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Levy, E.

Publications and source records attributed to Levy, E..

15 recordsLinked to original sources

Atonosomes, compartments involved in membrane tension decrease

To ensure survival, cells need to buffer the effects of environmental stress on their plasma membrane, yet the structural mechanisms by which this is acutely achieved remain largely unknown. Here, we propose atonosomes as a unifying identity for a class of previously observed but enigmatic, tension-responsive, plasma membrane-derived compartments that arise across contexts of acute and chronic membrane tension loss. Leveraging unprecedented high resolution cryo-FIB-ET imaging in yeast, we show that atonosomes are complex, organelle-containing structures bounded by membranes and cell wall material, spanning hundreds of nanometers, and displaying a remarkable morphological diversity. Atonosomes form within seconds in response to reduced plasma membrane tension, and their emergence appears to require no dedicated molecular machinery, arising instead as a direct consequence of membrane biophysics. Upon formation, they recruit key membrane-associated proteins, including TORC2, Slm1, and septins. Under conditions of chronic disruption of PM homeostasis, atonosomes become constitutively present. Their stability and reversibility are further modulated by the cell wall, whose polymerization state influences atonosome dynamics. Structural conservation in fungi and ichthyosporea, demonstrates that atonosomes are a conserved stress-triggered response of cell-wall enclosed organisms. Together, these findings establish atonosomes as a novel compartment that mediates cellular responses to plasma membrane tension variation, coupling membrane remodeling and lipid homeostasis to preserve cellular integrity under stress.

cell biology↗

Cell type-centric interaction networks define spatial architecture of intrahepatic cholangiocarcinoma

Tumor spatial organization critically shapes disease progression and therapeutic response, yet remains poorly defined. Intrahepatic cholangiocarcinoma (iCCA), a rare and aggressive liver malignancy with extensive stromal and immune remodeling, provides a compelling model to study tumor architecture. We generated a single-cell spatial atlas of 1 million cells from 131 iCCA patients using 53-plex spatial proteomics. To systemically characterize tumor spatial organization, we developed a graph-based deep learning framework to define cell type-centric interaction networks, identifying 41 distinct multicellular spatial patterns. Integration of these networks revealed higher-order tumor- and immune-enriched microenvironments associated with patient outcomes. Notably, neutrophil-associated tumor-enriched and tumor-desert microenvironments delineated patient groups with opposing clinical outcomes and distinct neutrophil states. These findings were validated by single-cell spatial transcriptomic profiling of 6 million cells from 162 iCCA patients. Together, this study defines the spatial architecture of iCCA and provides a comprehensive resource for exploring tumor spatial organization.

cancer biology↗

Endogenously generated Dutch-type Aβ nonfibrillar aggregates dysregulate presynaptic neurotransmission in the absence of detectable inflammation

Structured AbstractO_ST_ABSBackgroundC_ST_ABSAPPE693Q ("Dutch") transgenic mice develop aging-related learning deficits and accumulate endogenously generated nonfibrillar aggregates of A{beta} (NFA-A{beta}) and APP -carboxy terminal fragments. NFA-A{beta} correlates with synaptic loss and memory deficits more closely than does fibrillar A{beta}. MethodsWe assessed the physiological, transcriptomic, ultrastructural, histological, and metabolic changes associated with the accumulation of NFA of Dutch A{beta} in brains of APPE693Q mice. ResultsAging-related accumulation of NFA-A{beta} in APPE693Q mice was revealed by A11 immunohistochemistry and cyclic D,L--peptide-FITC microscopy. Presynaptic termini of APPE693Q mice developed physiological abnormalities in post-tetanic potentiation, synaptic fatigue, synaptic vesicle replenishment, and an aging-related reduction in mitochondrial complex I activity. Single-cell RNA sequencing showed that excitatory neurons exhibited an altered transcriptomic profile involving "protein translation" and "oxidative phosphorylation". DiscussionAccumulation of NFA-A{beta} alters neuronal metabolism but does not activate inflammation. Depletion of all forms of A{beta} may be required to eliminate A{beta} toxicity with anti-amyloid antibodies.

neuroscience↗

EGF and IgA in maternal milk, donor milk and milk fortifiers in the Neonatal Intensive Care Unit setting

Human milk contains a variety of factors that positively contribute to neonatal health, including epidermal growth factor (EGF) and immunoglobulin A (IgA). When maternal milk cannot be the primary diet, maternal milk alternatives like donor human milk or formula can be provided. Donor human milk is increasingly provided to infants born preterm or low birth weight with the aim to supply immunological factors at similar concentrations to maternal milk. We sought to assess the concentrations of human EGF and IgA in the diet and stool of neonates between exclusive maternal milk, donor human milk, or formula-based diets. Using a prospective cohort study, we collected samples of diet and stool weekly from premature and low birth weight neonates starting at 10 days postnatal through five weeks of life while admitted to a neonatal intensive care unit (NICU). Compared to formula, there was significantly more EGF in both the milk and the stool of the infants fed human milk. Donor milk pooled from multiple donors contained similar concentrations of EGF and IgA to maternal milk, which was also significantly more compared to formula diets. Maternal milk fortified with fortifier derived from human milk contained significantly more EGF and IgA compared to unfortified maternal milk or maternal milk fortified with fortifier derived from bovine milk. Further analysis of human milk-derived fortifiers confirmed these fortifiers contained significant concentrations of EGF and IgA, contributing to an increased concentration of those factors when added to maternal milk compared to bovine milk-derived fortifiers. These findings illustrate how the choice of diet for a newborn, and even how that diet is modified through fortifiers or pasteurization before ingestion, impacts the beneficial biomolecules the infant receives from feeding.

immunology↗

Engineering ERα degraders with pleiotropic ubiquitin ligase ligands maximizes therapeutic efficacy by co-opting distinct effector ligases

Proximity-inducing compounds that modulate target protein homeostasis are an emerging therapeutic strategy [1]. While the inherent complexity of these bifunctional compounds poses challenges for rational design and bioavailability, their composition also provides opportunities to co-opt specific cellular proteins to maximize therapeutic impact. Here, we systematically evaluate the cellular efficacy, biophysical mechanisms, and therapeutic benefits of a series of bifunctional degrader compounds, that are all engineered with the Estrogen Receptor-alpha (ER)-inhibitor endoxifen linked to different bioactive ubiquitin ligase ligands. Bifunctional ER degraders that incorporate CRL4-CRBN-binding ligands promoted the most potent ER degradation, whereas those incorporating either CRL2-VHL- or IAP-binding ligands maximized the depth of ER degradation. Notably, ER degraders containing pan-IAP antagonist ligands significantly decreased the proliferation of ER-dependent cells relative to clinical-stage ER-degraders, including the SERDs fulvestrant and GDC-9545 and the bifunctional degrader ARV-471. Mechanistic studies revealed that pan-IAP antagonist-based ER degraders uniquely promote TNF-dependent cell death, unlike the clinical-stage comparators. Remarkably, the pan-IAP antagonist-ER-degraders co-opt distinct effector ligases to achieve dual therapeutic effects: they harness XIAP within tumor cells to promote ER degradation, and activate cIAP1/2 within tumor and immune cells to induce TNF that drives tumor cell death. Our studies demonstrate a broader concept that co-opting the discrete functions of a selected set of cellular effectors, while simultaneously modulating therapeutic target protein homeostasis, are dual strategies that can be leveraged to maximize the efficacy of induced proximity therapeutics.

cancer biology↗

Rapid and Sensitive Protein Complex Alignment with Foldseek-Multimer

Advances in computational structure prediction will vastly augment the hundreds of thousands of currently-available protein complex structures. Translating these into discoveries requires aligning them, which is computationally prohibitive. Foldseek-Multimer computes complex alignments from compatible chain-to-chain alignments, identified by efficiently clustering their superposition vectors. Foldseek-Multimer is 3-4 orders of magnitudes faster than the gold standard, while producing comparable alignments; allowing it to compare billions of complex-pairs in 11 hours. Foldseek-Multimer is open-source software: github.com/steineggerlab/foldseek, webserver: search.foldseek.com and the BFMD database.

bioinformatics↗

Tumor-targeted delivery of Tetanus toxoid by Listeria improves immunotherapy against ovarian cancer in mice

Ovarian cancer is known for its poor neoantigen expression and strong immunosuppression. Here, we utilized an attenuated non-pathogenic bacterium Listeria monocytogenes to deliver a highly immunogenic Tetanus Toxoid protein (Listeria-TT), as a neoantigen surrogate, into tumor cells through infection in a metastatic mouse ovarian cancer model (Id8p53-/-Luc). Gemcitabine (GEM) was added to reduce immune suppression. Listeria-TT+GEM treatments resulted in tumors expressing TT and reactivation of pre-existing CD4 and CD8 memory T cells to TT (generated early in life). These T cells were then attracted to the TT-expressing tumors now producing perforin and granzyme B. This correlated with a strong reduction in tumor burden, and significant improvement of the survival time compared to all control groups. Checkpoint inhibitors have little effect on ovarian cancer partly because of low neoantigen expression. Here we demonstrated that Listeria-TT+GEM+anti-PD1 was significantly more effective (efficacy and survival) than anti-PD1 or Listeria-TT+GEM alone. Of clinical interest, high doses of anti-PD1 (PD1H) (when added to Listeria-TT+GEM) were less effective than the low doses (PD1L). IHC and ELISPOT demonstrated that high doses of anti-PD1 inhibited T cell function in the TME. Using RNAseq, Differentially Expressed Genes (DEG) analysis and Genes Set Enrichment Analysis (GSEA) showed that gene expression levels and biological pathways were predominantly upregulated in the PD1H compared to the PD1L group, in correlation with low immune infiltration in tumors, more immune suppression, and more aggressive ovarian cancer. In summary, this study suggests that our approach may benefit ovarian cancer patients. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/561944v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@197942eorg.highwire.dtl.DTLVardef@8180d4org.highwire.dtl.DTLVardef@3113e6org.highwire.dtl.DTLVardef@116851_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract Human concept: Childhood vaccinations with the highly immunogenic tetanus toxoid (TT) generate TT-specific memory T cells, which circulate in the blood for life. After appearance of ovarian cancer (late in life), the patients will receive one high dose with Listeria-TT to deliver TT into tumor cells, followed by multiple low doses of Listeria-TT over a period of 2 weeks to restimulate the pre-existing memory T cells to TT. MDSC are involved in the delivery of Listeria-TT to the TME. Reactivated memory T cells will in turn destroy the tumor cells expressing TT. Multiple low doses of GEM will be added after TT has been delivered at the tumor site, which reduce immune suppression by eliminating MDSC and TAM (not shown here). Since individuals have seen TT earlier in life (during childhood vaccinations) and since TT is highly immunogenic (attracting T cells to the TME) but not expressed in normal cells, TT functions here as a vaccine recall antigen and as a neoantigen surrogate, respectively. C_FIG

cancer biology↗

Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo

Synaptic loss is an early event in the undersupplied but not yet irreversibly injured penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. In the present study, we aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse the potential beneficial effect of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

neuroscience↗

Tau filaments are tethered within brain extracellular vesicles in Alzheimer's disease

The abnormal assembly of tau protein in neurons is the pathological hallmark of multiple neurodegenerative diseases, including Alzheimers disease (AD). In addition, assembled tau associates with extracellular vesicles (EVs) in the central nervous system of patients with AD, which is linked to its clearance and prion-like propagation between neurons. However, the identities of the assembled tau species and the EVs, as well as how they associate, are not known. Here, we combined quantitative mass spectrometry, cryo-electron tomography and single-particle cryo-electron microscopy to study brain EVs from AD patients. We found filaments of truncated tau enclosed within EVs enriched in endo-lysosomal proteins. We observed multiple filament interactions, including with molecules that tethered filaments to the EV limiting membrane, suggesting selective packaging. Our findings will guide studies into the molecular mechanisms of EV-mediated secretion of assembled tau and inform the targeting of EV-associated tau as potential therapeutic and biomarker strategies for AD.

neuroscience↗

Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci.

BackgroundThe discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e.: two loxP sites flanking an exon or a critical DNA sequence of interest) remain the most challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. In the current manuscript, we describe a modification of the sequential electroporation procedure described by Horii et al (2017). We demonstrate production of conditional allele mouse models for eight different genes via one of two alternative strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). ResultsBy using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 to 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 to 25%. ConclusionOur modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.

molecular biology↗

Validated machine learning algorithm with sub-clonal sensitivity reveals widespread pan-cancer human leukocyte antigen loss of heterozygosity

Human leukocyte antigen loss of heterozygosity (HLA LOH) allows cancer cells to escape immune recognition by deleting HLA alleles, causing the suppressed presentation of tumor neoantigens that would otherwise bind to them. Despite its importance in immunotherapy response, few methods exist to detect HLA LOH, and their accuracy is not well understood. Here, we develop DASH (Deletion of Allele-Specific HLAs), a novel machine learning-based algorithm to detect HLA LOH from paired tumor-normal sequencing data. Through validation with cell line mixtures and patient-specific digital PCR, we demonstrate increased sensitivity compared to previously published tools and pave the way for clinical utility. Using DASH on 611 patients across 15 tumor types, we found that 18% of patients had HLA LOH. Moreover, we show inflated HLA LOH rates compared to genome-wide LOH and correlations between CD274 (PD-L1) expression and MSI status, suggesting the HLA LOH is a key immune resistance strategy.

bioinformatics↗

Proteomic analysis reveals the recruitment of intrinsically disordered regions to stress granules

Heat-stress triggers the formation of condensates known as stress granules (SGs), which store non-translating mRNA and stalled translation initiation complexes. To gain a better understanding of SGs, we identified yeast proteins that sediment after heat-shock by mass spectrometry. Heat-regulated proteins are biased toward a subset of abundant proteins that are significantly enriched in intrinsically disordered regions (IDRs). SG localization of over 80 heat-regulated proteins was confirmed using microscopy, including 32 proteins that were not known previously to localize to SGs. We find that several IDRs are sufficient to mediate SG recruitment. Moreover, the diffusive exchange of IDRs within SGs, observed via FRAP, can be highly dynamic while other components remain immobile. Lastly, we showed that the IDR of the Ubp3 deubiquitinase is critical for SG formation. This work confirms that IDRs play an important role in cellular compartmentalization upon stress, can be sufficient for SG incorporation, can remain dynamic in vitrified SGs, and play a vital role during heat-stress.\n\nSummaryThe authors provide an in-depth proteomic study of yeast heat stress granule (SG) proteins. They identified intrinsic disordered regions (IDRs) as one of the main features shared by these proteins and demonstrated IDRs can be sufficient for SG recruitment.

cell biology↗

Rainfall As A Significant Contributing Factor To Butterfly Seasonality Along A Climatic Gradient In The Neotropics

We analyzed the dynamics of multi-species butterfly communities along a climatic gradient with varying precipitation regimes for three consecutive years, and determine how climatic variables associate with observed butterfly seasonality. To provide a baseline for future studies of how climate change might affect these butterfly populations, we additionally explored the role of butterfly seasonality as a potential contributing factor for their susceptibility to climate variation. As far as we know, this represents the first study that simultaneously sampled and described seasonality patterns of tropical butterfly communities across ecosystems with varying climatic seasonality. A 3-year survey was carried out at three sites (i.e., wet, transition and dry forests) across a climatic gradient in western Ecuador. Butterflies were sampled using traps baited with rotting banana and prawn every two months from Nov 2010 to Sep 2013. Traps were set up at two heights, in the understory and canopy. In total, 7046 individuals of 212 species were sampled over 180 sampling days.\n\nButterfly communities exhibited conspicuous intra and inter-annual variation in temporal dynamics with certain elements (e.g., maximum abundance recorded) of seasonality patterns likely synchronized in seasonal forests (i.e., transition and dry forest) across years but not in aseasonal forests (i.e., wet forest). In addition, the highest numbers of species and individuals occurred during the wet season across all study sites and years; indeed, rainfall was significantly positively associated with temporal abundance. Likewise, butterfly species displaying stronger seasonality were significantly associated with higher rainfall periods in seasonal forests. Variation in precipitation regimes might significantly affect more seasonal species.

ecology↗

ASSEMBLY MECHANISMS OF NEOTROPICAL BUTTERFLY COMMUNITIES ALONG AN ENVIRONMENTAL GRADIENT

Our goal was to test the hypothesis that assembly processes that limit species similarity (i.e., competition) predominantly occur in more stable abiotic environments, whereas habitat filtering (i.e., habitat characteristics) is a major driver of community composition within more variable environments at regional (e.g., aseasonal vs seasonal forests) and local scales (e.g., understory vs. canopy). A combined approach of phylogenetic- and functional trait-based analyses using forewing length and aspect ratio as traits, were used to this hypothesis.\n\nA 3-year survey was carried out at three sites (i.e., wet, transition and dry forests) across a climatic gradient in western Ecuador. Transition and dry forests were considered as seasonal, whereas wet forest were considered aseasonal. Butterflies were sampled using traps baited with rotting banana and prawn every two months from Nov 2010 to Sep 2013. Traps were set up at two heights, in the understory and canopy. DNA was extracted to sequence the barcode section of the mitochondrial gene cytochrome oxidase 1 (COI) for phylogenetic analyses. Measurements of morphological traits, forewing length and aspect ratio were done using digital photographs of specimens.\n\nA total of 6466 specimens representing 142 species of Nymphalidae were recorded. Based on phylogenetic- and trait-based analyses, we rejected the hypothesis that assembly processes that limit species similarity (i.e., competition) are likely to predominantly occur in more stable abiotic environments, whereas habitat filtering can be a major driver of community composition within more variable environments at regional (i.e., aseasonal forest vs seasonal forests) and local scales (i.e., understory vs. canopy). My study of assembly mechanisms revealed the opposite pattern, with stronger evidence for the action of ecological filters in the assembly of butterfly communities from the wet aseasonal forests, and competition likely to be a major assembly process within dry seasonal forests. The present study therefore provided new insights into community assembly mechanisms in one of the richest butterfly faunas worldwide.

ecology↗

Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

systems biology↗