Personalized medicine and education: the challenge

Abstract Although personalized medicine appears to be a truism, medical doctors are still generally trained in an old-fashioned manner with a focus on reactive treatment. The aim of this paper is to emphasize the evolution of life sciences into a more predictive science, where the development of quantitative models is starting to take place. Personalized medicine is a consequence of such paradigm shift. To keep up with the change, the various actors within the health system must be trained in a completely different manner, focusing on the ability to work as part of a multidisciplinary team that includes medical doctors, nurses, engineers in medical imaging, and others who collect information from patients. In addition, these teams should include modelers that are able to integrate the flood of data into predictive and quantitative models. The challenge of implementing new training methods in line with the shift is a major bottleneck to the emergence and success of personalized medicine in our societies

Quiescence status of glioblastoma stem-like cells involves remodelling of Ca 2+ signalling and mitochondrial shape

International audienceQuiescence is a reversible cell-cycle arrest which allows cancer stem-like cells to evade killing following therapies. Here, we show that proliferating glioblastoma stem-like cells (GSLCs) can be induced and maintained in a quiescent state by lowering the extracellular pH. Through RNAseq analysis we identified Ca 2+ signalling genes differentially expressed between proliferating and quiescent GSLCs. Using the bioluminescent Ca 2+ reporter EGFP-aequorin we observed that the changes in Ca 2+ homeostasis occurring during the switch from proliferation to quiescence are controlled through store-operated channels (SOC) since inhibition of SOC drives proliferating GSLCs to quiescence. We showed that this switch is characterized by an increased capacity of GSLCs' mitochondria to capture Ca 2+ and by a dramatic and reversible change of mitochondrial morphology from a tubular to a donut shape. Our data suggest that the remodelling of the Ca 2+ homeostasis and the reshaping of mitochondria might favours quiescent GSLCs' survival and their aggressiveness in glioblastoma. Multiform glioblastoma (GBM) is the most aggressive brain tumours with very poor prognosis. Despite a combination of surgical resection, radiotherapy and temozolomide (TMZ)-based chemotherapy, more than 90% of the patients show recurrence and the mean survival period rarely exceeds 2 years 1. According to the cancer stem cell model, the GBM lethality is due to a small sub-population of tumour cells with stem-like properties, called Glioblastoma Stem-Like Cells (GSLCs). The GSLCs have been further characterized as slow-cycling or relatively quiescent cells 2 , identified in vivo in a mouse model of glioblastoma 3 and in human glioblastoma tumors 4. These quiescent GSLCs are highly resistant to TMZ treatment 5. Quiescence is a cell-cycle arrest state which differs from the one observed in differentiation or senescence by the fact that it is reversible. Transcriptional profiling data reveals that quiescent stem cells are characterized by a common gene signature with the down-regulation of genes associated with cell-cycle progression (i.e. CCNA2, CCNB1 and CCNE2) and the upregulation of genes classified as tumour suppressors, including the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the G0/G1 switch gene 2 (G0S2) 6,7. These data also show that quiescence is a G 0 phase and not a prolonged G 1 phase 8. Furthermore, quiescence is actively regulated by signals provided by the stem cell microenvironment. In glioblastoma tumours, quiescent stem-like tumour cells are found close to necrotic tissues, in specific niches characterized by an hypoxic 4,5,9 and acidic microenviron-ment 10,11. The role of the microenvironment in the control of GSLCs quiescence is still poorly understood. Considering that quiescence represents a strategy for GSLCs to evade killing, it is of utmost importance to better characterize the quiescent GSLCs and to understand what governs the transition from a proliferative to a quiescence state. Here, we performed transcriptomic analysis using RNAseq to establish the RNA signatures of proliferative and quiescent GSLCs. We showed that genes involved in Ca 2+ signalling are modulated in GSLCs and we explored the causal role of Ca 2+ during this transition. Our data points out the reversible remodelling of mitochondrial morphology from tubular to donut shape, associated with an increased capacity of mitochon-dria to capture Ca 2+ and with the modification of the kinetics of Ca 2+ influx through SOC. The remodelling o

Risk assessment for long and short range airborne transmission of SARS-CoV-2, indoors and outdoors

Preventive measures to reduce infection are needed to combat the COVID-19 pandemic and prepare for a possible endemic phase. Current prophylactic vaccines are highly effective to prevent disease but lose their ability to reduce viral transmission as viral evolution leads to increasing immune escape. Long term proactive public health policies must therefore complement vaccination with available nonpharmaceutical interventions (NPI) aiming to reduce the viral transmission risk in public spaces. Here, we revisit the quantitative assessment of airborne transmission risk, considering asymptotic limits that considerably simplify its expression. We show that the aerosol transmission risk is the product of three factors: a biological factor that depends on the viral strain, a hydrodynamical factor defined as the ratio of concentration in viral particles between inhaled and exhaled air, and a face mask filtering factor. The short range contribution to the risk, both present indoors and outdoors, is related to the turbulent dispersion of exhaled aerosols by air drafts and by convection (indoor), or by the wind (outdoors). We show experimentally that airborne droplets and CO$_2$ molecules present the same dispersion. As a consequence, the dilution factor, and therefore the risk, can be measured quantitatively using the CO$_2$ concentration, regardless of the room volume, the flow rate of fresh air and the occupancy. We show that the dispersion cone leads to a concentration in viral particles, and therefore a short range transmission risk, inversely proportional to the squared distance to an infected person and to the flow velocity. The aerosolization criterion derived as an intermediate result, which compares the Stokes relaxation time to the Lagrangian time scale, may find application for a broad class of aerosol-borne pathogens and pollutants.Comment: 12 pages, 4 figures; updated manuscrip

Generation and Behavior Characterization of CaMKIIβ Knockout Mice

The calcium/calmodulin-dependent protein kinase II (CaMKII) is abundant in the brain, where it makes important contributions to synaptic organization and homeostasis, including playing an essential role in synaptic plasticity and memory. Four genes encode isoforms of CaMKII (α, β, δ, γ), with CaMKIIα and CaMKIIβ highly expressed in the brain. Decades of molecular and cellular research, as well as the use of a large number of CaMKIIα mutant mouse lines, have provided insight into the pivotal roles of CaMKIIα in brain plasticity and cognition. However, less is known about the CaMKIIβ isoform. We report the development and extensive behavioral and phenotypic characterization of a CaMKIIβ knockout (KO) mouse. The CaMKIIβ KO mouse was found to be smaller at weaning, with an altered body mass composition. The CaMKIIβ KO mouse showed ataxia, impaired forelimb grip strength, and deficits in the rotorod, balance beam and running wheel tasks. Interestingly, the CaMKIIβ KO mouse exhibited reduced anxiety in the elevated plus maze and open field tests. The CaMKIIβ KO mouse also showed cognitive impairment in the novel object recognition task. Our results provide a comprehensive behavioral characterization of mice deficient in the β isoform of CaMKII. The neurologic phenotypes and the construction of the genotype suggest the utility of this KO mouse strain for future studies of CaMKIIβ in brain structure, function and development

Ca2+-Dependent Transcriptional Repressors KCNIP and Regulation of Prognosis Genes in Glioblastoma

Glioblastomas (GBMs) are the most aggressive and lethal primary astrocytic tumors in adults, with very poor prognosis. Recurrence in GBM is attributed to glioblastoma stem-like cells (GSLCs). The behavior of the tumor, including proliferation, progression, invasion, and significant resistance to therapies, is a consequence of the self-renewing properties of the GSLCs, and their high resistance to chemotherapies have been attributed to their capacity to enter quiescence. Thus, targeting GSLCs may constitute one of the possible therapeutic challenges to significantly improve anti-cancer treatment regimens for GBM. Ca2+ signaling is an important regulator of tumorigenesis in GBM, and the transition from proliferation to quiescence involves the modification of the kinetics of Ca2+ influx through store-operated channels due to an increased capacity of the mitochondria of quiescent GSLC to capture Ca2+. Therefore, the identification of new therapeutic targets requires the analysis of the calcium-regulated elements at transcriptional levels. In this review, we focus onto the direct regulation of gene expression by KCNIP proteins (KCNIP1–4). These proteins constitute the class E of Ca2+ sensor family with four EF-hand Ca2+-binding motifs and control gene transcription directly by binding, via a Ca2+-dependent mechanism, to specific DNA sites on target genes, called downstream regulatory element (DRE). The presence of putative DRE sites on genes associated with unfavorable outcome for GBM patients suggests that KCNIP proteins may contribute to the alteration of the expression of these prognosis genes. Indeed, in GBM, KCNIP2 expression appears to be significantly linked to the overall survival of patients. In this review, we summarize the current knowledge regarding the quiescent GSLCs with respect to Ca2+ signaling and discuss how Ca2+via KCNIP proteins may affect prognosis genes expression in GBM. This original mechanism may constitute the basis of the development of new therapeutic strategies

A general framework to characterize inhibitors of calmodulin: Use of calmodulin inhibitors to study the interaction between calmodulin and its calmodulin binding domains

AbstractThe prominent role of Ca2+ in cell physiology is mediated by a whole set of proteins involved in Ca2+-signal generation, deciphering and arrest. Among these intracellular proteins, calmodulin (CaM) known as a prototypical calcium sensor, serves as a ubiquitous carrier of the intracellular calcium signal in all eukaryotic cell types. CaM is assumed to be involved in many diseases including Parkinson, Alzheimer, and rheumatoid arthritis. Defects in some of many reaction partners of CaM might be responsible for disease symptoms. Several classes of drugs bind to CaM with unwanted side effects rather than specific therapeutic use. Thus, it may be more promising to concentrate at searching for pharmacological interferences with the CaM target proteins, in order to find tools for dissecting and investigating CaM-regulatory and modulatory functions in cells.In the present study, we have established a screening assay based on fluorescence polarization (FP) to identify a diverse set of small molecules that disrupt the regulatory function of CaM. The FP-based CaM assay consists in the competition of two fluorescent probes and a library of chemical compounds for binding to CaM.Screening of about 5300 compounds (Strasbourg Academic Library) by displacement of the probe yielded 39 compounds in a first step, from which 6 were selected. Those 6 compounds were characterized by means of calorimetry studies and by competitive displacement of two fluorescent probes interacting with CaM. Moreover, those small molecules were tested for their capability to displace 8 different CaM binding domains from CaM. Our results show that these CaM/small molecules interactions are not functionally equivalent.The strategy that has been set up for CaM is a general model for the development and validation of other CaM interactors, to decipher their mode of action, or rationally design more specific CaM antagonists. Moreover, this strategy may be used for other protein binding assays intended to screen for molecules with preferred binding activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium

Homodimerization of the Death-Associated Protein Kinase Catalytic Domain: Development of a New Small Molecule Fluorescent Reporter

Agence Nationale de Recherche (ANR); Centre National de la Recherche Scientifique (CNRS); University of StrasbourgBackground: Death-Associated Protein Kinase (DAPK) is a member of the Ca(2+)/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases. Methodology/Principal Findings: In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution. Conclusions/Significance: The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure

CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors

<p>Abstract</p> <p>Background</p> <p>Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.</p> <p>Methods</p> <p>We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas.</p> <p>Results</p> <p>A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide.</p> <p>Conclusions</p> <p>Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.</p

Limitations in a frataxin knockdown cell model for Friedreich ataxia in a high-throughput drug screen

<p>Abstract</p> <p>Background</p> <p>Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay.</p> <p>Methods</p> <p>Using a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA.</p> <p>Results</p> <p>The frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model.</p> <p>Conclusion</p> <p>Although no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future.</p