Biology subjects

Hernandez, A.

Publications and source records attributed to Hernandez, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Cancer associated fibroblasts drive epithelial to mesenchymal transition and classical to basal change in pancreatic ductal adenocarcinoma cells with loss of IL-8 expression

Pancreatic ductal adenocarcinoma (PDAC) carries an extremely poor prognosis, in part resulting from cellular heterogeneity that supports overall tumorigenicity. Cancer associated fibroblasts (CAF) are key determinants of PDAC biology and response to systemic therapy. While CAF subtypes have been defined, the effects of patient-specific CAF heterogeneity and plasticity on tumor cell behavior remain unclear. Here, multi-omics was used to characterize the tumor microenvironment (TME) in tumors from patients undergoing curative-intent surgery for PDAC. In these same patients, matched tumor organoid and CAF lines were established to functionally validate the impact of CAFs on the tumor cells. CAFs were found to drive epithelial-mesenchymal transition (EMT) and a switch in tumor cell classificiaton from classical to basal subtype. Furthermore, we identified CAF-specific interleukin 8 (IL-8) as an important modulator of tumor cell subtype. Finally, we defined neighborhood relationships between tumor cell and T cell subsets. Statement of SignificanceThis multidimensional analysis highlights the diverse role CAFs have in influencing other cell types in the TME, including epithelial-derived tumor and infiltrating immune cells. Our methods provide a platform for evaluating emerging therapeutic approaches and for studying mechanisms that dictate tumor behavior in a manner that reflects patient-specific heterogeneity.

cancer biology↗

bioRxiv2024

Fecal Metabolite Quantitation for Rapid Assessment of the Gut Microbiome

The intestinal microbiome is composed of myriad microbial species that produce metabolites that impact host health. While loss of bacterial species and beneficial metabolites from the fecal microbiome is associated with development of a range of diseases and medical complications, there are currently no diagnostic tests that rapidly identify individuals with microbiome deficiencies. Reduced concentrations of fecal butyrate and deoxycholic acid are associated with adverse clinical outcomes and result from the loss of a subset of health-associated bacterial species. We present a rapid diagnostic test based on 3-nitrophenylhydrazine derivatization and ultrahigh-performance liquid chromatography-mass spectrometry that measures fecal butyrate and deoxycholic acid concentrations as markers of microbiome function. A matrix-matched calibration curve was developed using a simulated fecal mixture to optimize accuracy and facilitate adherence to clinical laboratory regulations. The assay showed an analytical measurement range from 4.3-3030.1 {micro}M (LLOQ = 9.75 {micro}M) for butyrate and from 0.9- 64.9 {micro}M (LLOQ = 0.9 {micro}M) for deoxycholic acid. Precision demonstrated a coefficient of variation <15% at all QC levels tested. The assay can be performed in under an hour from extraction to provision of quantitative results, enabling the rapid identification of patients with defective microbiome function.

pathology↗

bioRxiv2024

Genomic, phenotypic and environmental correlates of speciation in the midwife toads (Alytes)

Speciation, i.e., the formation of new species, implies that diverging populations evolve genetic and phenotypic factors that promote reproductive isolation (RI), but the adaptive vs. neutral origin of these factors and their relative contributions across the speciation continuum remain elusive. Here we test which of genomic, bioacoustic, morphological and environmental differentiation best predicts RI across the midwife toads (genus Alytes), a diversification of Mediterranean amphibians. Hybrid zone analyses in the A. obstetricans complex support that without strong geographic barriers to dispersal, the extent of introgression (which should reflect the strength of RI) covaries with genomic divergence irrespective of other factors. Phenotypic divergence become important later along the continuum, namely between non-admixing species attributed to distinct subgenera. Our results suggest that by putatively causing intrinsic incompatibilities in hybrids, the genetic mutations accumulating randomly between allopatric populations act as the initial trigger of RI, while substantial ecological and behavioral differentiation is a long-term consequence of species divergence that ultimately promotes sympatry. Whereas speciation is usually claimed to be primarily adaptive, our study corroborates recent findings that new species may also be a neutral outcome of gradual phylogeographic divergence, which has practical implications for species delimitation.

evolutionary biology↗

bioRxiv2024

Characterization and Antiatherogenic Potential of P3R99 Monoclonal Antibody Against Sulfated Glycosaminoglycans: Physicochemical and Functional Insights

Atherosclerosis is initiated by the retention of ApoB-containing lipoproteins in the arterial wall, mediated by glycosaminoglycan chains of proteoglycans. At the Center for Molecular Immunology, we are developing the P3R99 monoclonal antibody (mAb) to target this process. This study characterizes new P3R99 mAb variants expressed in CHO-K1 and HEK-293 cell lines. We compared these variants with the parental mAb from NS0 cells using SDS-PAGE, size exclusion and cation exchange chromatography, dynamic light scattering, peptide mapping, far-UV circular dichroism, and PNGase F deglycosylation. All variants exhibited a molecular size of [~]150 kDa, [~]99% purity, and similar average particle sizes (12.5-13.7 nm). They displayed a high {beta}-sheet content (>40%) and basic amino acids on the surface, with minor differences in peptide maps compared to the parental mAb. Notable differences were found in the content of acidic and basic species and glycosylation profiles. NS0-derived P3R99 had lower G0F content (10.39%), higher G1F (38.29%) and G2F (30.44%) levels, with more terminal galactose (83.07%) and sialylation (15.33%). In contrast, CHO-K1 and HEK-293 variants showed similar glycosylation patterns. Despite these differences, the antigen and atherosclerotic lesion recognition properties of the mAb were unaffected in vitro. Biodistribution studies in Sprague Dawley rats (1 mg, IV, n=3) revealed preferential accumulation of the new P3R99 variants in aortas and reduced LDL arterial retention (1 mg, IP). Passive administration of the mAbs (2 mg every three days, three IV doses, n=6-7) in a Lipofundin 20%-induced atherosclerosis NZW rabbit model also demonstrated preferential accumulation in aortas and reduced atherosclerosis, with 60% of treated rabbits not developing lesions. These results suggest that the P3R99 mAb derived from CHO-K1 and HEK-293 cells retains its antiatherogenic properties despite structural differences from the NS0-derived mAb associated with the different expression systems.

immunology↗

bioRxiv2024

A failure to discriminate social from non-social touch at the circuit level may underlie social avoidance in autism

Social touch is critical for communication and to impart emotions and intentions. However, certain autistic individuals experience aversion to social touch, especially when it is unwanted. We used a novel social touch assay and Neuropixels probes to compare neural responses to social vs. non-social interactions in three relevant brain regions: vibrissal somatosensory cortex, tail of striatum, and basolateral amygdala. We find that wild type (WT) mice showed aversion to repeated presentations of an inanimate object but not of another mouse. Cortical neurons cared most about touch context (social vs. object) and showed a preference for social interactions, while striatal neurons changed their preference depending on whether mice could choose or not to interact. Amygdalar and striatal neurons were preferentially modulated by forced object touch, which was the most aversive. In contrast, the Fmr1 knockout (KO) model of autism found social and non-social interactions equally aversive and displayed more aversive facial expressions to social touch when it invaded their personal space. Importantly, when Fmr1 KO mice could choose to interact, neurons in all three regions did not discriminate social valence. Thus, a failure to differentially encode social from non-social stimuli at the circuit level may underlie social avoidance in autism.

neuroscience↗

bioRxiv2024

Impacts of Perinatal Nicotine Exposure on nAChR Expression and Glutamatergic Synaptic Transmission in the Mouse Auditory Brainstem

Exposure to nicotine in utero, often due to maternal smoking, significantly elevates the risk of auditory processing deficits in offspring. This study investigated the effects of chronic nicotine exposure during a critical developmental period on the functional expression of nicotinic acetylcholine receptors (nAChRs), glutamatergic synaptic transmission, and auditory processing in the mouse auditory brainstem. We evaluated the functionality of nAChRs at a central synapse and explored the impact of perinatal nicotine exposure (PNE) on synaptic currents and auditory brainstem responses (ABR) in mice. Our findings revealed developmentally regulated changes in nAChR expression in the medial nucleus of the trapezoid body (MNTB) neurons and presynaptic Calyx of Held terminals. PNE was associated with enhanced acetylcholine-evoked postsynaptic currents and compromised glutamatergic neurotransmission, highlighting the critical role of nAChR activity in the early stages of auditory synaptic development. Additionally, PNE resulted in elevated ABR thresholds and diminished peak amplitudes, suggesting significant impairment in central auditory processing without cochlear dysfunction. This study provides novel insights into the synaptic disturbances that contribute to auditory deficits resulting from chronic prenatal nicotine exposure, underlining potential targets for therapeutic intervention.

neuroscience↗

bioRxiv2024

Mapping the breast tumor microenvironment: proximity analysis reveals spatial relationships between macrophage subtypes and metastasis-initiating cancer cells

The development of metastasis, responsible for the majority of cancer-related fatalities, is the most dangerous aspect of breast cancer, the predominant malignancy affecting women. We previously identified specific cancer cell populations responsible for metastatic events which are cytokeratin-14 (CK14) and E-cadherin positive in luminal tumors, and E-cadherin and vimentin positive in triple-negative tumors. Since cancer cells evolve within a complex ecosystem comprised of immune cells and stromal cells, we sought to decipher the spatial interactions of these aggressive cancer cell populations within the tumor microenvironment (TME). We used imaging mass cytometry to detect 36 proteins in tumor microarrays containing paired primary and metastatic lesions from luminal or triple-negative breast cancers (TNBC), resulting in a dataset of 1,477,337 annotated cells. Focusing on metastasis-initiating cell populations, we observed close proximity to specific fibroblast and macrophage subtypes, a relationship maintained between primary and metastatic tumors. Notably, high CK14 in luminal cancer cells and high vimentin in TNBC cells correlated with close proximity to specific macrophage subtypes (CD163intCD206intPDL1intHLA-DR+ or PDL1highARG1high). Our in-depth spatial analysis elucidates that metastasis-initiating cancer cells exhibit with distinct cell populations within the TME, implicating the role of these cell-cell interactions in promoting metastasis.

cancer biology↗

bioRxiv2024

Chitosan induces salicylic acid local and systemically in banana plants and reduces colonization by the pathogen Fusarium oxysporum f. sp. cubense TR4

Banana (Musa spp.) is essential for food security. For its clonality is highly susceptible to pests and diseases. The wilt fungus Fusarium oxysporum f. sp. cubense Tropical Race 4 (FocTR4) threatens banana production worldwide. We notice that chitosan protects banana from biotic stress. Chitosan, a plant defence elicitor with antimicrobial activity, triggers salicylic acid (SA) and methyl salicylate biosynthesis and induces systemic acquired resistance (SAR) genes, mainly flavin-dependent monooxygenase 1 (FMO1), in banana. Chitosan promotes root exudation of SA and emission of methyl salicylate (MeSA). Banana germplasm, Petit Naine, Gros Michel, Enzirabahima, Yangambi Km5 and Foconah banana differ in their response to chitosan. Chitosan induces, in Foc resistant Yangambi Km5, ca. 7-fold FMO1. Preventive chitosan treatments together with the endophytic biocontrol fungus Pochonia chlamydosporia strain 123 reduce (ca. 4-fold) colonization of banana roots by FocTR4. Therefore, chitosan and beneficial endophytes can help to manage sustainably FocTR4 in banana.

plant biology↗

bioRxiv2023

Preventing NK cell activation in the damaged liver induced by cabozantinib/PD-1 blockade increases survival in hepatocellular carcinoma models

Combining multikinase inhibitors with immune checkpoint blockade improves tumor control in hepatocellular carcinoma (HCC), but survival benefits remain inconsistent. To define this discordance, we integrated COSMIC-312 with orthotopic and autochthonous murine HCC models with or without liver fibrosis. In patients treated with cabozantinib plus atezolizumab, baseline liver function stratified overall survival but not progression-free survival, indicating uncoupling of tumor control from survival. In contrast, sorafenib outcomes tracked with both endpoints, supporting a treatment-specific effect rather than a purely prognostic effect of liver status. In murine HCC models, cabozantinib plus PD-1 blockade induced comparable tumor regression regardless of liver condition, but improved survival only in mice with preserved liver function, whereas fibrotic hosts developed hepatotoxicity. Immune profiling revealed compartment-specific remodeling, with enhanced cytotoxic T-cell programs in tumors but expansion of NK-lineage innate lymphocytes with ILC1-like features in fibrotic liver. Depletion of NK1.1-positive cells reduced liver injury and restored survival without compromising antitumor efficacy, whereas CD4-positive or CD8-positive T-cell depletion did not protect from hepatotoxicity. Transcriptomic, single-cell, adoptive-transfer, and human in vitro studies supported a model in which the fibrotic liver niche promotes NK-to-ILC1-like reprogramming, hepatocyte stress signaling, and TNF/TRAIL-associated epithelial injury. Consistently, cabozantinib and nivolumab showed liver-predominant remodeling of CD56-positive innate lymphocyte-enriched populations in human HCC samples, and ex vivo TGF-beta induced ILC1-like phenotypic changes in human NK cells. These findings identify liver fibrosis as a host determinant that can limit the survival benefit of multikinase inhibitor immunotherapy by promoting innate immune-mediated hepatotoxicity despite preserved tumor control in HCC. One Sentence SummaryLiver fibrosis limits the survival benefit of multikinase inhibitor immunotherapy in hepatocellular carcinoma by promoting innate immune-mediated hepatotoxicity despite preserved tumor control.

cancer biology↗

bioRxiv2023

Immune landscape of tertiary lymphoid structures in hepatocellular carcinoma (HCC) treated with neoadjuvant immune checkpoint blockade

Neoadjuvant immunotherapy is thought to produce long-term remissions through induction of antitumor immune responses before removal of the primary tumor. Tertiary lymphoid structures (TLS), germinal center-like structures that can arise within tumors, may contribute to the establishment of immunological memory in this setting, but understanding of their role remains limited. Here, we investigated the contribution of TLS to antitumor immunity in hepatocellular carcinoma (HCC) treated with neoadjuvant immunotherapy. We found that neoadjuvant immunotherapy induced the formation of TLS, which were associated with superior pathologic response, improved relapse free survival, and expansion of the intratumoral T and B cell repertoire. While TLS in viable tumor displayed a highly active mature morphology, in areas of tumor regression we identified an involuted TLS morphology, which was characterized by dispersion of the B cell follicle and persistence of a T cell zone enriched for ongoing antigen presentation and T cell-mature dendritic cell interactions. Involuted TLS showed increased expression of T cell memory markers and expansion of CD8+ cytotoxic and tissue resident memory clonotypes. Collectively, these data reveal the circumstances of TLS dissolution and suggest a functional role for late-stage TLS as sites of T cell memory formation after elimination of viable tumor. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/562104v2_fig0.gif" ALT="Figure 0"> View larger version (30K): org.highwire.dtl.DTLVardef@859572org.highwire.dtl.DTLVardef@169ed2org.highwire.dtl.DTLVardef@11510f0org.highwire.dtl.DTLVardef@b39a4_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LSTHighlightsC_LSTO_LIIn patients with hepatocellular carcinoma (HCC), tertiary lymphoid structures (TLS) are induced by neoadjuvant immunotherapy and are associated with favorable clinical outcomes. C_LIO_LITLS within the same tumor demonstrate extensive sharing of expanded granzyme K and granzyme B-expressing CD8+ T effector memory clonotypes, but the B cell repertoires of individual TLS are almost wholly distinct, consistent with independent germinal center reactions. C_LIO_LIWithin areas of viable tumor, mature TLS are characterized by high expression of CD21 and CD23, BCL6+ germinal center B cells, and close interactions between DCLAMP+ mature dendritic cells and CXCR5-CXCR3+ CD4 T peripheral helper cells within a T cell zone adjacent to the B cell follicle. C_LIO_LIWithin areas of tumor regression, an involuted TLS morphology is identified that is notable for dissolution of the B cell germinal center, retention of the T cell zone, and increased T cell memory. C_LI

immunology↗

bioRxiv2023

Novel chP3R99 mAb reduces subendothelial retention of atherogenic lipoproteins in Insulin-Resistant rats: Acute treatment versus long-term protection as an idiotypic vaccine for atherosclerosis

BACKGROUNDAtherosclerosis is triggered by the retention of apolipoprotein B-containing lipoproteins by proteoglycans. In addition to LDL, remnant lipoproteins have emerged as pivotal contributors to this pathology, particularly in the context of insulin resistance and diabetes. We have previously reported anti-atherogenic properties of a monoclonal antibody (chP3R99) that recognizes sulfated glycosaminoglycans on arterial proteoglycans. METHODS AND RESULTSSolid-phase assays demonstrated that chP3R99 effectively blocked over 50% lipoprotein binding to chondroitin sulfate and vascular extracellular matrix in vitro. The pre-perfusion of chP3R99 (competitive effect) resulted in specific antibody-arterial accumulation and reduced fluorescent lipoprotein retention by [~]60% in insulin resistant JCR:LA-cp rats. This competitive reduction was dose-dependent (25 {micro}g/mL-250 {micro}g/mL), effectively decreasing deposition of cholesterol associated with lipoproteins. In a five-week vaccination study in insulin resistant rats with (200 {micro}g SC, once a week), chP3R99 reduced arterial lipoprotein retention, and was associated with the production of anti-chondroitin sulfate antibodies (Ab3) able to accumulate in the arteries (dot-blot). Neither the intravenous inoculation of chP3R99 (4.5 mg/kg), nor the immunization with this antibody displayed adverse effects on lipid or glucose metabolism, insulin resistance, liver function, blood cell indices, or inflammation pathways in JCR:LA-cp rats. CONCLUSIONSBoth acute (passive) and long-term administration (idiotypic cascade) of chP3R99 antibody reduced LDL and remnant lipoprotein interaction with proteoglycans in an insulin-resistant setting. These findings support the innovative approach of targeting pro-atherogenic lipoprotein retention by chP3R99 as a passive therapy or as an idiotypic vaccine for atherosclerosis. CLINICAL PERSPECTIVEO_ST_ABSWhat Is New?C_ST_ABSO_LIInnovative anti-atherosclerotic chP3R99 mAb interferes with proteoglycan binding of both LDL and remnant lipoproteins in vitro and in vivo. C_LIO_LIIn vivo kinetic studies that immunize with chP3R99 reveal a temporal induction of an anti-idiotypic antibody cascade in a model of insulin resistance (analogous to a vaccine). C_LIO_LIWe discovered that the idiotypic chP3R99 monoclonal antibody (Ab1) was able to induce protective anti-anti-idiotypic (Ab3) antibodies present in both sera as well as the aorta (target organ) in vivo. C_LI What Are The Clinical Implications?O_LIThe chP3R99 mAb has efficacy for reducing the arterial retention of both LDL and remnant-derived lipoproteins and may be relevant of those with Type-2 Diabetes and/or residual CVD risk. C_LIO_LIWe show efficacy of chP3R99 mAb under pro-inflammatory conditions and that it does not exacerbate other metabolic aberrations during insulin resistance. C_LIO_LIData support the targeting pro-atherogenic lipoprotein retention with chP3R99 as a passive therapy or as an idiotypic vaccine for atherosclerosis, complementary to lipid lowering approaches. C_LI

pathology↗

bioRxiv2023

Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures

CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the main pathogenic (A2G3)n and the main nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in supercoiled DNA reveals triplex H-DNA formation by the pathogenic repeat. Consistently, bioinformatic analysis of the S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs in vivo. Finally, the pathogenic, but not the non-pathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.

genetics↗

bioRxiv2023

Beyond the MEP Pathway: a novel kinase required for prenol utilization by malaria parasites

A promising treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation, by competition. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional gene knockouts were created to investigate the biological importance of the FOH/GGOH salvage pathway. The knockout parasites were viable but more susceptible to fosmidomycin, and their sensitivity to MEP inhibitors could not be rescued by the addition of prenols. Moreover, the knockout parasites lost their ability to use prenols for protein prenylation. These results demonstrate that FOH/GGOH salvage is an additional source of isoprenoids by malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme whose inhibition may potentiate the antimalarial efficacy of drugs that affect isoprenoid metabolism.

biochemistry↗

bioRxiv2023

Functional traits of young seedlings predict trade-offs in seedling performance in three neotropical forests.

O_LIUnderstanding the mechanisms that promote the coexistence of hundreds of species over small areas in tropical forest remains a challenge. Many tropical tree species are presumed to be functionally equivalent shade tolerant species that differ in performance trade-offs between survival in shade and the ability to quickly grow in sunlight. C_LIO_LIVariation in plant functional traits related to resource acquisition is thought to predict variation in performance among species, perhaps explaining community assembly across habitats with gradients in resource availability. Many studies have found low predictive power, however, when linking trait measurements to species demographic rates. C_LIO_LISeedlings face different challenges recruiting on the forest floor and may exhibit different traits and/or performance trade-offs than older individuals face in the eventual adult niche. Seed mass is the typical proxy for seedling success, but species also differ in cotyledon strategy (reserve vs photosynthetic) or other seedling traits. These can cause species with the same average seed mass to have divergent performance in the same habitat. C_LIO_LIWe combined long-term studies of seedling dynamics with functional trait data collected at a standard developmental stage in three diverse neotropical forests to ask whether variation in coordinated suites of traits predicts variation among species in demographic performance. C_LIO_LIAcross hundreds of species in Ecuador, Panama, and Puerto Rico, we found seedlings displayed correlated suites of leaf, stem, and root traits, which strongly correlated with seed mass and cotyledon strategy. Variation among species in seedling functional traits, seed mass, and cotyledon strategy were strong predictors of trade-offs in seedling growth and survival. C_LIO_LIOur findings highlight the importance of cotyledon strategy in addition to seed mass as a key component of seed and seedling biology. These results also underscore the importance of matching the ontogenetic stage of the trait measurement to the stage of demographic dynamics. C_LIO_LISynthesis: With strikingly consistent patterns across three tropical forests, we find strong evidence for the promise of functional traits to provide mechanistic links between seedling form and demographic performance. C_LI

ecology↗

bioRxiv2023

A novel head-fixed assay for social touch in mice uncovers aversive responses in two autism models.

Social touch, an important aspect of social interaction and communication, is essential to kinship across animal species. How animals experience and respond to social touch has not been thoroughly investigated, in part due to the lack of appropriate assays. Previous studies that examined social touch in freely moving rodents lacked the necessary temporal and spatial control over individual touch interactions. We designed a novel head-fixed assay for social touch in mice, in which the experimenter has complete control to elicit highly stereotyped bouts of social touch between two animals. The user determines the number, duration, context, and type of social touch interactions, while monitoring with high frame rate cameras an array of complex behavioral responses. We focused on social touch to the face because of their high translational relevance to humans. We validated this assay in two different models of autism spectrum disorder (ASD), the Fmr1 knockout model of Fragile X Syndrome and maternal immune activation mice. We observed increased avoidance, hyperarousal, and more aversive facial expressions to social touch, but not to object touch, in both ASD models compared to controls. Because this new social touch assay for head-fixed mice can be used to record neural activity during repeated bouts of social touch it should be of interest to neuroscientists interested in uncovering the underlying circuits.

neuroscience↗

bioRxiv2022

MyD88-Dependent Signaling Drives Toll-Like Receptor-Induced Trained Immunity in Macrophages

Immunocompromised populations are highly vulnerable to developing life-threatening infections. Strategies to protect patients with weak immune responses are urgently needed. Employing trained immunity, whereby innate leukocytes undergo reprogramming upon exposure to a microbial product and respond more robustly to subsequent infection, is a promising approach. Previously, we demonstrated that the TLR4 agonist monophosphoryl lipid A (MPLA) induces trained immunity and confers broad resistance to infection. TLR4 signals through both MyD88- and TRIF-dependent cascades, but the relative contribution of each pathway to induction of trained immunity is unknown. Here, we show that MPLA-induced resistance to Staphylococcus aureus infection is lost in MyD88-KO, but not TRIF-KO, mice. The MyD88-activating agonist CpG (TLR9 agonist), but not TRIF-activating Poly I:C (TLR3 agonist), protects against infection in a macrophage-dependent manner. MPLA- and CpG-induced augmentation of macrophage metabolism and antimicrobial functions is blunted in MyD88-, but not TRIF-KO, macrophages. Augmentation of antimicrobial functions occurs in parallel to metabolic reprogramming and is dependent, in part, on mTOR activation. Splenic macrophages from CpG-treated mice confirmed that TLR/MyD88-induced reprogramming occurs in vivo. TLR/MyD88-triggered metabolic and functional reprogramming was reproduced in human monocyte-derived macrophages. These data show that MyD88-dependent signaling is critical in TLR-mediated trained immunity.

immunology↗

bioRxiv2022

Distinct GSDMB protein isoforms and protease cleavage processes differentially control pyroptotic cell death and mitochondrial damage in cancer cells.

The formation of Gasdermin (GSDM) pores, leading to pyroptosis or other context-dependent consequences, is directly involved in multiple diseases. Gasdermin-B (GSDMB) plays complex and controversial roles in pathologies, with pyroptosis-dependent and independent functions. GSDMB is promising oncologic therapeutic target since it exhibits either antitumor function, when immunocyte-mediated Granzyme-A (GZMA) cleaves GSDMB releasing its cytotoxic N-terminal domain, or pro-tumoral activities (invasion, metastasis, and drug resistance). However, it is still unknown the precise regulatory mechanisms of GSDMB pyroptosis as well as the differential effects of the four translated GSDMB variants (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Here, we first prove that exon 6 translation (in the interdomain protein linker) is essential for pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in large series of breast cancer samples GSDMB2 expression, and not of exon6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Moreover, cellular, and biochemical analyses combined with confocal, live cell imaging, and electron microscopy studies, demonstrated that diverse GSDMB N-terminal constructs containing exon-6 induce mitochondrial damage (increased mitochondrial ROS, membrane potential loss and mitochondrial DNA release) together with pyroptotic membrane cell lysis. While exon-6 residues are not required for membrane or mitochondrial localization, we also identified other key residues for N-terminal domain cytotoxicity. Additionally, we demonstrated that all GSDMB variants share the cleavage sites for GZMA, Neutrophil Elastase (identified in this study) and caspases. Interestingly, whereas Neutrophil Elastase and caspases produce N-terminal fragments in all GSDMB isoforms with no pyroptotic activity, thus acting as a potential inhibitory mechanism, GZMA cleavage activates pyroptosis in an isoform-dependent way. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer and other pathologies and for the future design of GSDMB-targeted therapies.

molecular biology↗

bioRxiv2022

Ventral Hippocampal Infusions of C20:0 Ceramide Stimulate Microglia and Induce Anhedonia-like Behavior in Female and Male Rats

Increased long-chain C20:0 ceramides have been found in the serum of patients with depression. Moreover, ceramides are linked with increased microglia reactivity and inflammatory cytokine production, which are associated with depression. Since ceramides can readily cross the blood brain barrier, peripheral C20:0 ceramides could enter the brain, activate microglia, and induce depressive-like behavior. In this study, we determined whether localized infusion of C20:0 ceramides into the ventral hippocampus (VH) of rats is sufficient to activate microglia and induce depressive-like behaviors. Adult male and female rats received infusions of C20:0 ceramides or vehicle solution every other day for 2 weeks. After the third infusion, C20:0-infused animals showed reduced sucrose preference suggesting anhedonia-like behavior. However, 4-6 days after the last infusion, animals infused with C20:0 ceramides did not show anhedonia-like or despair-like behavior suggesting the behavioral effect was reversible. After the final behavioral test, C20:0-infused animals had more Iba-1+ microglia in the VH. Despite the increased number of VH microglia, the expression of inflammatory genes was not increased. In conclusion, our data suggest that localized increases in C20:0 ceramides in the VH are sufficient to induce microglia activation and reversible anhedonia-like behavior.

neuroscience↗