Biology subjects

Vocelle, D.

Publications and source records attributed to Vocelle, D..

4 recordsLinked to original sources

bioRxiv2025

Autoinduction expression systems by AHL-mediated quorum-sensing circuits in Synechococcus elongatus PCC 7942

Despite significant potential for cyanobacteria as sustainable bioproduction chases, there are limited examples of scaled cyanobacterial bioproduction. In part, this is because most cyanobacterial species are poorly adapted to bioreactor cultivation conditions and lack features that facilitate biomass growth and harvesting at scale. We explored quorum sensing (QS) pathways derived from heterotrophic microbes as a method for autoinduction of gene expression circuits coordinated to population density in cyanobacteria. Here, we integrated genetic modules designed to produce and detect the diffusible QS signal, acyl-homoserine lactones (AHLs), in the cyanobacterial model, Synechococcus elongatus PCC 7942 (S. elongatus). We demonstrate that S. elongatus heterologously produces sufficient AHL signals to activate gene expression in a dose-dependent and population density-responsive manner. A hybrid combination of AHL synthesis enzyme from Vibrio fischeri (Lux system) with the transcription factor receiver from Pseudomonas aeruginosa (Las system) provides an ideal activation ratio and mitigates toxicity observed with some AHL systems. As a proof of concept, we coupled the QS pathway to the expression of a cell division inhibitory gene, cdv3, facilitating late-phase cell elongation, cell sedimentation, and improved biomass recovery. Our findings provide a foundation for the development of auto-induction systems leverageable to improve cyanobacterial biotechnology applications.

microbiology↗

bioRxiv2024

The membrane-associated ubiquitin ligase MARCHF8 promotes cancer immune evasion by degrading MHC class I proteins

The loss of major histocompatibility complex class I (MHC-I) molecules has been proposed as a mechanism by which cancer cells evade tumor-specific T cells in immune checkpoint inhibitor (ICI)-refractory patients. Nevertheless, the mechanism by which cancer cells downregulate MHC-I is poorly understood. We report here that membrane-associated RING-CH-type finger 8 (MARCHF8), upregulated by human papillomavirus (HPV), ubiquitinates and degrades MHC-I proteins in HPV-positive head and neck cancer (HPV+ HNC). MARCHF8 knockdown restores MHC-I levels on HPV+ HNC cells. We further reveal that Marchf8 knockout significantly suppresses tumor growth and increases the infiltration of natural killer (NK) and T cells in the tumor microenvironment (TME). Furthermore, Marchf8 knockout markedly increases crosstalk between the cytotoxic NK cells and CD8+ T cells with macrophages and enhances the tumor cell-killing activity of CD8+ T cells. CD8+ T cell depletion in mice abrogates Marchf8 knockout-driven tumor suppression and T cell infiltration. Interestingly, Marchf8 knockout, in combination with anti-PD-1 treatment, synergistically suppresses tumor growth in mice bearing ICI-refractory tumors. Taken together, our finding suggests that MARCHF8 could be a promising target for novel immunotherapy for HPV+ HNC patients. One Sentence SummaryTargeting MARCHF8 restores MHC-I proteins, induces antitumor CD8+ T cell activity, and suppresses the growth of ICI-refractory tumors.

cancer biology↗

bioRxiv2024

Imaging flow cytometry enables label-free cell sorting of morphological variants from unculturable bacterial populations.

Bacterial populations often display remarkable morphological heterogeneity. Flow cytometric cell sorting (often called FACS) is an important tool for understanding this diversity. FACs allows researchers to obtain pure samples of each morphological variant (or morphotype) that is present within a mixed population of cells and thus permits each morphotype to be phenotyped. In FACS, cells are first labeled with fluorescent markers such as antibodies or transgenic constructs, and then separated out based on their possession of these labels. However, since the development of fluorescent labels requires a priori knowledge of bacterial biology, it is often impossible to apply FACS to understudied and/or unculturable bacteria. This challenge has limited our capacity to investigate the biology of bacterial size and shape in all but a small, largely culturable subset of bacterial taxa. Here, we present an innovative strategy that permits label-free cell sorting of bacterial morphotypes, using an unculturable, pleiomorphic pathogen (Pasteuria ramosa) as a model bacterium. We show that imaging flow cytometry (IFC) can be used to systematically identify light-scattering and autofluorescence "signatures" of bacterial morphotypes, on which basis cell sorting can be conducted. Critically, our IFC-enabled cell sorting strategy yields samples of sufficient purity (>90%) for common downstream analyses e.g., "-omics" analyses. Our work represents an innovative application of IFC and provides an economical, widely applicable solution to a central problem in the study of bacterial diversity. ImportanceBacteria come in many different shapes and sizes. Why this morphological variation exists is a longstanding question in microbiology, but it remains difficult to answer. To phenotype different morphological variants (morphotypes) within a bacterial population, we need to separate them from one another. This is normally achieved using flow cytometric cell-sorting, whereby morphotypes are labelled with fluorescent antibodies and separated on the basis of their differential fluorescence. Unfortunately, it is difficult to develop fluorescent-labels specific to unculturable or poorly studied bacteria because of the limited availability of appropriate molecular tools. Here, we demonstrate that imaging flow cytometry can be used to design and validate label-free cell sorting strategies. Recently, there has been a resurgence of interest in bacterial morphological diversity and a call to expand its study across the tree of life. Our work will help microbiologists to answer this call.

microbiology↗

bioRxiv2022

Phosphatidylserine-exposing medium/large extracellular vesicles: potential cancer biomarkers

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) also externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

cancer biology↗