Biology subjects

Melamed, J.

Publications and source records attributed to Melamed, J..

7 recordsLinked to original sources

bioRxiv2026

Human caspase-4 and caspase-5 assemble into non-canonical inflammasomes in response to overlapping and distinct LPS structures

Innate immune sensing of bacterial lipopolysaccharide (LPS) is critical to host defense, but a dysregulated response can lead to sepsis, a life-threatening condition associated with multiple organ failure and shock. LPS is sensed at the cell surface by Toll-Like Receptor 4 (TLR4) and in the cytosol by mouse caspase-11 (Casp11) or its human orthologs, caspase-4 and 5 (CASP4/5). In response to cytosolic LPS, Casp11 oligomerizes in a catalytically dependent manner into supramolecular organizing centers (SMOCs). We demonstrate that CASP4 and CASP5 also form catalytically dependent SMOCs in response to bacterial LPS in macrophages. Furthermore, we find that CASP4 and CASP5 differentially respond to distinct LPS structural variants that fail to activate CASP11 and that only activating LPS structures induce SMOC formation by the corresponding caspase. Notably, transgenic mice expressing CASP4 in lieu of Casp11 exert more effective control of Francisella novicida, a rodent pathogen whose LPS evades TLR4 and Casp11 detection. Conversely, CASP4-expressing mice are more susceptible to sepsis when exposed to E. coli expressing a lipid A variant that fails to activate TLR4 and Casp11. Together, these findings reveal human CASP4 and CASP5 have evolved to assemble into SMOCs in response to a broader range of LPS structures than mouse Casp11 and highlight the potential tradeoff between systemic inflammation and control of bacterial infection that may underline these differences. Our findings may inform the development of targeted therapeutic strategies for gram-negative bacterial infection and sepsis that are effective in humans.

immunology↗

bioRxiv2025

DMEM, or Opti-MEM, that is the Question: An Important Consideration for Extracellular Vesicle Isolation and their Downstream Applications.

Serum-free synthetic media are frequently used as an alternative to extracellular vesicle-depleted serum containing media (EV-DEP) for EV production. However, the impact of this medium on EV biogenesis, release, and composition remains poorly understood. Here, we comprehensively characterised EV release by MDA-MB-231 or HEK-293T cells cultured in EV-DEP DMEM versus a serum-free synthetic medium, Opti-MEM. In Opti-MEM culture, cells released significantly more CD9- and CD63-positive EVs compared to EV-DEP DMEM. Proteomic analysis revealed that EVs from EV-DEP DMEM contained more histones and bovine proteins. Cells cultured in Opti-MEM released a substantially higher proportion of small-EVs derived from the sphingomyelinase-MVB pathway (60%) compared to EV-DEP DMEM (30%). Conversely, cells cultured in EV-DEP DMEM were more reliant upon the ROCK kinase pathway to release ectosomes (45 %) compared to OptiMEM (15 %). Where cells cultured in EV-DEP DMEM employed RabGTPase-dependent mechanisms for MVB-derived EV release (Rab3d, Rab27a, and Rab27b), cells cultured in Opti-MEM did not. Given that cells cultured in Opti-MEM vs EV-DEP DMEM produce EVs with different protein signatures from distinct molecular pathways, the choice of medium should be carefully considered when designing EV studies - particularly if they are to be used in therapeutic or immunological experiments.

cell biology↗

bioRxiv2025

Modeling Fibrosis with MASH Patient Liver-Derived Organoids.

Metabolic dysfunction-associated steatohepatitis (MASH) can lead to liver fibrosis and cirrhosis ultimately leading to liver transplantation or death. Therapeutic options for MASH-associated fibrosis are limited in part because of the lack of good model systems. To address this challenge, we developed a 3D MASH liver fibrosis model by using organoids derived from MASH patient liver co-cultured with human liver-derived hepatic stellate cells (HSC) and human peripheral blood monocytes (MC). Spontaneous self-assembly resulted in fibrotic scar-like 3D structures with senescent parenchymal cells, proliferating collagen secreting myofibroblasts (MFB) and proinflammatory TREM2+ scar-associated macrophages (MP). Single cell RNA sequencing suggested high similarity with MASH patient liver fibrotic scars. Lipid nanoparticles (LNPs) formulated with anti-YAP1siRNA could specifically and efficiently knockdown YAP1 in the MFBs, resulting in MFB senescence, a desirable therapeutic goal. This MASH patient liver-derived fibrosis model opens novel avenues towards testing treatments for MASH-associated liver fibrosis with reduced adverse effects.

bioengineering↗

bioRxiv2025

MAIT cell responses to intracellular and extracellular pathogens are mediated by distinct antigen presenting cells

Mucosal-associated invariant T (MAIT) cells recognize microbial derivatives of riboflavin synthesis presented by the MHC class I-related (MR1) molecule. Although these metabolites are highly conserved among bacteria, the cells that present them remain unknown. Here, we show type-17 MAIT cells respond to diverse isolates of the extracellular pathogen Acinetobacter baumannii and promote bacterial clearance. Both hematopoietic and non-hematopoietic cells mediate MR1 presentation within the lungs and mediastinal lymph nodes (meLNs). Conversely, the type-1 MAIT cell response to the intracellular pathogen Francisella tularensis requires MR1 presentation by type-2 conventional dendritic cells (cDC2s) within meLNs and ablation of these cells or their expression of MR1 renders animals more susceptible to the infection. Although MR1 is broadly expressed at homeostasis, A. baumannii enhances MR1 on macrophages and fibroblasts, while F. tularensis increases expression on cDC2s. These results demonstrate that microbial tropism dictates which APCs mediate MR1 presentation of metabolites, revealing alternative therapeutic approaches.

immunology↗

bioRxiv2024

Antibiotic use in early life subsequently impairs MAIT cell-mediated immunity

Mucosal-associated invariant T (MAIT) cells are predominantly located in barrier tissues where they rapidly respond to pathogens and commensals by recognizing microbial derivatives of riboflavin synthesis. Early-life exposure to these metabolites imprints the abundance of MAIT cells within tissues, so we hypothesized that antibiotic use during this period may abrogate their development. We identified antibiotics that deplete riboflavin-synthesizing commensals and revealed an early period of susceptibility during which antibiotic administration impaired MAIT cell development. The reduction in MAIT cell abundance rendered mice more susceptible to pneumonia, while MAIT cell-deficient mice were unaffected by early-life antibiotics. Concomitant administration of a riboflavin-synthesizing commensal during antibiotic treatment was sufficient to restore MAIT cell development and immunity. Our work demonstrates that transient depletion of riboflavin-synthesizing commensals in early life can adversely affect responses to subsequent infections.

immunology↗

bioRxiv2023

Neoadjuvant Therapy Differentially Remodels Carcinoma and Tumor Microenvironment in Pancreatic Ductal Adenocarcinoma: Insights from Spatially Resolved Transcriptomics

PurposeNeoadjuvant therapy (NAT) is increasingly being used for pancreatic ductal adenocarcinoma (PDAC) treatment. However, its distinct effects on carcinoma cells and the tumor microenvironment (TME) are not fully understood. This study employs spatial transcriptomics and single-cell RNA sequencing to investigate how NAT differentially remodels PDACs carcinoma cells and TME. Experimental DesignWe used spatial transcriptomics to compare gene expression profiles in carcinoma cells and the TME between NAT-treated and NAT-naive PDAC patients and correlated with their clinicopathologic features. Complementary single-nucleus RNA sequencing (snRNA-seq) analysis was conducted to validate our findings and identify cell types driving NAT-induced gene expression alterations. ResultsWe found NAT not only induces apoptosis and inhibits proliferation in carcinoma cells but also significantly remodels the TME. Notably, NAT induces a coordinated upregulation of multiple key complement genes (C3, C1S, C1R, C4B and C7) in the TME, making the complement pathway one of the most significantly affected pathways by NAT. Patients with higher TME complement expression following NAT exhibit improved overall survival; more immunomodulatory and neurotrophic cancer-associated fibroblasts (CAFs); more CD4+ T cells, monocytes and mast cells; and lower immune exhaustion gene expression. snRNA-seq analysis demonstrates C3 complement upregulation specifically in CAFs but not in other stroma cell types. ConclusionsOur findings indicate that NAT may reduce immunosuppression in PDAC by enhancing complement production and signaling within the TME. These findings suggest that local complement dynamics could serve as a novel biomarker for prognosis, evaluating treatment response and resistance, and guiding therapeutic strategies in NAT-treated PDAC patients. Translational RelevanceAs neoadjuvant therapy (NAT) increasingly becomes the preferred approach in treating resectable and borderline resectable pancreatic ductal adenocarcinoma (PDAC), there is a growing demand for novel biomarkers specifically tailored for post-NAT PDAC patients. Our study focused on how NAT differentially remodels the tumor cells and the tumor microenvironment (TME). We demonstrate that NAT can enhance local complement production and signaling in PDACs TME, which is associated with reduced immune exhaustion and improved overall survival. Our results highlight the importance of local complement dynamics in influencing treatment response, resistance, and overall clinical outcomes in PDAC. This new mechanism provides new opportunities in biomarker development which could facilitate more accurate prognostication and precise treatment stratification, particularly for patients undergoing NAT in PDAC.

genomics↗

bioRxiv2019

Drug resistant gut bacteria mimic a host mechanism for anticancer drug clearance

Pharmaceuticals are the top predictor of inter-individual variations in gut microbial community structure1, consistent with in vitro evidence that host-targeted drugs inhibit gut bacterial growth2 and are extensively metabolized by the gut microbiome3,4. In oncology, bacterial metabolism has been implicated in both drug efficacy5,6 and toxicity7,8; however, the degree to which bacterial drug sensitivity and metabolism can be driven by conserved pathways also found in mammalian cells remains poorly understood. Here, we show that anticancer fluoropyrimidine drugs inhibit the growth of diverse gut bacterial strains by disrupting pyrimidine metabolism, as in mammalian cells. Select bacteria metabolized 5-fluorouracil (5-FU) to its inactive metabolite dihydrofluorouracil (DHFU), mimicking the major host pathway for drug clearance. The preTA operon was necessary and sufficient for 5-FU inactivation in Escherichia coli, exhibited high catalytic efficiency for the reductive reaction, decreased the bioavailability and efficacy of oral fluoropyrimidine treatment in mice, and was prevalent in the gut microbiomes of colorectal cancer patients prior to and during treatment. The observed conservation of both the targets and pathways for metabolism of therapeutics across domains highlights the need to distinguish the relative contributions of human and microbial cells to drug disposition9, efficacy, and side effect profiles.

microbiology↗