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Hernandez, V.

Publications and source records attributed to Hernandez, V..

At least 19 recordsLinked to original sources

Deconvolution of the mechanisms of T cell drug response in multiple myeloma induction therapy

Lenalidomide (Revlimid), bortezomib (Velcade), and dexamethasone, are used alone or in combination (RVd therapy) as first-line therapies for hematologic malignancies including the plasma cell dyscrasia, Multiple Myeloma (MM). The effects of RVd treatment on tumor cells have been documented, but little is known about their impacts on "healthy" immune cells. New therapeutics, including chimeric antigen receptor T cell (CAR-T) and bispecific T-cell engagers (BiTEs) depend on robust T cell function but these are often harvested for cellular therapies after or in tandem with RVd therapy. Understanding the molecular effects of these drugs on healthy T cells is therefore of particular importance. Since the molecular effects of each drug includes regulation of key transcription factors, we used a tri-modal, single-cell assay (TEA-seq), simultaneously profiling mRNA transcripts, cell surface proteins, and chromatin accessibility in primary human peripheral blood T cells treated for 4, 24, or 72 hours in vitro. Synergies and conflicts potentially arising from combinatorial therapy were identified and validated by flow cytometry. Doublet and triplet drug combinations were used to assess effects on cell-surface marker expression and on T cell activation. Our results suggest that T cell function can be optimized by the administration of these drugs individually and that sequential administration may offer an opportunity to enhance T cell function by affecting localization and altering T cell states. This work provides those interested in immunology, oncology, and cell therapies an opportunity to explore molecular pathways that could be manipulated by these commonly used drugs to modify T cell function.

immunology↗

PACAPergic BAC engages basolateral amygdalar and anterodorsal thalamic networks during looming-threat learning and memory in a labyrinth

The bed nucleus of the anterior commissure (BAC) is a small pituitary adenylate cyclase-activating polypeptide (PACAP)-rich glutamatergic cell population located at the intersection of the anterior commissure, stria terminalis, stria medullaris, and fornix. Here, we provide functional neuroanatomical characterization of the BAC using PACAP-Cre mice and Cre-dependent viral tracing. BAC axons traverse these major forebrain conduits to engage distributed limbic and diencephalic networks, with dense innervation of the posterior basolateral amygdala (pBLA) and anterior dorsal thalamic nucleus (AD), both implicated in emotional processing and spatial orientation. To determine the functional significance of the BAC, we developed a novel looming-threat memory test (LTMT) paradigm in which mice learned the location of a shelter within a dual-maze labyrinth before exposure to an overhead looming stimulus. Chemogenetic inhibition of BAC PACAP neurons impaired shelter-directed escape, increased freezing behavior, prolonged escape trajectories, and disrupted efficient safety-seeking responses. Selective deletion of PACAP from BAC neurons also blocked key aspects of the looming-induced behavioral phenotype. Fos mapping revealed robust neuronal activation by the looming stimulus, within the AD, pBLA, and dorsal periaqueductal gray (dPAG). BAC silencing blocked Fos induction. PACAPBAC depletion blocked fos induction in AD, but not in pBLA or dPAG. Preliminary ex-vivo recordings further indicated that PACAP modulates the intrinsic excitability of AD (and pBLA) neurons. Together, these findings identify the BAC as a previously unrecognized PACAPergic forebrain hub that links emotional and spatial-orientation networks to coordinate looming-threat learning, memory, and adaptive defensive behavior.

neuroscience↗

Multi-omic profiling of human antibody-secreting cells reveals diverse subsets sustain durable humoral immunity

Antibody-secreting cells (ASCs) provide humoral immunity that can mediate lifelong protection against pathogens. Current classifications cannot delineate the heterogenous functionalities, tissue residencies, and lifespans of human ASC subsets, impeding clinical translation. We applied multi-omic sequencing, spatial proteomics, and functional assays to discover and characterize human bone marrow (BM) ASC subsets. We identified two peripheral subsets (ASCp) also present in blood and three BM-resident subsets (ASCr), comprising a maturation continuum associated with increased mitochondrial networking, diminished antibody secretion, differential transcription factor motif accessibility, and preferential co-localization in homotypic niches. CD19+9+ASCr and CD19-ASCr exhibited poor recovery years after BM transplantation, indicating a strong dependence on supportive niches. Childhood vaccine antigens were recognized by long-lived ASCr subsets in adults and by immature HLA-DR+ASCp, implying ASCs can differentiate without recent antigen exposure. Our results provide new insights into ASC identity, maturation, and longevity and a generalizable framework for study and manipulation of human ASCs.

immunology↗

Myeloma and therapy reshape the bone marrow niche to durably constrain immune reconstitution and vaccine responsiveness

Infections are the most common cause of non-relapse mortality in multiple myeloma (MM), but the basis of persistent immune dysfunction is obscured by patient heterogeneity and complex treatment regimens, including autologous stem cell transplant (ASCT). We performed longitudinal multi-omic profiling of matched bone marrow and peripheral blood from MM patients across diagnosis, induction, ASCT, and recovery. We found the tumor imposes a compartment-specific immune program where the marrow exhibits metabolic and inflammatory changes that bias hematopoiesis and alter cytotoxic effector programs not mirrored in blood. Adaptive immune reconstitution is impaired up to two years post-ASCT. Half of patients fail to mount IgG responses to high-dose non-adjuvanted influenza vaccine, a defect overcome by the lipid nanoparticle (LNP) adjuvanted COVID mRNA vaccine, which elicited responses in all patients, supporting adjuvanted influenza vaccine strategies in MM. Together these findings define how myeloma and its treatment durably reshape immunity from the marrow outward. HighlightsO_LIMultiple Myeloma marrow and blood show opposing metabolic and inflammatory states C_LIO_LIInduction therapy selects durable myeloma plasma-cell transcriptional states C_LIO_LIB cell and follicular helper T deficits blunt antigen responses after transplant C_LIO_LICOVID-19 vaccination builds immune memory with variable responses to flu vaccination C_LI eTOCMultiple myeloma and its treatment leave a lasting imprint on the bone marrow niche. By profiling bone marrow and blood longitudinally at diagnosis, through induction, autologous transplant, and recovery, we show that marrow-local metabolic and inflammatory constraints persist and help explain why influenza vaccination often fails while mRNA vaccination succeeds.

cancer biology↗

Analysis of the spatiotemporal dynamics of behavior in domestic dogs with free locomotion under appetitive Pavlovian contingencies

Spatiotemporal dynamics of behavior is key to understanding the organism-environment relationship. While often implicitly addressed, its relation to Pavlovian contingencies remains understudied in domestic dogs. This study quantitatively examined the spatial dimension of behavior of three freely moving dogs under pairing and extinction of a tone-food Pavlovian contingency. In pairing, a tone (CS) was paired with food delivery (US) on a fixed-time 60-second (FT 60 s) schedule; in extinction, only the tone was presented. Locomotion was recorded using two-dimensional tracking based on center of mass. During pairing, dogs moved closer to the dispenser, covered greater distances, displayed extended trajectories, and showed a conditional approach pattern to the dispenser during CS presentation. In extinction, they stayed closer to the owner or room periphery, traveled shorter distances, and exhibited more restricted trajectories. These findings show that spatial segments (dispenser area) are integrated into the CS-US relationship, demonstrate the usefulness of continuous recording of spatial behavior in the analysis of Pavlovian contingencies, and suggest potential application in contexts relevant to animal welfare. HighlightsO_LISpatial behavior remains largely unexplored in studies of Pavlovian contingencies C_LIO_LIDog locomotion was continuously tracked during pairing and extinction phases C_LIO_LIDistinct spatial patterns emerged across pairing and extinction phases C_LIO_LIIncorporating space into CS-US relations clarifies behavioral organization C_LIO_LITracking tools expand the experimental and applied scope of Pavlovian research C_LI

animal behavior and cognition↗

Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

Choline is a methyl-rich nutrient used in lipid synthesis and catabolized to support one-carbon metabolism. Choline consumption in most humans remains less than the suggested adequate intake, yet how systemic metabolism compensates for choline deficiency is not fully described. Here, we use in vivo stable isotope tracing to explore the fate of choline in mammalian tissues. We find that choline is catabolized in the liver to support both the methionine cycle and the mitochondrial folate cycle in addition to its role in lipid synthesis. When dietary methyl donors are deficient, surprisingly, we find maintained systemic choline and methionine fluxes, but diminished contribution of choline to the folate cycle. To compensate for dietary methyl deficiency, systemic flux of serine is doubled by increased kidney synthesis which supplies one-carbon units for increased methionine synthesis in the liver. Our study suggests that systemic one-carbon flexibility can compensate for nutritional methyl deficiency by inter-organ nutrient exchange.

physiology↗

Novel multi-transmitter calyx-like giant synapse revealed in the extended amygdala

We have discovered a highly specialized innervation of the forebrain by pituitary adenylate cyclase activating polypeptide (PACAP) immunohistochemistry originating from the brain stem that uses glutamate, acetylcholine and PACAP and other peptides as neurotransmitters. The parent neurons of the axons are in the Kolliker-Fuse nucleus and their terminals form calyx-like multi-release site synapses in the rodent forebrain extended amygdala similar to the calyx of Held in the auditory brain stem. The latter is a giant, excitatory, cup-like axo-somatic high-fidelity synapse. The PACAP-positive terminals also form enveloping axo-somatic specialization with mixed glutamatergic and cholinergic molecular identities, co-expressing VGluT1, VGluT2, VAChT, and the neuropeptides PACAP, CGRP, and neurotensin, together with calretinin in the presynaptic compartment. We identified a distinct neuronal subpopulation in the pontine Kolliker-Fuse region of the parabrachial complex that gives rise to these calyceal terminals, which engulf PKC/GluD1 somata in the capsular central amygdala and oval bed nucleus of the stria terminalis. Strikingly, GluD1 immunolabeling is concentrated at axo-somatic contact zones apposed to VAChT presynaptic vesicle clusters zones but is absent from postsynaptic densities of conventional type I synapses within the same terminals. The results demonstrate a previously unrecognized multimodal calyx-like synapse in the forebrain, with parallel fast ionotropic and modulatory peptidergic neurotransmission mechanisms, which is a substrate for high-fidelity signal transmission within viscerosensory -emotional circuits. Significance StatementA high-fidelity axo-somatic glutamatergic synapse is exemplified by the calyx of Held in the auditory brainstem, but is unknown in the forebrain. Here we reveal a multimodal calyceal synapse in the extended amygdala formed by glutamatergic afferents of the pontine Kolliker-Fuse nucleus that contain also several neuropeptides. These giant terminals co-package glutamate, acetylcholine, PACAP, CGRP and neurotensin. They envelop select PKC/GluD1 neurons in the capsular central amygdala and oval BNST. These unique forebrain synapses link viscerosensory input from the hindbrain to limbic autonomic centers. Such high-fidelity multimodal transmission is likely to underlie the precise synchronization of emotional and homeostatic control circuits.

neuroscience↗

Multi-lineage transcriptional and cell communication signatures define pathways in individuals at-risk for developing rheumatoid arthritis that initiate and perpetuate disease

Rheumatoid arthritis (RA) is a systemic autoimmune disease arising from loss of tolerance and autoantibody development in at-risk individuals. Targeted therapies yield variable responses and current preventive strategies delay but do not stop disease onset. We propose that a set of transcription factors (TFs) and their downstream pathways regulate inflammatory cell communication networks in at-risk populations and RA. These networks enable multiple pathogenic cell types and mediators and could account for variable responses to targeted agents. To test this hypothesis, we identified anti-citrullinated protein antibody (ACPA)-positive at-risk individuals, patients with early and established RA and healthy controls. Single cell chromatin accessibility and transcriptomic profiles from blood mononuclear r cells were integrated and identified share pathogenic mechanisms, especially SUMOylation, RUNX2, YAP1, NOTCH3, and {beta}-Catenin Pathways. Surprisingly, this signature was found in multiple cell types. Individualized gene expression patterns were then confirmed in RA synovium. Cell communication analysis revealed that multiple lineages can deliver a core set of pro-inflammatory mediators to receiver cells. Longitudinal analysis showed that the signature cell types evolve in individual at-risk participants. Cell-type-specific signature pathways could contribute to the differing clinical responses to targeted therapies. This study describes how a common clinical phenotype could arise from multiple pathogenic mechanisms.

systems biology↗

Movement path as an ethological lens into interval timing

Interval timing behavior is traditionally investigated in operant chambers based on the very focal responses of the subjects (e.g., head entry to the magazine, lever press). These measures are blind to the movement trajectory of the animals and capture only a tiny segment and some-times an idiosyncratic component of the animals behavior. In other words, the state of the temporal expectancy is not observable at every time point in the trial. On the other hand, in nature, temporal expectancies guide actions in a much more complex fashion. For instance, an animal might approach a food patch at different degrees as a function of the expected time of food availability (e.g., nectar collection behavior). The current study aimed to investigate interval timing in a more naturalistic fashion by analyzing the movement trajectory of rats in fixed time (FT-30s) vs. variable time (VT-30s) schedules in modified open field equipment. We observed a temporally patterned movement in FT but not in a VT schedule. In the FT schedule, rats moved away from the reward grid after consuming the presented water and were farthest from it for around 15 seconds, after which they started to approach the reward grid again. There was no such temporal patterning of movement trajectory in the VT schedule. Temporal control in the FT schedule was stronger in the Wall compared to the Center condition. Our results show that movement trajectory may reflect instantaneous temporal expectancy.

animal behavior and cognition↗

Systemic inflammation and lymphocyte activation precede rheumatoid arthritis

Some autoimmune diseases, including rheumatoid arthritis (RA), are preceded by a critical subclinical phase of disease activity. Proactive clinical management is hampered by a lack of biological understanding of this subclinical at-risk state and the changes underlying disease development. In a cross-sectional and longitudinal multi-omics study of peripheral immunity in the autoantibody-positive at-risk for RA period, we identified systemic inflammation, proinflammatory-skewed B cells, expanded Tfh17-like cells, epigenetic bias in naive T cells, TNF+IL1B+ monocytes resembling a synovial macrophage population, and CD4 T cell transcriptional features resembling those suppressed by abatacept (CTLA4-Ig) in RA patients. Our findings characterize pathogenesis prior to clinical diagnosis and suggest the at-risk state exhibits substantial immune alterations that could potentially be targeted for early intervention to delay or prevent autoimmunity. We provide a suite of tools at https://apps.allenimmunology.org/aifi/insights/ra-progression/ to facilitate exploration and enhance accessibility of this extensive dataset. One Sentence SummaryACPA+ at-risk individuals show RA-like inflammation and multi-compartment immune dysregulation during transition to clinically active RA

immunology↗

Damage-induced IL-18 stimulates thymic NK Cells limiting endogenous tissue regeneration

Interleukin-18 is an acute phase pro-inflammatory molecule crucial for mediating viral clearance by activating Th1 CD4+, cytotoxic CD8+ T, and NK cells. Here, we show that mature IL-18 is generated in the thymus following numerous distinct forms of tissue damage, all of which cause caspase-1-mediated immunogenic cell death. We report that IL-18 stimulated cytotoxic NK cells limit endogenous thymic regeneration, a critical process that ensures restoration of immune competence after acute insults like stress, infection, chemotherapy, and radiation. NK cells suppressed thymus recovery by aberrantly targeting thymic epithelial cells (TECs), which act as the master regulators of organ function and regeneration. Together these studies reveal a novel pathway regulating tissue regeneration in the thymus and offer IL-18 as a potential therapeutic target to boost thymic function. Moreover, given the enthusiasm for IL-18 as a cancer immunotherapy for its capacity to elicit a type-1 immune response, these findings also offer insight into potential off-target effects.

immunology↗

Longitudinal Multi-omic Immune Profiling Reveals Age-Related Immune Cell Dynamics in Healthy Adults

The generation and maintenance of protective immunity is a dynamic interplay between host and environment that is impacted by age. Understanding fundamental changes in the healthy immune system that occur over a lifespan is critical in developing interventions for age-related susceptibility to infections and diseases. Here, we use multi-omic profiling (scRNA-seq, proteomics, flow cytometry) to examined human peripheral immunity in over 300 healthy adults, with 96 young and older adults followed over two years with yearly vaccination. The resulting resource includes scRNA-seq datasets of >16 million PBMCs, interrogating 71 immune cell subsets from our new Immune Health Atlas. This study allows unique insights into the composition and transcriptional state of immune cells at homeostasis, with vaccine perturbation, and across age. We find that T cells specifically accumulate age-related transcriptional changes more than other immune cells, independent from inflammation and chronic perturbation. Moreover, impaired memory B cell responses to vaccination are linked to a Th2-like state shift in older adults memory CD4 T cells, revealing possible mechanisms of immune dysregulation during healthy human aging. This extensive resource is provided with a suite of exploration tools at https://apps.allenimmunology.org/aifi/insights/dynamics-imm-health-age/ to enhance data accessibility and further the understanding of immune health across age.

immunology↗

Binding profiles for 954 Drosophila and C. elegans transcription factors revealtissue specific regulatory relationships

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here we present the culmination of the modERN (model organism Encyclopedia of Regulatory Networks) consortium that systematically assayed TF binding events in vivo in two major model organisms, Drosophila melanogaster (fly) and Caenorhabditis elegans (worm). We describe key features of these datasets, comprising 604 TFs identifying 3.6M sites in the fly and 350 TFs identifying 0.9 M sites in the worm. Applying a machine learning model to these data identifies sets of TFs with a prominent role in promoting target gene expression in specific cell types. TF binding data are available through the ENCODE Data Coordinating Center and at https://epic.gs.washington.edu/modERNresource, which provides access to processed and summary data, as well as widgets to probe cell type-specific TF-target relationships. These data are a rich resource that should fuel investigations into TF function during development.

genomics↗

De-suppression of mesenchymal cell identities and variable phenotypic outcomes associated with knockout of Bbs1.

Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue specific, and gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane trafficking and intraflagellar transport (IFT) adaptor protein complex formed by 8 BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically-defined clones. Our results suggest that BBS1 is required for suppression of mesenchymal cell identities as IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, and BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively this work suggests that the dynamic stability of the BBSome is essential for suppression of mesenchymal cell identities as epithelial cells differentiate.

genetics↗

Effects of Aerobic Exercise in Hepatic Lipid Droplet-Mitochondria interaction in Non-alcoholic Fatty Liver Disease

Lipid Droplets (LD) are highly dynamic storage organelles. In the liver, its accumulation causes non-alcoholic fatty liver (NAFL) that can progress to a more severe disease stage, nonalcoholic steatohepatitis (NASH). In hepatic and non-hepatic tissues LD interacts with mitochondria impacting lipid homeostasis. However, whether exercise modulates this interaction in the liver has not been studied yet. Our objective is to determine whether exercise modifies LD-mitochondria interaction in hepatocytes and if this interaction has an association with the severity of the disease. Two different models of NAFLD, a high fat diet (HFD) to evaluate NAFL and a methionine choline deficient diet (MCD) to evaluate NASH, were used to analyze the effects of aerobic exercise in the liver. Our results in the NAFL model showed that exercise decreased the severity of the disease and improved physical capacity compared to sedentary HFD mice. In this regard, although exercise increased the number of LD in hepatocytes, LD were smaller in size than in the sedentary HFD mice. Notably, while sedentary HFD mice increased hepatic lipid droplet (LD)-mitochondria interaction, in exercised animals, this interaction was decreased. Additionally, exercise decreased the size of the LD bound to mitochondria, and this peridroplet mitochondria (PDM) exhibited higher basal respiration and ATP synthesis capacity than PDM from sedentary HFD mice. Besides, we found a positive correlation that predicts the severity of NAFL between LD-mitochondria interaction in the liver and plasmatic ALT transaminases. This correlation is also positive between hepatic LD-mitochondria interaction and the area under the glucose tolerance test curve in this model. Our results in the NASH model resemble, to a greater extent, what we observed in the NAFL model. In NASH, exercise also reduced collagen accumulation, decreased LD-mitochondria interaction, and reduced the size of LD coupled to mitochondria compared to sedentary MCD mice. In all, our results show that aerobic exercise decreases LD-mitochondria interaction in hepatocytes and this interaction is associated with less severity of NAFL and NASH. We propose that exercise provokes an improvement of NAFLD by reduction of the hepatic LD-mitochondria interaction that in turn increase peridroplet mitochondria activity. HighlightsO_LILipid droplet (LD)-mitochondria interaction is increased in high-fat diet-induced NAFLD and choline-methionine deficient diet induced-NASH. C_LIO_LIExercise decreased LD-mitochondria interaction and is associated with reduced plasmatic ALT transaminase levels and glucose tolerance test in HFD induced-NAFLD. C_LIO_LIExercise decreases LD-mitochondria interaction, decreasing peridroplet Mitochondria (PDM) with possible lipogenic function, which induces a decrease in the LD bound to mitochondria (M-LD) in HFD-induced NAFLD. C_LIO_LIExercise decreased LD-mitochondria interaction and collagen accumulation in MCD induced-NASH. C_LI

physiology↗

Distinct Heterogeneity in the Naive T cell Compartments of Children and Adults

AbstractThe naive T cell compartment undergoes multiple changes across age that associate with altered susceptibility to infection and autoimmunity. In addition to the acquisition of naive-like memory T cell subsets, mouse studies describe substantial molecular reprogramming of the naive compartment in adults compared with adolescents. However, these alterations are not well delineated in human aging. Using a new trimodal single cell technology (TEA-seq), we discovered that the composition and transcriptional and epigenetic programming of the naive T cell compartment in children (11-13 yrs) is distinct from that of older adults (55-65 yrs). Naive CD4 T cells, previously considered relatively resistant to aging, exhibited far more pronounced molecular reprogramming than naive CD8 T cells, in which alterations are preferentially driven by shifts in naive-like memory subsets. These data reveal the complex nature of the naive T cell compartment that may contribute to differential immune responses across the spectrum of human age. One Sentence SummaryThe naive CD8 and CD4 T cell compartments in humans are heterogeneous and impacted differently with age, in which naive CD8 T cell subsets dramatically shift in composition and true naive CD4 T cells display significant molecular re-programming.

immunology↗

Beyond single discrete responses: An integrative and multidimensional analysis of behavioral dynamics assisted by Machine Learning

Behavioral systems, understanding it as an emergent system comprising the environment and organism subsystems, include spatial dynamics as a primary dimension in natural settings. Nevertheless, under the standard approaches, the experimental analysis of behavior is based on the single response paradigm and the temporal distribution of discrete responses. Thus, the continuous analysis of spatial behavioral dynamics has been a scarcely studied field. The technological advancements in computer vision have opened new methodological perspectives for the continuous sensing of spatial behavior. With the application of such advancements, recent studies suggest that there are multiple features embedded in the spatial dynamics of behavior, such as entropy, and that they are affected by programmed stimuli (e.g., schedules of reinforcement), at least, as much as features related to discrete responses. Despite the progress, the characterization of behavioral systems is still segmented, and integrated data analysis and representations between discrete responses and continuous spatial behavior are exiguous in the Experimental Analysis of Behavior. Machine Learning advancements, such as t-SNE, variable ranking, provide invaluable tools to crystallize an integrated approach for analyzing and representing multidimensional behavioral data. Under this rationale, the present work: 1) proposes a multidisciplinary approach for the integrative and multilevel analysis of behavioral systems, 2) provides sensitive behavioral measures based on spatial dynamics and helpful data representations to study behavioral systems, and 3) reveals behavioral aspects usually ignored under the standard approaches in the experimental analysis of behavior. To exemplify and evaluate our approach, the spatial dynamics embedded in phenomena relevant to behavioral science, namely water-seeking behavior, and motivational operations, are examined, showing aspects of behavioral systems hidden until now.

animal behavior and cognition↗

Sterol 14α-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major

Sterol 14--demethylase (C14DM) is a key enzyme in the biosynthesis of sterols and the primary target of azoles. In Leishmania major, genetic or chemical inactivation of C14DM leads to accumulation of 14-methylated sterol intermediates and profound plasma membrane abnormalities including increased fluidity and failure to maintain ordered membrane microdomains. These defects likely contribute to the hypersensitivity to heat and severely reduced virulence displayed by the C14DM-null mutants (c14dm-). In addition to plasma membrane, sterols are present in intracellular organelles. In this study, we investigated the impact of C14DM ablation on mitochondria. Our results demonstrate that c14dm- mutants have significantly higher mitochondrial membrane potential than wild type parasites. Such high potential leads to the buildup of reactive oxygen species in the mitochondria, especially under nutrient-limiting conditions. Consistent with these mitochondrial alterations, c14dm- mutants show impairment in respiration and are heavily dependent on glucose uptake and glycolysis to generate energy. Consequently, these mutants are extremely sensitive to glucose deprivation and such vulnerability can be rescued through the supplementation of glucose or glycerol. In addition, the accumulation of oxidants may also contribute to the heat sensitivity exhibited by c14dm-. Finally, genetic or chemical ablation of C14DM causes increased susceptibility to pentamidine, an antimicrobial agent with activity against trypanosomatids. In summary, our investigation reveals that alteration of sterol synthesis can negatively affect multiple cellular processes in Leishmania parasites and make them vulnerable to clinically relevant stress conditions. AUTHOR SUMMARYSterols are well recognized for their stabilizing effects on the plasma membrane, but their functions in intracellular organelles are under explored, which hampers the development of sterol synthesis inhibitors as drugs. Our previous studies have demonstrated significant plasma membrane instability in the sterol biosynthetic mutant c14dm- in Leishmania major, a pathogenic protozoan responsible for cutaneous leishmaniasis causing 1-1.5 million infections a year. While the plasma membrane defects have undoubtedly contributed to the reduced virulence exhibited by c14dm- mutants, it was not clear whether other cellular processes were also affected. In this study, we revealed profound mitochondrial dysfunctions and elevated level of reactive oxygen species in c14dm- mutants. These sterol mutants rely heavily on glycolysis to generate energy and are extremely sensitive to glucose restriction. In addition, the accumulation of oxidants appears to be responsible (at least in part) for the previously observed heat sensitivity in c14dm- mutants. Thus, genetic or chemical inactivation of C14DM can influence the functions of cellular organelles beyond the plasma membrane. These findings shed light on the mechanism of action for azole compounds and provide new insight into the roles of sterol biosynthesis in Leishmania parasites.

microbiology↗