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Mechanical impact-induced primary injury after traumatic brain injury (TBI) leads to acute microglial pro-inflammatory activation and consequently mediates neurodegeneration, which is a major secondary brain injury mechanism. However, the detailed pathologic cascades have not been fully elucidated, partially because of the pathologic complexity in animal TBI models. Although there are severalin vitroTBI models, none of them closely mimic post-TBI microglial activation. In the present study, we aimed to establish anin vitroTBI model, specifically reconstituting the pro-inflammatory activation and associated neurodegeneration following TBI. We proposed three sets of experiments. First, we established a needle scratch injured neuron-induced microglial activation and neurodegenerationin vitromodel of TBI. Second, we compared microglial pro-inflammatory cytokines profiles between thein vitroTBI model and TBI in male mice. Additionally, we validated the role of injured neurons-derived damage-associated molecular patterns in amplifying microglial pro-inflammatory pathways using thein vitroTBI model. Third, we applied thein vitromodel for the first time to characterize the cellular metabolic profile of needle scratch injured-neuron-activated microglia, and define the role of metabolic reprogramming in mediating pro-inflammatory microglial activation and mediated neurodegeneration. Our results showed that we successfully established a novelin vitroTBI model, which closely mimics primary neuronal injury-triggered microglial pro-inflammatory activation and mediated neurodegeneration after TBI. Thisin vitromodel provides an advanced and highly translational platform for dissecting interactions in the pathologic processes of neuronal injury-microglial activation-neuronal degeneration cascade, and elucidating the detailed underlying cellular and molecular insights after TBI.SIGNIFICANCE STATEMENTMicroglial activation is a key component of acute neuroinflammation that leads to neurodegeneration and long-term neurologic outcome deficits after TBI. However, it is not feasible to truly dissect primary neuronal injury-induced microglia activation, and consequently mediated neurodegenerationin vivo. Furthermore, there is currently lacking ofin vitroTBI models closely mimicking the TBI primary injury-mediated microglial activation. In this study, we successfully established and validated a novelin vitroTBI model of microglial activation, and for the first time, characterized the cellular metabolic profile of microglia in this model. This novel microglial activationin vitroTBI model will help in elucidating microglial inflammatory activation and consequently associated neurodegeneration after TBI.

Insects, unlike vertebrates, are widely believed to lack male-biased sex steroid hormones1. In the malaria mosquito Anopheles gambiae, the ecdysteroid 20-hydroxyecdysone (20E) appears to have evolved to both control egg development when synthesized by females2 and to induce mating refractoriness when sexually transferred by males3. Because egg development and mating are essential reproductive traits, understanding how Anopheles females integrate these hormonal signals can spur the design of new malaria control programs. Here we reveal that these reproductive functions are regulated by distinct sex steroids through a sophisticated network of ecdysteroid-activating/inactivating enzymes. We identify a male-specific oxidized ecdysteroid, 3-dehydro-20E (3D20E), which safeguards paternity by turning off female sexual receptivity following its sexual transfer and activation by dephosphorylation. Notably, 3D20E transfer also induces expression of a reproductive gene that preserves egg development during Plasmodium infection, ensuring fitness of infected females. Female-derived 20E does not trigger sexual refractoriness but instead licenses oviposition in mated individuals once a 20E-inhibiting kinase is repressed. Identifying this male-specific insect steroid hormone and its roles in regulating female sexual receptivity, fertility and interactions with Plasmodium parasites suggests the possibility for reducing the reproductive success of malaria-transmitting mosquitoes.

Insecticide resistance is under strong selective pressure inAnophelesmosquitoes due to widespread usage of insecticides in vector control strategies. Resistance mechanisms likely cause changes that profoundly affect mosquito physiology, yet it remains poorly understood how selective pressures imposed by insecticides may alter the ability of the mosquito to host and transmit aPlasmodiuminfection. From pyrethroid-resistant field-derivedAnopheles gambiae s.l. mosquitoes, we performed selection experiments to establish resistant (RES) and susceptible (SUS) colonies by either selection for, or loss of, insecticide resistance. We show increased prevalence, intensity, and oocyst growth rate ofPlasmodium falciparuminfection in RES females compared to SUS. The increase in infection intensity in RES females was not associated with the presence of thekdrL1014F mutation, and was not impacted by inhibition of Cytochrome P450s. The lipid transporter lipophorin (Lp), which was upregulated in RES compared to SUS, was at least partly implicated in the increased intensity ofP. falciparumbut not directly in the insecticide resistance phenotype. Interestingly, we observed that althoughP. falciparuminfections were not affected when RES females were exposed to permethrin, these females had decreased lipid abundance in the fat body following exposure, pointing to a possible role for lipid mobilization in response to damage caused by insecticide challenge. The finding that selection for insecticide resistance can increaseP. falciparuminfection intensities and growth rate reinforces the need to assess the overall impact on malaria transmission dynamics of selective pressures mosquitoes experience during repeated insecticide challenge.Significance StatementInsecticide resistance poses a severe threat for malaria control. Resistance to pyrethroid insecticides, the active component of most insecticide-treated nets, is now widespread in sub-Saharan Africa, reducing the efficacy of these crucial tools. Despite significant research characterizing insecticide resistance mechanisms, it remains unknown how these traits influencePlasmodium falciparuminfections in malaria-transmittingAnophelesmosquitoes. We established a pyrethroid-resistant and pyrethroid-susceptible population ofAnopheles gambiaederived from the same genetic background and performed experimental infections withP. falciparum. We found that the pyrethroid-resistant population was more supportive of malaria parasites compared to the susceptible population. This was not caused by well-known insecticide resistance mechanisms, but linked with a lipid transporter, lipophorin, which may play an indirect role in resistance.

SummaryDNA damage causes genomic instability underlying many human diseases. Traditional approaches to DNA damage analysis provide minimal insights into the spectrum of disease-driving DNA lesions and the mechanisms causing imbalances in damage formation and repair. Here we used untargeted mass spectrometry-based adductomics1to discover 114 putative DNA lesions and modifications consistently detected in humans and two independent analyses in rats, showing species-, tissue-, age-, and sex-biases. As evidence of methodologic rigor, 10 selected adductomic signals were structurally validated as epigenetic marks: 5-MdC, 5-HMdC, 5-FdC; DNA damage products:N2-CMdG, 1,N6ε-dA, 3,N4-εdC, M1dG,O6/N2-MdG, and 8-Oxo-dG; and established analytical artifacts: cyclobutane dimers of 2’-deoxycytosine. With steady-state levels of putative DNA adducts integrating multiple cell types in each tissue, there was strong age-dependent variation for many putative adducts, includingN2-CMdG, 5-HMdC, and 8-Oxo-dG in rats and 1,N6ε-dA in human heart, as well as sex biases for 67 putative adducts in rat tissues. These results demonstrate the potential of untargeted adductomic analysis for defining DNA adducts as disease determinants, assigning substrates to DNA repair pathways, discovering new metabolically-driven DNA lesions, and quantifying inter-individual variation in DNA damage and repair across populations.

Semivolatile oxygenated organic compounds (SV-OVOCs) are important atmospheric species, in particular for the production and chemistry of atmospheric particulate matter and related impacts on air quality and climate. In this study, SV-OVOCs were collected in the horizontal plane of the roughness layer over the tropical forest in the central Amazon during the wet season of 2018. A sampler mounted to a copter-type, hovering unmanned aerial vehicle was used. Underlying the collection region, a plateau forest transitioned into a slope forest across several hundred meters. The concentrations of pinonic and pinic acids, which are monoterpene oxidation products, had no statistical difference over the two forests. By comparison, across the study period, differences in the concentration of 2-methyltetrols, which are products of isoprene oxidation, ranged from −70% to +480% over the two forests. The chemical lifetime of 2-methyltetrols in the atmosphere is sufficiently long that heterogeneity in the isoprene emission rate from the two forests followed by atmospheric oxidation does not explain the concentration heterogeneity of 2-methyltetrols. Standing waves and local meteorology also cannot account for the heterogeneity. Of the possibilities considered, the most plausible explanation is the direct emission from the forest of 2-methyltetrols produced through biological processes within the plants. Under this explanation, the rate of direct SV-OVOC emissions should be modulated by forest type and related environmental stressors. Direct emissions of SV-OVOCs should be more broadly considered for constraining and improving models of atmospheric composition, transport, and chemistry over tropical forests.

Poison frogs bioaccumulate alkaloids for chemical defense from their arthropod diet. Although many alkaloids are accumulated without modification, some poison frog species can metabolize pumiliotoxin (PTX 251D) into the more potent allopumiliotoxin (aPTX 267A). Despite extensive research characterizing the chemical arsenal of poison frogs, the physiological mechanisms involved in the sequestration and metabolism of individual alkaloids remain unclear. We first performed a feeding experiment with the Dyeing poison frog (Dendrobates tinctorius) to ask if this species can metabolize PTX 251D into aPTX 267A and what gene expression changes are associated with PTX 251D exposure in the intestines, liver, and skin. We found that D. tinctorius can metabolize PTX 251D into aPTX 267A, and that PTX 251D exposure changed the expression level of genes involved in immune system function and small molecule metabolism and transport. To better understand the functional significance of these changes in gene expression, we then conducted a series of high-throughput screens to determine the molecular targets of PTX 251D and identify potential proteins responsible for metabolism of PTX 251D into aPTX 267A. Although screens of PTX 251D binding human voltage-gated ion channels and G-protein coupled receptors were inconclusive, we identified human CYP2D6 as a rapid metabolizer of PTX 251D in a cytochrome P450 screen. Furthermore, a CYP2D6-like gene had increased expression in the intestines of animals fed PTX, suggesting this protein may be involved in PTX metabolism. These results show that individual alkaloids can modify gene expression across tissues, including genes involved in alkaloid metabolism. More broadly, this work suggests that specific alkaloid classes in wild diets may induce physiological changes for targeted accumulation and metabolism.

Much of the world’s biodiversity is held within tropical rainforests, which are increasingly fragmented by agricultural practices. In these threatened landscapes, there are many organisms that acquire chemical defenses from their diet and are therefore intimately connected with their local food webs. Poison frogs (Family Dendrobatidae) are one such example, as they acquire alkaloid-based chemical defenses from their diet of leaf litter ants and mites. It is currently unknown how habitat fragmentation impacts chemical defense across trophic levels, from arthropods to frogs. We examined the chemical defenses and diets of the Diablito poison frog (Oophaga sylvatica), and the diversity of their leaf litter ant communities in secondary forest and reclaimed cattle pasture. Notably, this research was performed in collaboration with two high school science classrooms. We found that the leaf litter of forest and pasture frog habitats differed significantly in ant community structure. We also found that forest and pasture frogs differed significantly in diet and alkaloid profiles, where forest frogs contained more of specific alkaloids and ate more ants in both number and volume. Finally, ant species composition of frog diets resembled the surrounding leaf litter, but diets were less variable. This suggests that frogs tend to consume particular ant species within each habitat. To better understand how ants contribute to the alkaloid chemical profiles of frogs, we chemically profiled several ant species and found some alkaloids to be common across many ant species while others are restricted to a few species. At least one alkaloid (223H) found in ants from disturbed sites was also found in skins from pasture. Our experiments are the first to link anthropogenic land use changes to dendrobatid poison frog chemical defenses through variation in leaf litter communities, which has implications for conservation management of these threatened amphibians.

Understanding how bactericidal antibiotics kill bacteria remains an open question. Previous work has proposed that primary drug-target corruption leads to increased energetic demands, resulting in the generation of reactive metabolic byproducts (RMBs), particularly reactive oxygen species, that contribute to antibiotic-induced cell death. Studies have challenged this hypothesis by pointing to antibiotic lethality under anaerobic conditions. Here, we show that treatment of Escherichia coli with bactericidal antibiotics under anaerobic conditions leads to changes in the intracellular concentrations of central carbon metabolites, as well as the production of RMBs, particularly reactive electrophilic species (RES). We show that antibiotic treatment results in DNA double-strand breaks and membrane damage and demonstrate that antibiotic lethality under anaerobic conditions can be decreased by RMB scavengers, which reduce RES accumulation and mitigate associated macromolecular damage. This work indicates that RMBs, generated in response to antibiotic-induced energetic demands, contribute in part to antibiotic lethality under anaerobic conditions.

Brilliantly-colored birds are a model system for research into evolution and sexual selection. Red, orange, and yellow carotenoid-colored plumages have been considered honest signals of condition; however, sex differences in feather pigments and microstructures are not well understood. Here, we show that microstructures, rather than carotenoid pigments, seem to be a major driver of male–female color differences in the social, sexually-dimorphic tanager genus Ramphocelus. We comprehensively quantified feather (i) color (using spectrophotometry), (ii) pigments (using liquid chromatography–mass spectrometry (LC–MS)), and (iii) microstructures (using scanning electron microscopy (SEM) and finite-difference time-domain (FDTD) optical modeling). Males have significantly more saturated color patches than females. However, our exploratory analysis of pigments suggested that males and females have concordant carotenoid pigment profiles across all species (MCMCglmm model, female:male ratio = 0.95). Male, but not female, feathers have elaborate microstructures which amplify color appearance. Oblong, expanded feather barbs in males enhance color saturation (for the same amount of pigment) by increasing the transmission of optical power through the feather. Dihedral barbules (vertically-angled, strap-shaped barbules) in males reduce total reflectance to generate “super black” and “velvet red” plumage. Melanin in females explains some, but not all, of the male–female plumage differences. Our results suggest that a widely cited index of honesty, carotenoid pigments, cannot fully explain male appearance. We propose that males are selected to evolve amplifiers—in this case, microstructures that enhance appearance—that are not necessarily themselves linked to quality.

The neurovascular unit (NVU) regulates metabolic homeostasis as well as drug pharmacokinetics and pharmacodynamics in the central nervous system. Metabolic fluxes and conversions over the NVU rely on interactions between brain microvascular endothelium, perivascular pericytes, astrocytes and neurons, making it difficult to identify the contributions of each cell type. Here we model the human NVU using microfluidic organ chips, allowing analysis of the roles of individual cell types in NVU functions. Three coupled chips model influx across the blood–brain barrier (BBB), the brain parenchymal compartment and efflux across the BBB. We used this linked system to mimic the effect of intravascular administration of the psychoactive drug methamphetamine and to identify previously unknown metabolic coupling between the BBB and neurons. Thus, the NVU system offers an in vitro approach for probing transport, efficacy, mechanism of action and toxicity of neuroactive drugs.

At deep-sea hydrothermal vents, primary production is carried out by chemolithoautotrophic microorganisms, with the oxidation of reduced sulfur compounds being a major driver for microbial carbon fixation. Dense and highly diverse assemblies of sulfur-oxidizing bacteria (SOB) are observed, yet the principles of niche differentiation between the different SOB across geochemical gradients remain poorly understood. In this study niche differentiation of the key SOB was addressed by extensive sampling of active sulfidic vents at six different hydrothermal venting sites in the Manus Basin, off Papua New Guinea. We subjected 33 diffuse fluid and water column samples and 23 samples from surfaces of chimneys, rocks and fauna to a combined analysis of 16S rRNA gene sequences, metagenomes and real-time in situ measured geochemical parameters. We found Sulfurovum Epsilonproteobacteria mainly attached to surfaces exposed to diffuse venting, while the SUP05-clade dominated the bacterioplankton in highly diluted mixtures of vent fluids and seawater. We propose that the high diversity within Sulfurimonas- and Sulfurovum-related Epsilonproteobacteria observed in this study derives from the high variation of environmental parameters such as oxygen and sulfide concentrations across small spatial and temporal scales.

Summary Cancer relapse begins when malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the byproducts of the massive cell death in the surrounding region. In acute myeloid leukemia, complete remissions are common, but few are cured. We tracked leukemia cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells, and conducted unbiased metabolomics, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine and glutathione generation, but not the mitochondrial tricarboxylic acid cycle. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual leukemia-initiating cells and improved survival in leukemia mouse models and patient-derived xenografts. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce metabolic collapse in cancer cells to overcome chemoresistance.

Cancer relapse begins at the moment malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the by-products of massive surrounding cell death. In acute myeloid leukemia (AML), complete remissions are common, but few are cured. We tracked AML cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells and conducted unbiased metabolic analysis, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine synthesis and glutathione generation, but not oxidative phosphorylation. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual cells and improved survival in AML mouse models. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce a metabolic collapse in cancer cells to overcome chemoresistance.

Extensive characterisations of the zebrafish genome and proteome have established a foundation for the use of the zebrafish as a model organism; however, characterisation of the zebrafish lipidome has not been as comprehensive. In an effort to expand current knowledge of the zebrafish sphingolipidome, a Parallel Reaction Monitoring (PRM)-based liquid chromatography–mass spectrometry (LC–MS) method was developed to comprehensively quantify zebrafish ceramides. Comparison between zebrafish and a human cell line demonstrated remarkable overlap in ceramide composition, but also revealed a surprising lack of most sphingadiene-containing ceramides in the zebrafish. PRM analysis of zebrafish embryogenesis identified developmental stage-specific ceramide changes based on long chain base (LCB) length. A CRISPR-Cas9-generated zebrafish model of Farber disease exhibited reduced size, early mortality, and severe ceramide accumulation where the amplitude of ceramide change depended on both acyl chain and LCB lengths. Our method adds an additional level of detail to current understanding of the zebrafish lipidome, and could aid in the elucidation of structure-function associations in the context of lipid-related diseases.

Red, orange, and yellow carotenoid-colored plumages have been considered honest signals of condition. We comprehensively quantified carotenoid signals in the social, sexually-dimorphic tanager genusRamphocelususing scanning electron microscopy (SEM), finite-difference time-domain (FDTD) optical modeling, liquid chromatography–mass spectrometry (LC-MS), and spectrophotometry. Despite males having significantly more saturated color patches, males and females within a species have equivalent amounts and types of carotenoids. Male, but not female, feathers have elaborate microstructures which amplify color appearance. Expanded barbs enhance color saturation (for the same amount of pigment) by increasing the transmission of optical power through the feather. Dihedral barbules (vertically-angled, strap-shaped barbules) reduce total reflectance to generate “super black” plumage, an optical illusion to enhance nearby color. Dihedral barbules paired with red carotenoid pigment produce “velvet red” plumage. Together, our results suggest that a widely cited index of honesty—carotenoid pigments—cannot fully explain male appearance. We propose that males are selected to evolve amplifiers of honest signals—in this case, microstructures that enhance appearance —that are not necessarily themselves linked to quality.

The functional state of the neurovascular unit (NVU), composed of the blood-brain barrier and the perivasculature that forms a dynamic interface between the blood and the central nervous system (CNS), plays a central role in the control of brain homeostasis and is strongly affected by CNS drugs. Human primary brain microvascular endothelium, astrocyte, pericyte, and neural cell cultures are often used to study NVU barrier functions as well as drug transport and efficacy; however, the proteomic and metabolomic responses of these different cell types are not well characterized. Culturing each cell type separately, using deep coverage proteomic analysis and characterization of the secreted metabolome, as well as measurements of mitochondrial activity, the responses of these cells under baseline conditions and when exposed to the NVU-impairing stimulant methamphetamine (Meth) are analyzed. These studies define the previously unknown metabolic and proteomic profiles of human brain pericytes and lead to improved characterization of the phenotype of each of the NVU cell types as well as cell-specific metabolic and proteomic responses to Meth.

Parental provisioning of offspring with physiological products occurs in many animals. Within amphibians, maternal provisioning has evolved multiple times, including in South American dendrobatid and Malagasy mantellid poison frogs. In some of these species, mothers feed unfertilized eggs to their developing tadpoles for several months until tadpoles complete metamorphosis. We conducted field studies in Ecuador and Madagascar to ask whether convergence at the behavioral level provides similar benefits to offspring and whether nursing behavior relies on shared neural mechanisms across frogs and vertebrates more broadly. At an ecological level, we found that nursing allows poison frog mothers to provide chemical defenses to their tadpoles in both species. At the level of brain regions, nursing behavior was associated with increased neural activity in the lateral septum and preoptic area in both species, demonstrating recruitment of shared brain regions in the convergent evolution of maternal care within frogs and across vertebrates. In contrast at a molecular level, only mantellids showed increased oxytocin neuron activity akin to that in nursing mammals. Taken together, our findings demonstrate that convergently evolved maternal provisioning behavior provides similar benefits to offspring and relies on similar brain regions. However, the molecular mechanisms underlying the convergence in nursing behavior may be different, suggesting evolutionary versatility in the mechanisms promoting maternal behavior.

Deep-sea hydrothermal vents are highly dynamic habitats characterized by steep temperature and chemical gradients. The oxidation of reduced compounds dissolved in the venting fluids fuels primary production providing the basis for extensive life. Until recently studies of microbial vent communities have focused primarily on chemolithoautotrophic organisms. In our study, we targeted the change of microbial community compositions along mixing gradients, focusing on distribution and capabilities of heterotrophic microorganisms. Samples were retrieved from different venting areas within the Menez Gwen hydrothermal field, taken along mixing gradients, including diffuse fluid discharge points, their immediate surroundings and the buoyant parts of hydrothermal plumes. High throughput 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization, and targeted metagenome analysis were combined with geochemical analyses. Close to diffuse venting orifices dominated by chemolithoautotrophic Epsilonproteobacteria, in areas where environmental conditions still supported chemolithoautotrophic processes, we detected microbial communities enriched for versatile heterotrophic Alpha- and Gammaproteobacteria. The potential for alkane degradation could be shown for several genera and yet uncultured clades. We propose that hotspots of chemolithoautotrophic life support a 'belt' of heterotrophic bacteria significantly different from the dominating oligotrophic microbiota of the deep sea.

Advanced bioproduct synthesis via reductive metabolism requires coordinating carbons, ATP and reducing agents, which are generated with varying efficiencies depending on metabolic pathways. Substrate mixtures with direct access to multiple pathways may optimally satisfy these biosynthetic requirements. However, native regulation favouring preferential use precludes cells from co-metabolizing multiple substrates. Here we explore mixed substrate metabolism and tailor pathway usage to synergistically stimulate carbon reduction. By controlled cofeeding of superior ATP and NADPH generators as ‘dopant’ substrates to cells primarily using inferior substrates, we circumvent catabolite repression and drive synergy in two divergent organisms. Glucose doping in Moorella thermoacetica stimulates CO2 reduction (2.3 g gCDW−1 h−1) into acetate by augmenting ATP synthesis via pyruvate kinase. Gluconate doping in Yarrowia lipolytica accelerates acetate-driven lipogenesis (0.046 g gCDW−1 h−1) by obligatory NADPH synthesis through the pentose cycle. Together, synergistic cofeeding produces CO2-derived lipids with 38% energy yield and demonstrates the potential to convert CO2 into advanced bioproducts. This work advances the systems-level control of metabolic networks and CO2 use, the most pressing and difficult reduction challenge. Bioproduct synthesis via reductive metabolism occurs with different efficiencies according to the availability of carbons, ATP and reducing agents. To maximize overall product synthesis efficiency, the authors develop a substrate cofeeding strategy, which circumvents catabolite repression and drives synergy in lipid synthesis from CO2 using two microbes.

Poison frogs acquire chemical defenses from the environment for protection against potential predators. These defensive chemicals are lipophilic alkaloid toxins that are sequestered by poison frogs from dietary arthropods and stored in skin glands. Despite decades of research focusing on identifying poison frog toxins, we know relatively little about how environmental variation and subsequent arthropod availability impacts toxicity in poison frogs. We investigated how seasonal environmental variation influences poison frog toxin profiles through changes in the diet of the Climbing Mantella (Mantella laevigata). We collected M. laevigata females on the Nosy Mangabe island reserve in Madagascar during the wet and dry seasons and tested the hypothesis that seasonal differences in rainfall is associated with changes in the diet and skin toxin profiles of M. laevigata. The arthropod diet of each frog was characterized into five groups (i.e. ants, termites, mites, insect larvae, or ‘other’) using visual identification and cytochrome oxidase 1 DNA barcoding. We found that frog diet differed between the wet and dry seasons, where frogs had a more diverse diet in the wet season and consumed a higher percentage of ants in the dry season. To determine if seasonality was associated with variation in frog defensive chemical composition, we used gas chromatography / mass spectrometry to quantify toxins from individual skin samples. Although the assortment of identified toxins was similar across seasons, we detected significant differences in the abundance of certain alkaloids, which we hypothesize reflects seasonal variation in the diet of M. laevigata. We suggest that these variations could originate from seasonal changes in either arthropod leaf litter composition or changes in frog behavioral patterns. Although additional studies are needed to understand the consequences of long-term environmental shifts, this work suggests that toxin profiles are relatively robust against short-term environmental perturbations.

Chemoresistance is the difference between remission and cure in cancer patients and is particularly evident in acute myeloid leukemia (AML) where complete remissions are common, but few are cured. Genetic analysis in patients points to residual founder clones persisting through therapy and ultimately causing relapse. However, most of these genetic alterations are currently not amenable to targeted therapies. We hypothesized that residual chemoresistant cells must pass through extreme metabolic challenges when under selection from chemotherapy and surrounded by massive cell death. Our goal was to define the distinctive metabolic profile of AML cells as they pass through that time of extreme selection pressure and determine whether metabolic pathway manipulation would reveal an exploitable vulnerability for therapy. We used in vivo imaging to define the kinetics of leukemic growth, response to chemotherapy and relapse in the mouse. We reasoned that the moment of maximal response immediately following chemotherapy represents a time when the metabolic milieu is maximally hostile and therefore a distinctive selection moment that the cells ultimately causing relapse must pass through. We captured cells at that time and conducted unbiased metabolic profiling, which revealed a metabolite profile that was distinct from AML cells during pre-chemo growth or late post-chemo relapse. Metabolite enrichment analysis revealed hyperactivation of glutamine metabolism during the extreme selection period, with chemoresistant cells showing high levels of glutamine, glutamate and aspartate. Inhibition of glutamine metabolism using the glutamine analog 6-diazo-5-oxo-L-norleucine specifically during the window of maximal selection pressure reduced the number of chemoresistant cells and improved survival in mouse models of AML. Unexpectedly, we found that residual AML cells were not sensitive to inhibition of the enzyme glutaminase, a target currently under clinical investigation for AML. In vivo metabolic tracer analysis instead revealed that chemoresistant AML cells mainly depend on glutamine nitrogen to fuel pyrimidine synthesis. Aspartate, another essential metabolite for pyrimidine synthesis, can be made from glutamine through the Krebs cycle. Interestingly, we detected almost no glutamine carbon entering the Krebs cycle in vivo even though labeled aspartate was found, indicating a different aspartate source. We found that aspartate levels in peripheral blood plasma are very low compared to those in bone marrow plasma, and explored the possibility of a local bone marrow source. Single cell RNA sequencing of bone marrow stromal cells (BMSC) revealed that CXCL12-positive BMSC express high levels of aspartate synthesis genes and transporters, which further increased in the presence of AML cells. In vitro co-cultures showed that BMSC convert glutamine to aspartate, and transfer this to AML cells. Genetic inhibition of aspartate synthesis in BMSC sensitized AML cells to chemotherapy, showing the importance of this metabolic crosstalk. We propose that induction of a timed metabolic collapse targeting AML cells both directly and indirectly through the bone marrow niche can prevent development of chemoresistance and improve treatment outcomes. Figure Disclosures Scadden: Novartis: Other: Sponsored research; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership; Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Few studies have directly measured sulfate reduction at hydrothermal vents, and relatively little is known about how environmental or ecological factors influence rates of sulfate reduction in vent environments. A better understanding of microbially mediated sulfate reduction in hydrothermal vent ecosystems may be achieved by integrating ecological and geochemical data with metabolic rate measurements. Here we present rates of microbially mediated sulfate reduction from three distinct hydrothermal vents in the Middle Valley vent field along the Juan de Fuca Ridge, as well as assessments of bacterial and archaeal diversity, estimates of total biomass and the abundance of functional genes related to sulfate reduction, and in situ geochemistry. Maximum rates of sulfate reduction occurred at 90 °C in all three deposits. Pyrosequencing and functional gene abundance data revealed differences in both biomass and community composition among sites, including differences in the abundance of known sulfate-reducing bacteria. The abundance of sequences for Thermodesulfovibro-like organisms and higher sulfate reduction rates at elevated temperatures suggests that Thermodesulfovibro-like organisms may have a role in sulfate reduction in warmer environments. The rates of sulfate reduction presented here suggest that--within anaerobic niches of hydrothermal deposits--heterotrophic sulfate reduction may be quite common and might contribute substantially to secondary productivity, underscoring the potential role of this process in both sulfur and carbon cycling at vents.

We studied the effect of a developing Skeletonema marinoi/Phaeocystis spp. bloom on Calanus finmarchicus hatching success, early naupliar survival and metabolism. Our focus was (1) on the development of reproductive rates during a bloom initiation, peak and decline in relation to the production of potentially toxic algal metabolites and (2) on the proportional importance of female nutrition versus naupliar food environment for the production of viable nauplii. Despite polyunsaturated aldehyde (PUA) production by both S. marinoi and Phaeocystis sp., we did not observe any harmful effects on hatching success or naupliar survival and condition in any stages of the short-term (

We introduce a rigorously validated protocol based on extraction, derivatisation and GC/MS for the analysis of diatom metabolomes. Using this methodology we characterised general patterns of the metabolism of the diatom Skeletonema marinoi during different growth phases. Canonical analysis of principal coordinate revealed clearly that the intracellular metabolites differ between exponential, stationary and declining phase. In addition, diurnal variation during the exponential phase was observed. A detailed analysis of the metabolic changes is presented and discussed in the context of previous physiological studies of diatoms. The observed variability in metabolites has a significant consequence for further physiological and ecological studies. Investigations have to take into account that diatom metabolism is a highly dynamic process and that food quality, chemical defence and also the production of signal molecules might be dependent on different growth phases or diurnal variations. The introduced protocol is in general suitable for the monitoring of microalgae and has also the potential to be applied to complex plankton communities.

Three strains of the chain-forming diatom Skeletonema marinoi, differing in their production of polyunsaturated aldehydes (PUA) and nutritional food components, were used in experiments on feeding, egg production, hatching success, pellet production, and behavior of three common planktonic copepods: Acartia tonsa, Pseudocalanus elongatus, and Temora longicornis. The three different diatom strains (9B, 1G, and 7J) induced widely different effects on Acartia tonsa physiology, and the 9B strain induced different effects for the three copepods. In contrast, different strains induced no or small alterations in the distribution, swimming behavior, and turning frequency of the copepods. 22:6(n-3) fatty acid (DHA) and sterol content of the diet typically showed a positive effect on either egg production (A. tonsa) or hatching success (P. elongatus), while other measured compounds (PUA, other long-chain polyunsaturated fatty acids) of the algae had no obvious effects. Our results demonstrate that differences between strains of a given diatom species can generate effects on copepod physiology, which are as large as those induced by different algae species or groups. This emphasizes the need to identify the specific characteristics of local diatoms together with the interacting effects of different mineral, biochemical, and toxic compounds and their potential implications on different copepod species.

Mild winters are modifying the plankton spring bloom composition so that diatoms are decreasing and dinoflagellates increasing. We used two common spring bloom phytoplankton species, a diatom and a dinoflagellate to study the effects of changing bloom composition on the reproduction of the calanoid copepod Acartia bifilosa Giesbrecht, a dominant species in the northern Baltic Sea. Egg production was significantly higher when copepods were fed with Scrippsiella hangoei (Schiller) Larsen dinoflagellates or a mixture of Scrippsiella and Skeletonema marinoi Sarno and Zingone diatoms than when they were provided with Skeletonema only. This effect was observed despite the fact that the Skeletonema strain did not produce polyunsaturated aldehydes (PUAs) and its nutritional quality was high according to polyunsaturated fatty acid (PUFAs) and sterol measurements, and moderate according to mineral (C:N and C:P) measurements. When offered mixtures of Skeletonema and Scrippsiella, copepods ingested both, even when the other one was rare. This indicates potential positive effect of multispecies diets not verified in this study. Here we show that increasing dinoflagellate:diatom ratio might have a positive effect on copepod reproduction.

Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.

Copepods dominate the biomass of marine zooplankton and through their prey selection they act as top-down regulators of planktonic communities. We investigated feeding preference of copepods in the presence of the diatom Skeletonema marinoi at different time points throughout the development of a bloom and a culture. Quantitative PCR gut content assessment revealed that the food uptake of the copepod Calanus spp. on mixed diets and on artificially induced mesocosm blooms was selective. Uptake of S. marinoi was highest during the post-bloom phase in the mesocosms even if the abundance of this alga was already low. In laboratory assays, copepods showed a greater preference for S. marinoi in the late stationary phase than for cultures of the same strain under exponentially growing culture conditions. The copepods thus discriminate between different growth phases of a single algal species in both laboratory and field settings. In parallel, we monitored cellular metabolites of the diatom using a metabolomic approach. Complex changes in the metabolic profile occur during development of a culture. Since no obvious effect of nutrient quality and cell size was involved, we suggest that changes in (info)chemicals within or surrounding S. marinoi regulate selective feeding by zooplankton.

We studied the patterns of waterborne metabolites released by diatom cultures during different growth phases. A carefully optimized enrichment procedure for metabolites from seawater medium facilitated the generation of large-scale sample sets that could be used for metabolic profiling. Samples from cartridge enrichments were directly submitted to ultraperformance liquid chromatography (UPLC) coupled with time-of-flight mass spectrometry (TOF-MS). Metabolic profiling revealed distinct patterns of metabolite release depending on the growth phases of the major bloom-forming diatoms Skeletonema marinoi and Thalassiosira pseudonana. Principal component analysis (PCA) enabled the recognition of specific metabolites that were only temporarily released by growing algal cultures. The exometabolome of the cells was complex and variable. The kinetics of metabolite release of some compounds resembles those of signal molecules involved in quorum sensing by bacteria. The introduced exometabolome investigation will help unravel the implications of release patterns of potential infochemicals for plankton ecology.

Polyunsaturated aldehydes (PUAs) have been extensively studied for their potential role in phytoplankton defence against grazers, but recently a potential infochemical role has been identified as well. We present here the first evidence of a synchronised release of PUAs from intact cells of a culture of the diatom Skeletonema marinoi. The release occurs transiently before the culture changes to the decline phase, providing a time-limited potential signal. When cultures are exposed during different stages of growth to PUAs in concentrations found in the medium, no general responses were found. However, a weak effect on cell concentration is observed exclusively when cultures in the late stationary phase are challenged with PUAs. Our results support for the first time the experimentally postulated hypothesis that PUA can play a role as infochemicals in mediating plankton interactions.

Deep-sea hydrothermal vents are highly dynamic habitats characterized by steep temperature and chemical gradients. The oxidation of reduced compounds dissolved in the venting fluids fuels primary production providing the basis for extensive life. Until recently studies of microbial vent communities have focused primarily on chemolithoautotrophic organisms. In our study, we targeted the change of microbial community compositions along mixing gradients, focusing on distribution and capabilities of heterotrophic microorganisms. Samples were retrieved from different venting areas within the Menez Gwen hydrothermal field, taken along mixing gradients, including diffuse fluid discharge points, their immediate surroundings and the buoyant parts of hydrothermal plumes. High throughput 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization, and targeted metagenome analysis were combined with geochemical analyses. Close to diffuse venting orifices dominated by chemolithoautotrophic Epsilonproteobacteria, in areas where environmental conditions still supported chemolithoautotrophic processes, we detected microbial communities enriched for versatile heterotrophic Alpha- and Gammaproteobacteria. The potential for alkane degradation could be shown for several genera and yet uncultured clades. We propose that hotspots of chemolithoautotrophic life support a 'belt' of heterotrophic bacteria significantly different from the dominating oligotrophic microbiota of the deep sea.

Degradation of unsaturated fatty acids through the peroxisomal β-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core β-oxidation cycle. The auxiliary enzyme Δ3,5,Δ2,4-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) Δ3,5,Δ2,4-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have Δ3,5,Δ2,4-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the β-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the Δ3,5,Δ2,4-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has Δ3,5,Δ2,4-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.

Oxidation-reduction reactions underlie energy generation in nearly all life forms. Although most organisms use soluble oxidants and reductants, some microbes can access solid-phase materials as electron-acceptors or -donors via extracellular electron transfer. Many studies have focused on the reduction of solid-phase oxidants. Far less is known about electron uptake via microbial extracellular electron transfer, and almost nothing is known about the associated mechanisms. Here we show that the iron-oxidizing photoautotroph Rhodopseudomonas palustris TIE-1 accepts electrons from a poised electrode, with carbon dioxide as the sole carbon source/electron acceptor. Both electron uptake and ruBisCo form I expression are stimulated by light. Electron uptake also occurs in the dark, uncoupled from photosynthesis. Notably, the pioABC operon, which encodes a protein system essential for photoautotrophic growth by ferrous iron oxidation, influences electron uptake. These data reveal a previously unknown metabolic versatility of photoferrotrophs to use extracellular electron transfer for electron uptake.

The capacity to make in situ geo-referenced measurements of methane concentration and stable isotopic composition (δ 13 CCH4) would greatly improve our under- standing of the distribution and type of methane sources in the environment, allow refined determination of the extent to which microbial production and consumption contributes to methane cycling, and enable the testing of hypotheses about the sensitivity of methane cycling to changes in environmental conditions. In particular, characterizing biogeochemical meth- ane cycling dynamics in the deep ocean is hampered by a number of challenges, especially in environments where high methane concentrations preclude intact recovery of undis- turbed samples. To that end, we have developed an in situ analyzer capable of δ 13 CCH4 measurements in the deep ocean. Here we present data from laboratory and field studies in which we characterize the instrument's analytical capabilities and performance and provide the first in situ stable isotope based characterization of the influence of anaerobic methane oxidation on methane flux from seep sediments. These data illustrate how in situ measurements can permit finer-scale analyses of variations in AOM activity, and facilitate advances in using δ 13 CCH4 and other isotopic systems to interrogate biogeochemical cycles in the deep sea and other remote or challenging environments.

Chemosynthetic primary production supports hydrothermal vent ecosystems, but the extent of that productivity and its governing factors have not been well constrained. To better understand anaerobic primary production within massive vent deposits, we conducted a series of incubations at 4, 25, 50 and 90 °C using aggregates recovered from hydrothermal vent structures. We documented in situ geochemistry, measured autochthonous organic carbon stable isotope ratios and assessed microbial community composition and functional gene abundances in three hydrothermal vent chimney structures from Middle Valley on the Juan de Fuca Ridge. Carbon fixation rates were greatest at lower temperatures and were comparable among chimneys. Stable isotope ratios of autochthonous organic carbon were consistent with the Calvin–Benson– Bassham cycle being the predominant mode of carbon fixation for all three chimneys. Chimneys exhibited marked differences in vent fluid geochemistry and microbial community composition, with structures being differentially dominated by gamma (c) or epsilon (e) proteobacteria. Similarly, qPCR analyses of functional genes representing different carbon fixation pathways showed striking differences in gene abundance among chimney structures. Carbon fixation rates showed no obvious correlation with observed in situ vent fluid geochemistry, community composition or functional gene abundance. Together, these data reveal that (i) net anaerobic carbon fixation rates among these chimneys are elevated at lower temperatures, (ii) clear differences in community composition and gene abundance exist among chimney structures, and (iii) tremendous spatial heterogeneity within these environments likely confounds efforts to relate the observed rates to in situ microbial and geochemical factors. We also posit that microbes typically thought to be mesophiles are likely active and growing at cooler temperatures, and that their activity at these temperatures comprises the majority of endolithic anaerobic primary production in hydrothermal vent chimneys.

Egg and faecal pellet production and egg hatching success of the calanoid copepod Calanus finmarchicus were monitored over a period of 14 days (14–28 April, 2008) while fed water from 4 differently treated mesocosms and ambient water. Two of the mesocosms used were inoculated with the polyunsaturated aldehyde (PUA)-producing diatom Skeletonema marinoi, while 2 received only nutrient additions with or without silica. The mesocosms developed blooms of S. marinoi, mixed diatoms or the haptophyte Phaeocystis pouchetii, respectively. Faecal pellet production of C. finmarchicus increased with increasing food availability. Egg production increased with time in all mesocosms to a maximum single female production of 232 eggs female−1 day−1 (average of 90 eggs female−1 day−1) and followed the development of ciliates and P. pouchetii, but was not affected by the observed high (up to 15 nmol L−1) PUA production potential of the phytoplankton. The hatching success of the eggs produced on the mesocosm diets was high (78–96%) and was not affected by either aldehydes in the maternal diet or exposure to the dissolved aldehydes in the water.

We surveyed the role of chemically mediated interactions between the 2 diatom species Skeletonema costatum and Thalassiosira weissflogii. S. costatum promoted the growth of T. weiss- flogii in non-contact co-cultures, allowing an exchange of exuded metabolites. In contrast, cell counts of S. costatum were not affected by T. weissflogii. Metabolic profiling of the co-culture medium revealed a significant change in diatom-derived metabolites in comparison with those of monoculture controls. Several compounds detected in monocultures were not present in the co-culturing set-up, indicating either a transformation or uptake of released metabolites by the competing species. In addition, metabolic profiling of intracellular metabolites revealed that the biochemical processes of both diatoms changed in the presence of the co-cultured species. The present study illustrates that chemical cross-talk between diatom species is possible and that these types of chemical interactions lead to physiological responses and might even result in changed cell abundances.

Diatoms have evolved a silicified cell wall that provides an efficient barrier against herbivores. These microalgae also produce chemical compounds such as polyunsaturated aldehydes (PUAs) that can potentially impair recruitment and cause malformations in the offspring of such grazers. We measured silica content as an indication of cell wall thickness, organic nutrient cell quotas, PUAs and polyunsaturated fatty acid cell content in Skeletonema marinoi grown under N-, P- and Si-limitation in continuous cultures. A 7.5 fold increase in PUA production was observed (27.5 fmol cell(-1)) in Si-limited cells with respect to the controls, while Si content decreased by 50%. PUA production decreased in nitrogen-limited cells to 0.14 fmol cell(-1) and increased 3 times in phosphorus-limited cells (7.53 fmol cell(-1)), while silica content increased by 20% in both cases. The substrate to product ratio suggested that production of the PUAs heptadienal and octadienal was limited by the amount of substrate under P- and Si-limitation, and by enzyme activity under N-limitation. Octatrienal production was likely limited by enzyme activity in all growth conditions. The high PUA levels produced under Si-limitation, when cells have a thinner cell wall, suggests a compensatory alternation of mechanical and chemical defense mechanisms.

Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom.