROS production rises in murine macrophages exposed to swollen conidia of the fungal pathogen Aspergillus fumigatus compared to untreated macrophages, or those treated with resting conidia. Furthermore, the exposure of macrophages to swollen conidia increases the activity of complex II of the respiratory chain and raises mitochondrial membrane potential. These alterations in mitochondria of infected macrophages suggest that mitoROS are produced via reverse electron transport (RET). Significantly, preventing mitoROS generation via RET by treatment with rotenone, or a suppressor of site IQ electron leak, S1QEL1.1, lowers the production of pro-inflammatory cytokines TNF-α and IL-1β in macrophages exposed to swollen conidia of A. fumigatus. Rotenone and S1QEL1.1 also reduces the fungicidal activity of macrophages against swollen conidia. Moreover, we have established that elevated recruitment of NADPH oxidase 2 (NOX2, also called gp91phox) to the phagosomal membrane occurs prior to the increase in mitoROS generation. Using macrophages from gp91phox-/- mice, we have further demonstrated that NOX2 is required to regulate cytokine secretion by RET-associated mitoROS in response to infection with swollen conidia. Taken together, these observations demonstrate the importance of RET-mediated mitoROS production in macrophages infected with A. fumigatus.