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Unlike Quality by Testing approach, where products were tested only after drug manufacturing, Quality by Design (QbD) is a proactive control quality paradigm, which handles risks from the early development steps. In QbD, regression models built from experimental data are used to predict a risk mapping called Design Space in which the developers can identify values of critical input factors leading to acceptable probabilities to meet the efficacy and safety specifications for the expected product. These empirical models are often limited to quantitative responses. Moreover, in practice the smallness and incompleteness of datasets degrade the quality of predictions. In this study, a Bayesian approach including variable selection, parameter estimation and model quality assessment is proposed and assessed using a real case study devoted to the development of a Cationic Nano-Lipid Structures for siRNA Transfection. Two original model structures are also included to describe both binary and percentage response variables. The results confirm the practical relevance and applicability of the Bayesian implementation of the QbD analysis.

Les organoïdes sont des assemblages multicellulaires auto-organisés résultant de la prolifération et de la différenciation principalement des cellules souches. Des organoïdes d’îlots pancréatiques–récemment nommés Langerhanoïdes–ont été développés, dans l’espoir d’apporter une solution supplémentaire aux patients diabétiques. Pour construire des organoïdes ressemblant à des îlots humains, les cellules, les biomatériaux et la structure tridimensionnelle sont trois éléments clés à considérer. Le potentiel des Langerhanoïdes en tant que plateforme pour des études de développement, la modélisation des maladies, le dépistage de la toxicité et les traitements personnalisés et préventifs pour le diabète semble évident. Nous augurons qu’ils auront aussi un impact clinique substantiel dans un proche avenir et offriront de nouvelles perspectives thérapeutiques pour les patients.

Androgen receptor (AR) signalling is a key prostate cancer (PC) driver, even in advanced ‘castrate-resistant’ disease (CRPC). To systematically identify microRNAs (miRs) modulating AR activity in lethal disease, hormone-responsive and -resistant PC cells expressing a luciferase-based AR reporter were transfected with a miR inhibitor library; 78 inhibitors significantly altered AR activity. Upon validation, miR-346, miR-361-3p and miR-197 inhibitors markedly reduced AR transcriptional activity, mRNA and protein levels, increased apoptosis, reduced proliferation, repressed EMT, and inhibited PC migration and invasion, demonstrating additive effects with AR inhibition. Corresponding miRs increased AR activity through a novel and anti-dogmatic mechanism of direct association with AR 6.9 kb 3′UTR and transcript stabilisation. In addition, miR-346 and miR-361-3p modulation altered levels of constitutively active AR variants, and inhibited variant-driven PC cell proliferation, so may contribute to persistent AR signalling in CRPC in the absence of circulating androgens. Pathway analysis of AGO-PAR-CLIP-identified miR targets revealed roles in DNA replication and repair, cell cycle, signal transduction and immune function. Silencing these targets, including tumour suppressors ARHGDIA and TAGLN2, phenocopied miR effects, demonstrating physiological relevance. MiR-346 additionally upregulated the oncogene, YWHAZ, which correlated with grade, biochemical relapse and metastasis in patients. These AR-modulatory miRs and targets correlated with AR activity in patient biopsies, and were elevated in response to long-term enzalutamide treatment of patient-derived CRPC xenografts. In summary, we identified miRs that modulate AR activity in PC and CRPC, via novel mechanisms, and may represent novel PC therapeutic targets.

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multiangle acquisitions on 3D cell cultures embedded in extracellular matrices. Lens-free microscopy acquisitions present some inherent issues such as the lack of phase information on the sensor plane and a limited angular coverage. We developed and compared three different algorithms based on the Fourier diffraction theorem to obtain fully 3D reconstructions. These algorithms present an increasing complexity associated with a better reconstruction quality. Two of them are based on a regularized inverse problem approach. To compare the reconstruction methods in terms of artefact reduction, signal-to-noise ratio, and computation time, we tested them on two experimental datasets: an endothelial cell culture and a prostate cell culture grown in a 3D extracellular matrix with large reconstructed volumes up to ∼5  mm^3 with a resolution sufficient to resolve isolated single cells. The lens-free reconstructions compare well with standard microscopy.

Biased DNA segregation is a mitotic event in which the chromatids carrying the original template DNA strands and those carrying the template copies are not segregated randomly into the two daughter cells. Biased segregation has been observed in several cell types, but not in human mesenchymal stem cells (hMSCs), and the factors affecting this bias have yet to be identified. Here, we have investigated cell adhesion geometries as a potential parameter by plating hMSCs from healthy donors on fibronectin-coated micropatterns. On symmetric micropatterns, the segregation of sister chromatids to the daughter cells appeared random. In contrast, on asymmetric micropatterns, the segregation was biased. This sensitivity to asymmetric extracellular cues was reproducible in cells from all donors but was not observed in human skin-derived fibroblasts or in a fibroblastic cell line used as controls. We conclude that the asymmetry of cell adhesion is a major factor in the regulation of biased DNA segregation in hMSCs.

La cellule est un système infiniment complexe et même si la biologie moléculaire a permis des avancées remarquables, les biologistes cherchent aujourd’hui à aborder cette complexité à l’échelle du système, espérant ainsi mieux décrypter les mystères cellulaires. L’inférence des réseaux de régulation transcriptionnelle, de leur topologie, de leur évolution dynamique, de leur rôle dans les grandes fonctions biologiques est au coeur de cette approche. Nous proposons une démarche systémique, fondée sur l’intégration de données à large échelle telles que les profils d’expression, l’analyse par puce des produits d’immunoprécipitation de chromatine, et l’ARN interférence à haut débit pour inférer ces réseaux et caractériser les phénotypes associés. A plus long terme, la compréhension des propriétés des réseaux pourrait permettre la découverte de nouvelles cibles thérapeutiques et la mise à jour de nouvelles stratégies médicamenteuses.

The aim of the present study was to characterize human side population (SP) epidermal keratinocytes isolated from primary cell cultures. For that purpose, keratinocytes were isolated from normal adult breast skin samples and the Hoechst 33342 exclusion assay described for hematopoietic cells was adapted to keratinocytes. Three types of keratinocytes were studied: the SP, the main population (MP), and the unsorted initial population. SP keratinocytes represented 0.16% of the total population. In short‐term cultures, they exhibited an increased colony‐forming efficiency and produced more actively growing colonies than did unsorted and MP keratinocytes. In long‐term cultures, SP cells exhibited an extensive expansion potential, performing a mean of 44 population doublings for up to 12 successive passages after cell sorting. Moreover, even in long‐term cultures, SP keratinocytes were able to form a pluristratified epidermis when seeded on a dermal substrate. Unsorted and MP keratinocytes promoted a reduced expansion: mean values of 14 population doublings for five passages and 12 population doublings for four successive passages, respectively. To further characterize SP cells, cDNA microarrays were used to identify their molecular signature. Transcriptome profiling showed that 41 genes were differentially expressed in SP (vs. MP) cells, with 37 upregulated genes and only four downregulated genes in SP cells. The majority of these genes were functionally related to the regulation of transcription and cell signaling. In conclusion, SP human keratinocytes isolated from primary cultures exhibited both short‐ and long‐term high proliferative potential, formed a pluristratified epidermis, and were characterized by a specific gene expression profile.

Abstract: Hydroxyurea (HU) is a specific inhibitor of ribonucleotide reductase and thus impairs dNTP synthesis and DNA replication. The long-term transcriptional response of yeast cells to hydroxyurea was investigated using DNA microarrays containing all yeast coding sequences. We show that the redox-responsive Yap regulon and the iron-mobilization Aft regulon are activated in yeast cells treated with HU. Yap1 accumulates in the nucleus in response to HU, but HU activation of the Yap regulon was only partially dependent on Yap1 and yap1Δ mutants were not hypersensitive to HU. In contrast, deletion of the AFT1 and AFT2 transcription factor genes blocked the HU activation of a subset of the Aft regulon and the aft1Δ aft2Δ double mutant was hypersensitive to HU in an iron-suppressible manner. These results highlight the importance of the redox and iron mobilization regulons in the cellular response to HU.

With the use of Hoechst staining techniques, we have previously shown that the C2C12 myogenic cell line contains a side population (SP) that is largely increased in the presence of fibroblast growth factor 6 (FGF6). Here, we compared transcriptional profiles from SP and main population (MP) cells from either C2C12 or FGF6-expressing C2C12. Expression profiles of SPs show that these cells are less differentiated than MPs and display some similarities to stem cells. Moreover, principal component analysis made it possible to distinguish specific contributions of either FGF6 or differentiation effects on gene expression profiles. This demonstrated that FGF6-expanded SPs were similar to parental C2C12-derived SPs. Conversely, FGF6-treated MPs differed from parental MPs and were more related to SP cells. These results show that FGF6 pushed committed myogenic cells toward a more immature phenotype resulting in the accumulation of cells with a SP phenotype. We propose that FGF6 conditioning could provide a way to expand the pool of immature cells by myoblast dedifferentiation.

Our knowledge of the molecular mechanisms that regulate hematopoiesis in physiologic and pathologic conditions is limited. Using a molecular approach based on cDNA microarrays, we demonstrated the emergence of an alternative pathway for mature bone marrow cell recovery after the programmed and reversible eradication of CD41+ cells in transgenic mice expressing a conditional toxigene targeted by the platelet αIIb promoter. The expression profile of the newly produced CD41+ cells showed high levels of transcripts encoding Ezh2, TdT, Rag2, and various immunoglobulin (Ig) heavy chains. In this context, we identified and characterized a novel population of Lin-Sca-1hic-Kit- cells, with a lymphoid-like expression pattern, potentially involved in the reconstitution process. Our study revealed novel transcriptional cross talk between myeloid and lymphoid lineages and identified gene expression modifications that occur in vivo under these particular stress conditions, opening important prospects for therapeutic applications.

À l’interface du vivant et de l’inerte, se développe un ensemble de nouvelles technologies regroupées sous le terme générique de biopuces. Grâce à la miniaturisation, nous pouvons imaginer que, demain, de nombreuses études biologiques et médicales se feront avec des biopuces qui permettront d’accroître de plusieurs ordres de grandeur le parallélisme des analyses, les vitesses de réaction des tests et leur débit, tout en réduisant les coûts. Cette évolution a démarré avec l’apparition des puces à ADN et se poursuit aujourd’hui avec, entre autres, les puces à cellules qui permettent d’accélérer considérablement l’étude des gènes de fonctions inconnues et leurs implications potentielles dans différentes maladies. Bien que la technologie en soit encore à ses prémices, il est vraisemblable que les puces à cellules feront évoluer la biologie et la médecine de manière significative.

Cytochemical localization of Concanavalin A binding sites in protoplasts of Candida tropicalis, investigated with glycosylated-ferritin and electron microscopy, showed that the lectin was specifically bound to the external protoplast surface. Thus, the plasma membranes have been labelled with 125I-Concanavalin A and followed through the isolation procedure. Relative distribution of 125I-radioactivity and azide-insensitive ATPase activity in the obtained fractions, suggested that this enzyme was an equivocal plasma membrane marker. Despite the presence of internal Concanavalin A binding sites, Concanavalin A could be used unambiguously as an exogenous plasma membrane marker of intact protoplasts.

Cardiac glycosides kill cancer cells in a way that stimulates the immune response.

In attached oat leaves the levels of adenine nucleotides decreased during leaf development and senescence. However, the energy charge (EC) only decreased from 0.90 in 4-cm leaves to 0.80 in senescent leaves. In detached leaves the levels of adenine nucleotides increased for 48 h, in association with an increase in RNase activity and a decrease in levels of RNA. The EC remained high until late senescence when levels of adenine nucleotides fell to about 30% of initial values. A decrease in energy parameters induced by transfer from light to darkness and from high (21%) to low (0.5% and anoxia) concentrations of oxygen resulted in an increase in membrane permeability. Oxidative stress (above 0.5% O2 induced an increase in levels of malondialdehyde (MDA) and then in permeability, associated with a decrease in levels of adenine nucleotides. Oxidative stress provoked by 0.05 and 0.10 M H2O2 caused a more rapid decrease in energy parameters than O2. Under oxidative stress (above 0.5% O2) there is, first of all, an increase in membrane permeability and then a decrease in energy parameters, which in turn are involved with senescence via increases in oxidation of membranes and degradation of energy-producing systems.