The Cytotoxic Effect of the BCL-2 Family of Proteins in Breast Cancer Cells

Breast cancer is the second leading cause of death amongst women ages 20 to 59. Despite advancements in cancer therapies, more research is necessary to improve the diagnoses and treatment of several types of breast cancer. Paclitaxel (Taxol) is a commonly utilized anti-cancer drug for various types of solid tumors. However, the molecular mechanism utilized by paclitaxel to induce cell death is still elusive. Previous studies in our laboratory have shown that the pro-apoptotic BCL-2 family protein, BAK (BCL-2 homologous antagonist/killer) plays an important role in paclitaxel-induced cell death. In untreated breast cancer cells, BAK is associated with the anti-apoptotic BCL-2 family protein MCL-1 (myeloid leukemia cell differentiation protein). BAK is activated with paclitaxel treatment in concert with loss of MCL-1 expression. In addition, it has been shown that the pro-apoptotic BH3-only BCL-2 family protein Noxa, specifically interacts with MCL-1 to inactivate MCL-1 function. Based on these observations, we hypothesized that modulation of Noxa/MCL-1 axis could mimic paclitaxel-induced cell death. Here, we found that down-regulation of MCL-1 induced cell death in all breast cancer cell lines that we tested, but not in a non-transformed breast epithelial cell line. In contrast, Noxa overexpression induced MCL-1 degradation and cell death in some cell lines (Noxa-sensitive), while in others Noxa overexpression neither changed MCL-1 levels nor induced cell death (Noxa-resistant). Noxa strongly interacted with MCL-1 in the Noxa-sensitive cell line, but not in the Noxa-resistant cell line. Based on these findings, the overexpression of Noxa might have two different mechanistic effects on MCL-1 levels in the breast cancer cell lines (induction of MCL-1 degradation or no effect on MCL-1). In Noxa-sensitive cells, the finding could be used as a potential therapeutic strategy for the treatment of breast cancer

Dynamics and diversity of bacteria associated with the disease vectors Aedes aegypti and Aedes albopictus

Aedes aegypti and Aedes albopictus develop in the same aquatic sites where they encounter microorganisms that infuence their life history and capacity to transmit human arboviruses. Some bacteria such as Wolbachia are currently being considered for the control of Dengue, Chikungunya and Zika. Yet little is known about the dynamics and diversity of Aedes-associated bacteria, including larval habitat features that shape their tempo-spatial distribution. We applied large-scale 16S rRNA amplicon sequencing to 960 adults and larvae of both Ae. aegypti and Ae. albopictus mosquitoes from 59 sampling sites widely distributed across nine provinces of Panama. We fnd both species share a limited, yet highly variable core microbiota, refecting high stochasticity within their oviposition habitats. Despite sharing a large proportion of microbiota, Ae. aegypti harbours higher bacterial diversity than Ae. albopictus, primarily due to rarer bacterial groups at the larval stage. We fnd signifcant diferences between the bacterial communities of larvae and adult mosquitoes, and among samples from metal and ceramic containers. However, we fnd little support for geography, water temperature and pH as predictors of bacterial associates. We report a low incidence of natural Wolbachia infection for both Aedes and its geographical distribution. This baseline information provides a foundation for studies on the functions and interactions of Aedes-associated bacteria with consequences for bio-control within Panama.Aedes aegypti and Aedes albopictus develop in the same aquatic sites where they encounter microorganisms that infuence their life history and capacity to transmit human arboviruses. Some bacteria such as Wolbachia are currently being considered for the control of Dengue, Chikungunya and Zika. Yet little is known about the dynamics and diversity of Aedes-associated bacteria, including larval habitat features that shape their tempo-spatial distribution. We applied large-scale 16S rRNA amplicon sequencing to 960 adults and larvae of both Ae. aegypti and Ae. albopictus mosquitoes from 59 sampling sites widely distributed across nine provinces of Panama. We fnd both species share a limited, yet highly variable core microbiota, refecting high stochasticity within their oviposition habitats. Despite sharing a large proportion of microbiota, Ae. aegypti harbours higher bacterial diversity than Ae. albopictus, primarily due to rarer bacterial groups at the larval stage. We fnd signifcant diferences between the bacterial communities of larvae and adult mosquitoes, and among samples from metal and ceramic containers. However, we fnd little support for geography, water temperature and pH as predictors of bacterial associates. We report a low incidence of natural Wolbachia infection for both Aedes and its geographical distribution. This baseline information provides a foundation for studies on the functions and interactions of Aedes-associated bacteria with consequences for bio-control within Panama

Effet du cluster de microARN C19MC au cours des infections virales congénitales

Congenital viral infections caused by TORCH pathogens affect women worldwide and are associated with neonatal developmental disabilities. For instance, the Zika virus (ZIKV) has been linked to severe birth defects, including microcephaly. Similarly, the human Cytomegalovirus (hCMV) is the most prevalent viral infection acquired by the fetus, resulting in neurological damage. The ZIKV and the hCMV infect placental cells, directly affecting the neonate's development. In turn, the placenta employs extracellular vesicles (EVs) to interact with its environment, and it has been demonstrated that their C19MC microRNA cargo is dysregulated during pathological pregnancies. The primate-specific C19MC microRNA cluster shows a dynamic expression pattern throughout pregnancy, with levels rising toward the end of the first trimester and falling rapidly after parturition. Using an innovative Knock-Out (KO) model for the C19MC microRNA cluster generated by the CRISPR/Cas9 technique by J. Cavaillé's team, we investigated the antiviral function of this cluster during congenital viral infections. Experiments that involved real-time quantitative PCR, western blotting, flow cytometry, and immunofluorescence techniques showed that KO cells infected with the hCMV and the ZIKV are more susceptible to viral infections than their wild-type (WT) counterparts. Additionally, TCID50 assay and flow cytometry analysis showed a significantly higher viral infectivity of the supernatants containing infectious particles released from infected KO cells than from WT cells. Additionally, we used the RT2 profiler qPCR arrays to examine KO and WT cells' immunological and autophagy responses to viral infections. Distinct genetic differences in expression suggest that the C19MC cluster influences the reaction to ZIKV infections. We also explored the possibility of a paracrine antiviral mechanism of action; we tested whether or not this antiviral effect might be transmitted to recipient cells (Vero cells) via EVs. Flow cytometry data showed a transferable antiviral impact in Vero cells exposed to conditioned media from WT cells compared to KO cells. Nonetheless, more studies are required to confirm these findings further. Our research offers a unique KO-model to evaluate the multidimensional function of the C19MC miRNA cluster during congenital viral infections and whether or not it might operate in part through EVs. The development of improved and more accurate prognostic and therapeutic methods relies on a thorough understanding of the antiviral significance of this cluster.Les infections virales congénitales touchent les femmes du monde entier et sont associées à des troubles du développement néonatal. Par exemple, le virus Zika (ZIKV) a été associé à de graves anomalies congénitales, dont la microcéphalie. De même, le cytomégalovirus humain (hCMV) est l'infection virale la plus répandue chez le fœtus et entraîne des troubles neurodéveloppementaux. Le ZIKV et le hCMV infectent les cellules placentaires, affectant directement le développement du nouveau-né. Le placenta utilise des vésicules extracellulaires (EVs) pour interagir avec son environnement, et il a été démontré que leur contenu en microARN, notamment ceux du cluster C19MC, est dérégulé au cours des grossesses pathologiques. Le cluster de microARN C19MC, spécifique aux primates, présente un profil d'expression dynamique tout au long de la grossesse, avec des niveaux qui augmentent vers la fin du premier trimestre et chutent rapidement après la naissance. En utilisant un modèle innovant de cellules Knock-Out (KO) pour le cluster C19MC généré par la technique CRISPR/Cas9 par l'équipe de J. Cavaillé, nous avons étudié la fonction antivirale de ce cluster au cours des infections virales congénitales. Des expériences impliquant des techniques de PCR quantitative en temps réel, de western blotting, de cytométrie de flux et d'immunofluorescence ont montré que les cellules KO infectées par le hCMV et le ZIKV sont plus sensibles aux infections virales que leurs homologues sauvages (WT). De plus, des tests TCID50 et l'analyse par cytométrie de flux ont montré une infectivité virale significativement plus élevée des surnageants contenant des particules infectieuses libérées par les cellules KO infectées que par les cellules WT. Nous avons utilisé des dispositifs de qPCR RT2 profiler pour examiner les réponses immunes innées antivirales et d'autophagie des cellules KO et WT à l'infection par le ZIKV. Des différences génétiques distinctes dans l'expression suggèrent que le cluster C19MC influence la réaction à l'infection. Nous avons également exploré la possibilité d'un mécanisme d'action antiviral paracrine; nous avons testé si cet effet antiviral pouvait être transmis à des cellules réceptrices (cellules Vero) via des EVs. Les données de cytométrie en flux ont montré un impact antiviral transférable dans les cellules Vero exposées au milieu conditionné des cellules WT par rapport aux cellules KO. Néanmoins, d'autres études sont nécessaires pour confirmer ces résultats. Notre recherche offre un modèle KO unique pour évaluer la fonction multidimensionnelle du cluster de microARN C19MC au cours d'infections virales congénitales et pour déterminer s'il peut ou non fonctionner en partie par le biais des EVs. Le développement de méthodes pronostiques et thérapeutiques améliorées et plus précises repose sur une compréhension approfondie des mécanismes d'action de ce cluster

Role of the C19MC miRNA cluster during viral congenital infections

Les infections virales congénitales touchent les femmes du monde entier et sont associées à des troubles du développement néonatal. Par exemple, le virus Zika (ZIKV) a été associé à de graves anomalies congénitales, dont la microcéphalie. De même, le cytomégalovirus humain (hCMV) est l'infection virale la plus répandue chez le fœtus et entraîne des troubles neurodéveloppementaux. Le ZIKV et le hCMV infectent les cellules placentaires, affectant directement le développement du nouveau-né. Le placenta utilise des vésicules extracellulaires (EVs) pour interagir avec son environnement, et il a été démontré que leur contenu en microARN, notamment ceux du cluster C19MC, est dérégulé au cours des grossesses pathologiques. Le cluster de microARN C19MC, spécifique aux primates, présente un profil d'expression dynamique tout au long de la grossesse, avec des niveaux qui augmentent vers la fin du premier trimestre et chutent rapidement après la naissance. En utilisant un modèle innovant de cellules Knock-Out (KO) pour le cluster C19MC généré par la technique CRISPR/Cas9 par l'équipe de J. Cavaillé, nous avons étudié la fonction antivirale de ce cluster au cours des infections virales congénitales. Des expériences impliquant des techniques de PCR quantitative en temps réel, de western blotting, de cytométrie de flux et d'immunofluorescence ont montré que les cellules KO infectées par le hCMV et le ZIKV sont plus sensibles aux infections virales que leurs homologues sauvages (WT). De plus, des tests TCID50 et l'analyse par cytométrie de flux ont montré une infectivité virale significativement plus élevée des surnageants contenant des particules infectieuses libérées par les cellules KO infectées que par les cellules WT. Nous avons utilisé des dispositifs de qPCR RT2 profiler pour examiner les réponses immunes innées antivirales et d'autophagie des cellules KO et WT à l'infection par le ZIKV. Des différences génétiques distinctes dans l'expression suggèrent que le cluster C19MC influence la réaction à l'infection. Nous avons également exploré la possibilité d'un mécanisme d'action antiviral paracrine; nous avons testé si cet effet antiviral pouvait être transmis à des cellules réceptrices (cellules Vero) via des EVs. Les données de cytométrie en flux ont montré un impact antiviral transférable dans les cellules Vero exposées au milieu conditionné des cellules WT par rapport aux cellules KO. Néanmoins, d'autres études sont nécessaires pour confirmer ces résultats. Notre recherche offre un modèle KO unique pour évaluer la fonction multidimensionnelle du cluster de microARN C19MC au cours d'infections virales congénitales et pour déterminer s'il peut ou non fonctionner en partie par le biais des EVs. Le développement de méthodes pronostiques et thérapeutiques améliorées et plus précises repose sur une compréhension approfondie des mécanismes d'action de ce cluster.Congenital viral infections caused by TORCH pathogens affect women worldwide and are associated with neonatal developmental disabilities. For instance, the Zika virus (ZIKV) has been linked to severe birth defects, including microcephaly. Similarly, the human Cytomegalovirus (hCMV) is the most prevalent viral infection acquired by the fetus, resulting in neurological damage. The ZIKV and the hCMV infect placental cells, directly affecting the neonate's development. In turn, the placenta employs extracellular vesicles (EVs) to interact with its environment, and it has been demonstrated that their C19MC microRNA cargo is dysregulated during pathological pregnancies. The primate-specific C19MC microRNA cluster shows a dynamic expression pattern throughout pregnancy, with levels rising toward the end of the first trimester and falling rapidly after parturition. Using an innovative Knock-Out (KO) model for the C19MC microRNA cluster generated by the CRISPR/Cas9 technique by J. Cavaillé's team, we investigated the antiviral function of this cluster during congenital viral infections. Experiments that involved real-time quantitative PCR, western blotting, flow cytometry, and immunofluorescence techniques showed that KO cells infected with the hCMV and the ZIKV are more susceptible to viral infections than their wild-type (WT) counterparts. Additionally, TCID50 assay and flow cytometry analysis showed a significantly higher viral infectivity of the supernatants containing infectious particles released from infected KO cells than from WT cells. Additionally, we used the RT2 profiler qPCR arrays to examine KO and WT cells' immunological and autophagy responses to viral infections. Distinct genetic differences in expression suggest that the C19MC cluster influences the reaction to ZIKV infections. We also explored the possibility of a paracrine antiviral mechanism of action; we tested whether or not this antiviral effect might be transmitted to recipient cells (Vero cells) via EVs. Flow cytometry data showed a transferable antiviral impact in Vero cells exposed to conditioned media from WT cells compared to KO cells. Nonetheless, more studies are required to confirm these findings further. Our research offers a unique KO-model to evaluate the multidimensional function of the C19MC miRNA cluster during congenital viral infections and whether or not it might operate in part through EVs. The development of improved and more accurate prognostic and therapeutic methods relies on a thorough understanding of the antiviral significance of this cluster

Usutu Virus Infects Human Placental Explants and Induces Congenital Defects in Mice

International audienceUsutu virus (USUV) is a neurotropic mosquito-borne flavivirus that has dispersed quickly in Europe these past years. This arbovirus mainly follows an enzootic cycle involving mosquitoes and birds, but can also infect other mammals, causing notably sporadic cases in humans. Although it is mainly asymptomatic or responsible for mild clinical symptoms, USUV has been associated with neurological disorders, such as encephalitis and meningoencephalitis, highlighting the potential health threat of this virus. Among the different transmission routes described for other flaviviruses, the capacity for some of them to be transmitted vertically has been demonstrated, notably for Zika virus or West Nile virus, which are closely related to USUV. To evaluate the ability of USUV to replicate in the placenta and gain access to the fetus, we combined the use of several trophoblast model cell lines, ex vivo human placental explant cultures from first and third trimester of pregnancy, and in vivo USUV-infected pregnant mice. Our data demonstrate that human placental cells and tissues are permissive to USUV replication, and suggest that viral transmission can occur in mice during gestation. Hence, our observations suggest that USUV could be efficiently transmitted by the vertical route

Dynamics and diversity of bacteria associated with the disease vectors Aedes aegypti and Aedes albopictus

[eng] Aedes aegypti and Aedes albopictus develop in the same aquatic sites where they encounter microorganisms that influence their life history and capacity to transmit human arboviruses. Some bacteria such as Wolbachia are currently being considered for the control of Dengue, Chikungunya and Zika. Yet little is known about the dynamics and diversity of Aedes-associated bacteria, including larval habitat features that shape their tempo-spatial distribution. We applied large-scale 16S rRNA amplicon sequencing to 960 adults and larvae of both Ae. aegypti and Ae. albopictus mosquitoes from 59 sampling sites widely distributed across nine provinces of Panama. We find both species share a limited, yet highly variable core microbiota, reflecting high stochasticity within their oviposition habitats. Despite sharing a large proportion of microbiota, Ae. aegypti harbours higher bacterial diversity than Ae. albopictus, primarily due to rarer bacterial groups at the larval stage. We find significant differences between the bacterial communities of larvae and adult mosquitoes, and among samples from metal and ceramic containers. However, we find little support for geography, water temperature and pH as predictors of bacterial associates. We report a low incidence of natural Wolbachia infection for both Aedes and its geographical distribution. This baseline information provides a foundation for studies on the functions and interactions of Aedes-associated bacteria with consequences for bio-control within Panama