Biology subjects

Boucher, J.

Publications and source records attributed to Boucher, J..

12 recordsLinked to original sources

bioRxiv2025

Characterization of HIV-1 particles co-purified with three extracellular vesicle subtypes from the Raji CD4 DCIR cell line, a hybrid model of CD4 T cells and dendritic cells

BackgroundHIV-1 proteins and RNA are packaged into extracellular vesicles (EVs) through interaction with the multivesicular endosomes of the EV biogenesis machinery. This interaction also allows functional or abortive viruses to exit the cells as exosomes, microvesicles or apoptotic vesicles. HIV-1 viral particles and EVs share significant similarities in size, composition, and molecular cargo, making their separation challenging. Current HIV-1 purification methods neglect the effects of EVs on virus infectivity, which could influence experiment outcomes. Here, we co-characterized HIV-1 particles co-purified with exosomes, microvesicles and apoptotic vesicles to determine their impact on HIV-1 infection. MethodsThe HIV-infected Raji CD4 DCIR cells supernatants were harvested 2 and 8 days after infection. The 2-day supernatant was treated with proteinase K to discard viral protein and HIV-1 RNA associated with proteins outside the EVs. The supernatants were fractionated into three pellets by differential centrifugation. The 3K pellet contained the largest EVs, such as apoptotic vesicles. The 17K and 100K pellets were respectively associated with microvesicles and exosomes. EVs and viral particles were co-characterized for their host and viral contents and the pellets obtained after 8 days post-infection were tested for infectivity. ResultsProteinase K treatment notably lowered HIV-1 RNA concentration in the EV pellet and did not affect viral p24 capsid protein concentration. The p24 protein was mostly found in the 17K pellet and HIV-1 RNA was the most abundant in the 100K pellet for both 2- and 8-day productions. Nevertheless, the 3K pellet had the highest infectivity when cells were infected with an equal quantity of virus (measured by p24) from each pellet. ConclusionProductively infected cells released functional viruses in the three EV subtypes, each exerting a distinct effect on virus infectivity. For future experiments, the presence of EVs in viral preparations should be taken into account, as they influence the progression of HIV-1 infection. HighlightsO_LIHIV-1 RNA and p24 capsid protein are packaged within apoptotic vesicles, microvesicles and exosomes released by Raji CD4 DCIR cells. C_LIO_LIInfectious particles co-precipitated with the 3K pellet from Raji CD4 DCIR cells are more infectious than other viruses associated with other EV pellets. C_LI

cell biology↗

bioRxiv2025

Immunosuppressive myeloid cells induce mesenchymal-like breast cancer stem cells by a mechanism involving membrane-bound TGF-β1.

Suppressive myeloid cells play a central role in cancer escape from anti-tumor immunity. Beyond their immunosuppressive function, these cells are capable of exerting multiple other pro-tumoral activities, including the promotion of cancer cell survival, invasion and metastasis. The ability of some myeloid subsets to induce cancer stemness has recently emerged. Here we demonstrated that human immunosuppressive myeloid cells, generated in vitro or isolated from breast cancer patients, promoted the acquisition of mesenchymal-like breast cancer stemness properties. This cancer-stemness-inducing function was restricted to a myeloid subset expressing the glycoprotein CD52. Single cell transcriptomic- and surface proteome-based interactome analysis pointed towards membrane-bound TGF-{beta}1 as a potential factor involved in cancer stemness induction. Functional inhibition of the TGF-{beta}1 pathway blocked the emergence of cancer stem cells induced by suppressive myeloid cells. These results therefore identified the underlying mechanisms of a new tumor-promoting function of immunosuppressive myeloid cells, which may potentially be targeted. HighlightsO_LIImmunosuppressive CD33highCD52+ myeloid cells induce mesenchymal-like cancer stem cells C_LIO_LICancer stemness induction requires membrane bound TGF-{beta}1 C_LIO_LIBlockade of the TGF-{beta}1 pathway prevents cancer stemness induction C_LI

cancer biology↗

bioRxiv2024

Exploring the Relationship Between Extracellular Vesicles, the Dendritic Cell Immunoreceptor and microRNA-155 in an In Vivo Model of HIV-1 Infection to Understand the Disease and Develop New Treatments

HIV-1 infection induces persistent immune system activation despite antiretroviral therapy. New immunomodulatory targets might be required to restore immune competence. The dendritic cells immunoreceptor (DCIR) can bind HIV-1 and regulate immune functions and extracellular vesicles (EVs) production. EVs have emerged as biomarkers and a non-invasive tool to monitor HIV-1 progression. In people living with HIV-1, an increase in the size and abundance of EVs is associated with a decline in the CD4/CD8 T cells ratio, a key marker of immune dysfunction. Analysis of host nucleic acids within EVs has revealed an enrichment of microRNA-155 (miR-155) during HIV-1 infection. Experiments have demonstrated that miR-155-rich EVs enhance HIV-1 infection in vitro. A humanized NSG-mice model was established to assess the in vivo impact of miR-155-rich EVs. Co-production of virus with miR-155-rich EVs heightened the viral load and lowered the CD4/CD8 ratio in the mice. Upon euthanasia, EVs were isolated from plasma for size and quantity assessment. Consistent with findings in individuals with HIV-1, increased EVs size and abundance were inversely correlated with the CD4/CD8 ratio. Next, by using the more closely related physiological virus co-product with EV-miR-155, we tested a DCIR inhibitor to limit infection and immune damage in a humanized mouse model. DCIR inhibition reduced infection and partially restored immune functions. Finally, viral particles and various EV subtypes can convey HIV-1 RNA. HIV-1 RNA was predominantly associated with large EVs (200-1000nm) rather than small EVs (50-200nm). Viral loads in large EVs strongly correlated with blood and tissue markers of immune activation. The humanized mice model has proven its applicability to studying the roles of EVs on HIV-1 infection and investigating the impact of DCIR inhibition. Author SummaryDespite more than 40 years of research, HIV remains a threat to public health around the world. People living with HIV are efficiently treated with antiretroviral therapy, but damage to the immune system persists and the causes remain unknown. Extracellular vesicles allow material, such as microRNA, to transfer between cells. Here, we evaluated the impact of one microRNA, microRNA-155, transported by extracellular vesicles, on HIV infection. Mice were grafted with a human immune system to allow infection by HIV. We showed that extracellular vesicles carrying microRNA-155 amplified mice infection. Extracellular vesicles also reflect the state of their cell of origin. Their analysis can reveal biomarkers to monitor HIV infection. Thus, HIV viral load was quantified in purified extracellular vesicles. We found that the measurement of HIV viral load in purified EVs is a more precise biomarker of disease progression than the traditional plasma viral load. Additionally, potential treatments like DCIR inhibitors improve our ability to manage HIV-1 by restoring the CD4/CD8 ratio, a critical element of the infection process. Overall, our study highlighted the importance of extracellular vesicle cargo in a humanized mouse model of HIV-1 infection, as well as the potential of targeting DCIR to restore the immune response. HighlightsO_LIMicroRNA-155 promotes HIV-1 infection of humanized NSG mice C_LIO_LIAbundance and size of total plasmatic EVs are biomarkers of immune dysfunction associated with HIV-1 infection C_LIO_LIDCIR inhibition limits HIV-1 infection of humanized NSG mice and attenuates immune impairment C_LIO_LIHIV-1 RNA enrichment in large EVs was associated with biomarkers of immune activation and dysfunction C_LI

immunology↗

bioRxiv2024

The 2023 wildfire season in Quebec: an overview of extreme conditions, impacts, lessons learned and considerations for the future.

The 2023 wildfire season in Quebec set records due to extreme warm and dry conditions, burning 4.5 million hectares and indicating persistent and escalating impacts associated with climate change. The study reviews the unusual weather conditions that led to the fires, discussing their extensive impacts on the forest sector, fire management, boreal caribou habitats, and particularly the profound effects on First Nation communities. The wildfires led to significant declines in forest productivity and timber supply, overwhelming fire management resources, and necessitating widespread evacuations. First Nation territories were dramatically altered, facing severe air quality issues and disruptions. While caribou impacts were modest across the province, the broader ecological, economical, and social repercussions were considerable. To mitigate future extreme wildfire seasons, the study suggests changes in forest management practices to increase forest resilience and resistance, adapting industrial structures to new timber supplies, and enhancing fire suppression and risk management strategies. It calls for a comprehensive, unified approach to risk management that incorporates the lessons from the 2023 fire season and accounts for ongoing climate change. The study underscores the urgent need for detailed planning and proactive measures to reduce the growing risks and impacts of wildfires in a changing climate.

ecology↗

bioRxiv2023

GalNAc-siRNA Mediated Knockdown of Ketohexokinase Versus Systemic, Small Molecule Inhibition of its Kinase Activity Exert Divergent Effects on Hepatic Metabolism in Mice on a HFD

Consumption of diets high in sugar and fat are well-established risk factors for the development of obesity and its metabolic complications, including non-alcoholic fatty liver disease. Metabolic dysfunction associated with sugar intake is dependent on fructose metabolism via ketohexokinase (KHK). Here, we compared the effects of systemic, small molecule inhibition of KHK enzymatic activity to hepatocyte-specific, GalNAc-siRNA mediated knockdown of KHK in mice on a HFD. Both modalities led to an improvement in liver steatosis, however, via substantially different mechanisms. KHK knockdown profoundly decreased lipogenesis, while the inhibitor increased the fatty acid oxidation pathway. Moreover, hepatocyte-specific KHK knockdown completely prevented hepatic fructose metabolism and improved glucose tolerance. Conversely, KHK inhibitor only partially reduced fructose metabolism, but it also decreased downstream triokinase. This led to the accumulation of fructose-1 phosphate, resulting in glycogen accumulation, hepatomegaly, and impaired glucose tolerance. In summary, KHK profoundly impacts hepatic metabolism, likely via both kinase-dependent and independent mechanisms. HIGHLIGHTSO_LIKHK knockdown or inhibition of its kinase activity differently target hepatic metabolism. C_LIO_LIKHK inhibitor increases F1P and glycogen accumulation as it also lowers triokinase. C_LIO_LIKHK knockdown completely prevents hepatic fructose metabolism and lipogenesis. C_LIO_LIE of wild type, but not mutant, kinase dead KHK-C increases glycogen accumulation. C_LI

molecular biology↗

bioRxiv2023

The CDK12 inhibitor SR-4835 functions as a molecular glue that promotes cyclin K degradation in melanoma

CDK12 is a transcriptional cyclin-dependent kinase (CDK) that interacts with cyclin K to regulate different aspects of gene expression. The CDK12-cyclin K complex phosphorylates several substrates, including RNA polymerase II (Pol II), and thereby regulates transcription elongation, RNA splicing, as well as cleavage and polyadenylation. Because of its implication in cancer, including breast cancer and melanoma, multiple pharmacological inhibitors of CDK12 have been identified to date, including THZ531 and SR-4835. While both CDK12 inhibitors affect Poll II phosphorylation, we found that SR-4835 uniquely promotes cyclin K degradation via the proteasome. Using loss-of-function genetic screening, we found that SR-4835 cytotoxicity depends on a functional CUL4-RBX1-DDB1 ubiquitin ligase complex. Consistent with this, we show that DDB1 is required for cyclin K degradation, and that SR-4835 promotes DDB1 interaction with the CDK12-cyclin K complex. Docking studies and structure-activity relationship analyses of SR-4835 revealed the importance of the benzimidazole side-chain in molecular glue activity. Together, our results indicate that SR-4835 acts as a molecular glue that recruits the CDK12-cyclin K complex to the CUL4-RBX1-DDB1 ubiquitin ligase complex to target cyclin K for degradation.

molecular biology↗

bioRxiv2023

Alpaca Single B Cell Interrogation and Heavy-Chain-Only Antibody Discovery on an Optofluidic Platform

In vivo discovery approaches for single domain antibodies such as VHH have been limited by the lack of methodologies available for camelid B cell interrogation. Here, we report a novel application of the Berkeley Lights Beacon(R) optofluidic platform to the discovery of heavy-chain-only antibodies by screening single B cells from alpacas immunized with two different targets. Custom methods for alpaca B cell enrichment, culture, on-Beacon IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH candidates from an alpaca immunized with either human prostate specific membrane antigen (PSMA) or a second blinded target, in a proof-of-concept study. PSMA-specific VHH hits discovered on the Beacon were recombinantly expressed as VHH-Fc, purified, and characterized using label-free techniques. All but one VHH-Fc bound PSMA with a single-digit nanomolar affinity, and four candidates were successfully humanized in silico using a rapid bulk humanization approach. In addition, next-generation repertoire sequencing was performed at longitudinal timepoints after immunization, uncovering additional variants within the clonal lineages of the validated hits discovered on the Beacon platform. The establishment of this single B cell VHH discovery workflow extends the powerful Beacon technology to enable rapid discovery of VHH directly from natural camelid immune repertoires.

immunology↗

bioRxiv2022

In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer

How the genetic landscape of a tumor governs the tumors response to immunotherapy remains largely elusive. Here, we established a direct in vivo CRISPR/Cas9 gene editing methodology to assess the immune-modulatory capabilities of 573 putative cancer genes associated with altered cytotoxic activity in human cancers. Using KrasG12D- and BrafV600E-driven mouse lung cancer models, we identify Serpinb9 and Adam2 as our top immune suppressive and immune enhancing genes, respectively. Mechanistically, we show that Serpinb9 ablation in KrasG12D- and BrafV600E-mutant lung tumor cells greatly enhances the efficacy of cytotoxic T-cells in vitro and in vivo. ADAM2 is a cancer testis antigen broadly expressed in human cancers such as lung adenocarcinoma (13.9%), renal (74.7%), prostate (72.4%), uterine (28.6%) and invasive breast (9.5%) cancer. In our mouse models, we show that Adam2 expression is induced in KrasG12D- but not BrafV600E-driven murine lung tumors and that its expression is further enhanced by immunotherapy. We show that loss of Adam2 significantly decreases KrasG12D-lung tumor burden but blocks the efficacy of cytotoxic T-cells. Consistently, Adam2 overexpression dramatically increases tumor growth and enhances immunotherapy efficacy. Mechanistically, we find that Adam2s oncogenic function depends on modulating the tumor immune microenvironment by restraining productive type I and type II interferon responses as well as cytokine signaling, reducing the presentation of tumor-associated antigen, and modulating surface expression of several immunoregulatory receptors within Kras-driven lung tumors. Adam2 expression also leads to reduced levels of immune checkpoint inhibitors such as Pd-l1, Lag3, Tigit and Tim3. This reduced exhaustion within the tumor microenvironment may explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy against Adam2 overexpressing lung tumors. Together, our study highlights the power of integrating cancer genomic with in vivo CRISPR/Cas9 screens to uncover how cancer-associated genetic alterations control responses to immunotherapies.

cancer biology↗

bioRxiv2021

Accelerated Antibody Discovery Targeting the SARS-CoV-2 Spike Protein for COVID-19 Therapeutic Potential

Rapid deployment of technologies capable of high-throughput and high-resolution screening is imperative for timely response to viral outbreaks. Risk mitigation in the form of leveraging multiple advanced technologies further increases the likelihood of identifying efficacious treatments in an aggressive timeline. In this study, we describe two parallel, yet distinct, in vivo approaches for accelerated discovery of antibodies targeting the SARS-CoV-2 spike protein. Working with human transgenic Alloy-GK mice, we detail a single B-cell discovery workflow to directly interrogate antibodies secreted from plasma cells for binding specificity and ACE2 receptor blocking activity. Additionally, we describe a concurrent accelerated hybridoma-based workflow utilizing a DiversimAb mouse model for increased diversity. The panel of antibodies isolated from both workflows revealed binding to distinct epitopes with both blocking and non-blocking profiles. Sequence analysis of the resulting lead candidates uncovered additional diversity with the opportunity for straightforward engineering and affinity maturation. By combining in vivo models with advanced integration of screening and selection platforms, lead antibody candidates can be sequenced and fully characterized within one to three months.

immunology↗

bioRxiv2020

Model-guided design of mammalian genetic programs

Genetically engineering cells to perform customizable functions is an emerging frontier with numerous technological and translational applications. However, it remains challenging to systematically engineer mammalian cells to execute complex functions. To address this need, we developed a method enabling accurate genetic program design using high-performing genetic parts and predictive computational models. We built multi-functional proteins integrating both transcriptional and post-translational control, validated models for describing these mechanisms, implemented digital and analog processing, and effectively linked genetic circuits with sensors for multi-input evaluations. The functional modularity and compositional versatility of these parts enable one to satisfy a given design objective via multiple synonymous programs. Our approach empowers bioengineers to predictively design mammalian cellular functions that perform as expected even at high levels of biological complexity.

synthetic biology↗

bioRxiv2017

Epidermal stem cells self-renew upon neighboring differentiation

Many adult tissues are dynamically sustained by the rapid turnover of stem cells. Yet, how cell fates such as self-renewal and differentiation are orchestrated to achieve long-term homeostasis remains elusive. Studies utilizing clonal tracing experiments in multiple tissues have argued that while stem cell fate is balanced at the population level, individual cell fate - to divide or differentiate - is determined intrinsically by each cell seemingly at random ( 1 2 3 4 5). These studies leave open the question of how cell fates are regulated to achieve fate balance across the tissue. Stem cell fate choices could be made autonomously by each cell throughout the tissue or be the result of cell coordination ( 6 7). Here we developed a novel live tracking strategy that allowed recording of every division and differentiation event within a region of epidermis for a week. These measurements reveal that stem cell fates are not autonomous. Rather, direct neighbors undergo coupled opposite fate decisions. We further found a clear ordering of events, with self-renewal triggered by neighbor differentiation, but not vice-versa. Typically, around 1-2 days after cell delamination, a neighboring cell entered S/G2 phase and divided. Functional blocking of this local feedback showed that differentiation continues to occur in the absence of cell division, resulting in a rapid depletion of the epidermal stem cell pool. We thus demonstrate that the epidermis is maintained by nearest neighbor coordination of cell fates, rather than by asymmetric divisions or fine-tuned cell-autonomous stochastic fate choices. These findings establish differentiation-dependent division as a core feature of homeostatic control, and define the relevant time and length scales over which homeostasis is enforced in epithelial tissues.

cell biology↗

bioRxiv2017

The eIF2α Kinase Heme Regulated Inhibitor (HRI) Protects The Host From Infection By Regulating Intracellular Pathogen Trafficking

Phosphorylation of eIF2 by its kinases is a stress response universally conserved among the eukaryota. Previously, we reported that the eIF2 kinases Heme Regulator Inhibitor (HRI) and Protein Kinase R (PKR) control distinct activities of diverse bacterial pathogens. Specifically for Listeria monocytogenes, it was shown that in HRI-deficient cells there was a reduction in the translocation of the pathogen to the cytosolic compartment as well as reduced loading of pathogen-derived antigens on MHC-1 complexes. Here we show that Hri -/- mice, as well as wild-type mice treated with a HRI inhibitor, are more susceptible to listeriosis. In the first few hours of L. monocytogenes infection, Hri -/- mice supported greater pathogen proliferation in the liver compared to that observed in Hri +/+ mice. This greater susceptibility of Hri -/- mice was not due to deficits in immune cell development as proportions and numbers of innate and adaptive cell compartments were largely normal and these mice could mount potent antibody responses to a model T cell-dependent antigen. Using in vitro cellular infection assays, we show that the rate of pathogen efflux from infected Hri -/- macrophages and fibroblasts is significantly higher than that observed in infected Hri +/+ cells. In contrast to the stark differences between Hri +/+ and Hri -/- cells in the infection dynamics of virulent L. monocytogenes, HRI was entirely dispensable for killing non-virulent strains of L. monocytogenes. These results suggest that in wild-type cells, HRI helps ensure the cellular confinement of virulent L. monocytogenes and loading of cytosolic-derived antigens on MHC-1 complexes that limit pathogen spreading and activating innate immune responses, respectively.

immunology↗