A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons

IntroductionHuman induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process.MethodsIn this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter.ResultsThe retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types.DiscussionOur results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures

Understanding the nervous system: Lessons from Frontiers in Neurophotonics

The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada

Expression of the extracellular domain of endoglin in mammalian cells and bacteria

grantor: University of TorontoClass I histocompatibility molecules, consisting of a heavy chain, a light chain (ß2-microglobulin) and peptide, are assembled in the endoplasmic reticulum before being exported to the Golgi apparatus and the cell surface where they are surveyed by cytotoxic T cells. Assembly of the heavy/light chain heterodimer with peptide occurs in the peptide loading complex, which consists of the transporter associated with antigen processing, tapasin, ERp57, calnexin and calreticulin. In the first part of this thesis, I have used a mutational approach to investigate the physical organization of the peptide loading complex with a particular emphasis on tapasin. Several residues were identified to be involved in the interaction between H-2 D d and tapasin and mutations at these positions resulted in phenotypes of low surface expression, altered endoplasmic reticulum to Golgi transport and mildly affected peptide loading. These complex observations, analyzed in the context of similar studies performed with HLA-A2 and H-2 Ld suggest that the organization of the peptide loading complex can vary depending on the specific class I molecule examined. In the second part of this thesis, I investigated later events in the biogenesis of class I molecules, namely their export from the endoplasmic reticulum to the Golgi apparatus. I found that the putative cargo receptor Bap31 binds to two allotypes of mouse class I molecules with the interaction initiated at the time of heavy chain association with ß2-microglobulin and maintained until the class I molecule has left the endoplasmic reticulm. Consistent with an important role in recruiting class I molecules to transport vesicles, I showed that in the absence of Bap31 and its binding partner Bap29, there is a loss of class I colocalization with p137, a component of mammalian COPII coats. This observation is also associated with a delay in class I traffic from the endoplasmic reticulum to the Golgi. These observations are consistent with the view that class I molecules are largely recruited to endoplasmic reticulum exit sites by Bap29/31 and that Bap29/31 is a cargo receptor for major histocompatibility complex class I molecules., Endoglin is a homodimeric membrane glycoprotein predominantly expressed on endothelial cells. Cloning and sequencing of betaglycan, the transforming growth factor (TGF)-$\beta$ receptor III, had revealed homology with endoglin, particularly in the cytoplasmic domain. These observations led to the identification of endoglin as component of the TGF-$\beta$ receptor complex. Although betaglycan can be released from the plasma membrane by enzymatic cleavage, no soluble form of endoglin has been identified. The purpose of the current study was to produce a recombinant soluble form of endoglin to be used in functional and structural studies. We engineered several constructs of endoglin, including natural variants, and expressed them in COS-1 cells. Although all fragments were detected intracellularly, only $\Delta$586, corresponding to the complete extracellular domain was secreted. Our results demonstrate that a recombinant soluble form of endoglin can be secreted in a stable and dimeric form by COS-1 cells. (Abstract shortened by UMI.)M.Sc

Under the Radar Screen: How Bugs Trick Our Immune Defenses

In this course, we will explore the specific ways by which microbes defeat our immune system and the molecular mechanisms that are under attack (phagocytosis, the ubiquitin/proteasome pathway, MHC I/II antigen presentation). Through our discussion and dissection of the primary research literature, we will explore aspects of host-pathogen interactions. We will particularly emphasize the experimental techniques used in the field and how to read and understand research data. Technological advances in the fight against microbes will also be discussed, with specific examples. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching

Women with obesity in cervical cancer screening. The double penalty: Underscreening and income inequalities

International audienc

Data_Sheet_1_A robust and reliable methodology to perform GECI-based multi-time point neuronal calcium imaging within mixed cultures of human iPSC-derived cortical neurons.docx

IntroductionHuman induced pluripotent stem cells (iPSCs), with their ability to generate human neural cells (astrocytes and neurons) from patients, hold great promise for understanding the pathophysiology of major neuropsychiatric diseases such as schizophrenia and bipolar disorders, which includes alterations in cerebral development. Indeed, the in vitro neurodifferentiation of iPSCs, while recapitulating certain major stages of neurodevelopment in vivo, makes it possible to obtain networks of living human neurons. The culture model presented is particularly attractive within this framework since it involves iPSC-derived neural cells, which more specifically differentiate into cortical neurons of diverse types (in particular glutamatergic and GABAergic) and astrocytes. However, these in vitro neuronal networks, which may be heterogeneous in their degree of differentiation, remain challenging to bring to an appropriate level of maturation. It is therefore necessary to develop tools capable of analyzing a large number of cells to assess this maturation process. Calcium (Ca2+) imaging, which has been extensively developed, undoubtedly offers an incredibly good approach, particularly in its versions using genetically encoded calcium indicators. However, in the context of these iPSC-derived neural cell cultures, there is a lack of studies that propose Ca2+ imaging methods that can finely characterize the evolution of neuronal maturation during the neurodifferentiation process.MethodsIn this study, we propose a robust and reliable method for specifically measuring neuronal activity at two different time points of the neurodifferentiation process in such human neural cultures. To this end, we have developed a specific Ca2+ signal analysis procedure and tested a series of different AAV serotypes to obtain expression levels of GCaMP6f under the control of the neuron-specific human synapsin1 (hSyn) promoter.ResultsThe retro serotype has been found to be the most efficient in driving the expression of the GCaMP6f and is compatible with multi-time point neuronal Ca2+ imaging in our human iPSC-derived neural cultures. An AAV2/retro carrying GCaMP6f under the hSyn promoter (AAV2/retro-hSyn-GCaMP6f) is an efficient vector that we have identified. To establish the method, calcium measurements were carried out at two time points in the neurodifferentiation process with both hSyn and CAG promoters, the latter being known to provide high transient gene expression across various cell types.DiscussionOur results stress that this methodology involving AAV2/retro-hSyn-GCaMP6f is suitable for specifically measuring neuronal calcium activities over multiple time points and is compatible with the neurodifferentiation process in our mixed human neural cultures.</p

Combined influence of immigration status and income on cervical cancer screening uptake

The regular performance of Pap tests for cervical cancer screening reduces this disease's incidence and mortality. Income inequalities have been reported for this screening, partly because in some countries women must advance or even pay out-of-pocket costs. Because immigrant status is also associated with low Pap test uptake, we aimed to analyze the combined impact of immigrant status and low income on cervical cancer underscreening. This study, based on the French CONSTANCES cohort, uses data from the cohort questionnaires and linked health insurance fund data about Pap test reimbursement. To measure income inequalities in screening, we calculated a Slope Index of Inequality (SII) by linear regression, taking into account the migration status of participants. The majority of the 70,614 women included in the analysis were not immigrants (80.2%), while 12.9% were second-generation immigrants, and 6.9% first-generation immigrants. The proportion of underscreening increased with immigrant status, from 19.5% among nonimmigrants to 23.6% among the second generation, and 26.5% among the first (P < 0.01). The proportion of underscreening also increased as income level decreased. The income gradient rose significantly from 14% among nonimmigrants to 21% in second-generation immigrants and 19% in the first generation (P < 0.01). Among first-generation migrants, the shorter the duration of residence, the higher the SII. Women who are first- or second-generation immigrants are simultaneously underscreened and subject to a more unfavorable economic gradient than native French women born to native French parents. The accumulation of several negative factors could be particularly unfavorable to screening uptake