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Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.

The ionotropic GABAA receptors represent the main target for different groups of widely used drugs having hypnotic and anxiolytic effects. So far, most approaches used to assess GABA activity invol...

Significance Although converging evidence point to α-synuclein aggregation and Lewy body (LB) formation as central events in Parkinson’s disease, the molecular mechanisms that regulate these processes and their role in disease pathogenesis remain elusive. Herein, we describe a neuronal model that reproduces the key events leading to the formation of inclusions that recapitulate the biochemical, structural, and organizational features of bona fide LBs. This model allowed us to dissect the molecular events associated with the different stages of LB formation and how they contribute to neuronal dysfunctions and degeneration, thus providing a powerful platform for evaluating therapeutics targeting α-synuclein aggregation and LB formation and to identify and validate therapeutic targets for the treatment of Parkinson’s disease., Parkinson’s disease (PD) is characterized by the accumulation of misfolded and aggregated α-synuclein (α-syn) into intraneuronal inclusions named Lewy bodies (LBs). Although it is widely believed that α-syn plays a central role in the pathogenesis of PD, the processes that govern α-syn fibrillization and LB formation remain poorly understood. In this work, we sought to dissect the spatiotemporal events involved in the biogenesis of the LBs at the genetic, molecular, biochemical, structural, and cellular levels. Toward this goal, we further developed a seeding-based model of α-syn fibrillization to generate a neuronal model that reproduces the key events leading to LB formation, including seeding, fibrillization, and the formation of inclusions that recapitulate many of the biochemical, structural, and organizational features of bona fide LBs. Using an integrative omics, biochemical and imaging approach, we dissected the molecular events associated with the different stages of LB formation and their contribution to neuronal dysfunction and degeneration. In addition, we demonstrate that LB formation involves a complex interplay between α-syn fibrillization, posttranslational modifications, and interactions between α-syn aggregates and membranous organelles, including mitochondria, the autophagosome, and endolysosome. Finally, we show that the process of LB formation, rather than simply fibril formation, is one of the major drivers of neurodegeneration through disruption of cellular functions and inducing mitochondria damage and deficits, and synaptic dysfunctions. We believe that this model represents a powerful platform to further investigate the mechanisms of LB formation and clearance and to screen and evaluate therapeutics targeting α-syn aggregation and LB formation.

Current treatments for retinoblastoma, the most common intraocular malignancy, rely on a limited number of drugs repurposed from other pediatric cancer therapies. Drug toxicity and relapse of the disease necessitate new therapeutic strategies. The implementation of drug candidates in the clinic is usually followed by in vitro validation, which requires accurate and clinically relevant in vitro models. We report here a robust 3D tumoroid-based platform, for testing various clinical protocols, e.g. therapeutic agents following a single or repeated exposure, alone or in combination with focal therapy (thermotherapy). The platform, generated from the Y79 retinoblastoma cell line, consists of matrix-embedded tumoroids, which retain the key features of the disease and allow prolonged culture time. Following the optimization of growth conditions, the effect of single and repeated exposure to chemotherapeutic agents has been measured by high-throughput automated fluorescence imaging followed by quantification. The clinical conditions of chemothermotherapy have been recapitulated with a setup consisting of: an infrared diode laser (810 nm, 0.3 W) that heats the tumoroids in the presence of indocyanine green (50 µg/mL); a thermal camera to enable real-time temperature measurement; and an environment-controlled inverted microscope to ensure physiological conditions and precise focusing of the laser. Melphalan and carboplatin, two drugs currently used to treat retinoblastoma in clinical protocols of chemotherapy and chemothermotherapy respectively, have been applied to validate the system.Tumoroid viability and size progressively decreased over 4 weeks of repeated drug exposure with melphalan, reflecting the response of retinoblastoma in advanced clinical cases. Carboplatin treatment showed an activity enhancement in the tumoroid model similar to that observed in the clinic, when combined with focal therapy, demonstrating that the platform system closely recapitulates the clinical conditions. Inversely, the thermotherapy experimental protocol designed for 2D in vitro cell culture and commonly used to date, i.e. an incubation temperature increased to 42 °C for 1 h, did not show an improvement of the carboplatin cytotoxicity, confirming that our laser-based thermotherapy protocol provides a more accurate and clinically relevant drug response. Such an approach, combining physiologically relevant in vitro cancer models and investigation of drugs in well-established treatment modalities, can significantly reduce the number of new drug candidates that give false-positives or overestimated efficacies when evaluated in relevant in vivo models. It is expected that the platform will contribute to the implementation of new therapeutic strategies to treat retinoblastoma, and could also be adapted to other diseases for which repeated drug exposure and/or chemothermotherapy is relevant. Citation Format: Irina Sinenko, Fabien Kuttler, Valentin Simeonov, Alexandre Moulin, Christina Stathopoulos, Gerardo Turcatti, Adeline Berger, Francis Munier, Paul Dyson. Tumoroid-based screening platform to test focal, chemo- and combination therapy for retinoblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3541.

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the β-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses., Graphical abstract Image 1

We use quantitative phase digital holographic microscopy (QP-DHM) to image and quantify the beating movement of cardiomyocytes, derived from induced pluripotent stem cells (iPSCs), in control and drug-treated conditions. The development of an analysis algorithm has allowed extracting from the recorded quantitative phase signal (QPS) a set of several parameters that can efficiently characterize the cardiomyocytes beating patterns. Based on this approach, we monitored the effects of E-4031 (a class III antiarrhythmic drug) and isoprenaline (a common medication for bradycardia and heart block problems) on the cardiomyocyte beating patterns. Our results show that some effects specific to the mode of action of the drugs used can be identified. This stresses that QP-DHM can represent a promising label-free approach to identify new drug candidates by measuring their effects on iPSC-derived cardiomyocytes.

Coronaviruses (CoVs) circulate in humans and animals, and expand their host range by zoonotic and anthroponotic transmissions. Endemic human CoVs, such as 229E and OC43 cause limited respiratory disease, and elicit short term anti-viral immunity favoring recurrent infections. Yet, severe acute respir-atory syndrome (SARS)-CoV-2 spreads across the globe with unprecedented impact on societies and economics. The world lacks broadly effective and affordable anti-viral agents to fight the pandemic and reduce the death toll. Here, we developed an image-based multicycle replication assay for focus for-mation of α-coronavirus hCoV-229E-eGFP infected cells for screening with a chemical library of 5440 compounds arrayed in 384 well format. The library contained about 39% clinically used compounds, 26% in phase I, II or III clinical trials, and 34% in preclinical development. Hits were counter-selected against toxicity, and challenged with hCoV-OC43 and SARS-CoV-2 in tissue culture and human bronchial and nasal epithelial explant cultures from healthy donors. Fifty three compounds inhibited hCoV-229E-GFP, 39 of which at 50% effective concentrations (EC50) < 2μM, and were at least 2-fold separated from toxicity. Thirty nine of the 53 compounds inhibited the replication of hCoV-OC43, while SARS-CoV-2 was inhibited by 11 compounds in at least two of four tested cell lines. Six of the 11 compounds are FDA-approved, one of which is used in mouth wash formulations, and five are systemic and orally available. Here, we demonstrate that methylene blue (MB) and mycophenolic acid (MPA), two broadly available low cost compounds, strongly inhibited shedding of infectious SARS-CoV-2 at the apical side of the cultures, in either pre- or post-exposure regimens, with somewhat weaker effects on viral RNA release indicated by RT-qPCR measurements. Our study illustrates the power of full cycle screens in repurposing clinical compounds against SARS-CoV-2. Importantly, both MB and MPA reportedly act as immunosuppressants, making them interesting candidates to counteract the cytokine storms affecting COVID-19 patients.

Human adenoviruses (HAdVs) are fatal to immuno-suppressed individuals, but no effective anti-HAdV therapy is available. Here, we present a novel image-based high-throughput screening (HTS) platform, which scores the full viral replication cycle from virus entry to dissemination of progeny and second-round infections. We analysed 1,280 small molecular weight compounds of the Prestwick Chemical Library (PCL) for interference with HAdV-C2 infection in a quadruplicate, blinded format, and performed robust image analyses and hit filtering. We present the entire set of the screening data including all images, image analyses and data processing pipelines. The data are made available at the Image Data Resource (IDR, idr0081). Our screen identified Nelfinavir mesylate as an inhibitor of HAdV-C2 multi-round plaque formation, but not single round infection. Nelfinavir has been FDA-approved for anti-retroviral therapy in humans. Our results underscore the power of image-based full cycle infection assays in identifying viral inhibitors with clinical potential., Measurement(s) nucleus • number of infected nuclei • infection index (number of infected nuclei/number of nuclei) • Plaque • fluorescent reporter intensity • percent cell viability Technology Type(s) epifluorescence microscopy • compound toxicity assay Factor Type(s) compound treatment Sample Characteristic - Organism Human mastadenovirus C Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12594470

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. The human AdV (HAdV) species B and C, such as HAdV-C2, C5 and B14, cause respiratory disease, and constitute a health threat for immuno-compromised individuals. HAdV-Cs are well known for lysing cells, owing to the E3 CR1-β-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multi-round infection, Nelfinavir Mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of the human immuno-deficiency virus (HIV) aspartyl protease, and used to treat acquired immunodeficiency syndrome (AIDS). It is not effective against single round HAdV infections. Here, we show that Nelfinavir inhibits the lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighbouring uninfected cells. HAdV lacking ADP was insensitive to Nelfinavir, but gave rise to comet-shaped foci indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and B14p1 lacking ADP were highly sensitive to Nelfinavir, although HAdV-A31, B3, B7, B11, B16, B21, D8, D30 or D37 were less sensitive. Conspicuously, Nelfinavir uncovered slow-growing round-shaped HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of non-lytic cell-to-cell transmission. Our study demonstrates the repurposing potential of Nelfinavir with post-exposure efficacy against different HAdVs, and describes an alternative non-lytic cell-to-cell transmission mode of HAdV.Graphical AbstractFigure 1.

Human adenoviruses (HAdVs) are fatal to immuno-suppressed people, but no effective anti-HAdV therapy is available. Here, we present a novel image-based high-throughput screening (HTS) platform, which scores the full viral replication cycle from virus entry to dissemination of progeny. We analysed 1,280 small molecular weight compounds of the Prestwick Chemical Library (PCL) for interference with HAdV-C2 infection in a quadruplicate, blinded format, and included robust image analyses, and hit filtering. We present the entire set of the screening data including all the images, image analyses and data processing pipelines. The data are made available at the Image Data Repository (IDR) 1, accession number idr0081. Our screen identified Nelfinavir mesylate as an inhibitor of HAdV-C2 multi-round plaque formation, but not single round infection. Nelfinavir has been FDA-approved for anti-retroviral therapy in humans. Our results underscore the power of image-based full cycle infection assays in identifying viral inhibitors with clinical potential.

Simple Summary We determined the sensitivity of several conjunctival melanoma cell lines to kinase inhibitors as well as a few other inhibitors using an image-based high throughput drug screening assay with 542 compounds. All cell lines demonstrated sensitivity to cell cycle inhibition, especially with polo-like inhibitors. The response to MAPK pathway inhibition was better in the presence of BRAF(V600E) mutations, while the response to PI3k/mTOR inhibition was better in the presence of the NRAS(Q61L) mutation. Our study uncovers a large panel of new vulnerabilities in conjunctival melanoma and establishes the background for the expansion of therapeutic options in the management of this tumor. Recent evidence suggests that numerous similarities exist between the genomic landscapes of both conjunctival and cutaneous melanoma. Since alterations of several components of the MAP kinases, PI3K/mTOR, and cell cycle pathways have been reported in conjunctival melanoma, we decided to assess the sensitivity of conjunctival melanoma to targeted inhibition mostly of kinase inhibitors. A high content drug screening assay based on automated fluorescence microscopy was performed in three conjunctival melanoma cell lines with different genomic backgrounds with 489 kinase inhibitors and 53 other inhibitors. IC50 and apoptosis induction were respectively assessed for 53 and 48 compounds. The genomic background influenced the response to MAK and PI3K/mTOR inhibition, more specifically cell lines with BRAF (V600E) mutations were more sensitive to BRAF/MEK inhibition, while CRMM2 bearing the NRAS(Q61L) mutation was more sensitive to PI3k/mTOR inhibition. All cell lines demonstrated sensitivity to cell cycle inhibition, being more pronounced in CRMM2, especially with polo-like inhibitors. Our data also revealed new vulnerabilities to Hsp90 and Src inhibition. This study demonstrates that the genomic background partially influences the response to targeted therapy and uncovers a large panel of potential vulnerabilities in conjunctival melanoma that may expand available options for the management of this tumor.

In vitro differentiating adipocytes are sensitive to liquid manipulations and have the tendency to float. Assessing adipocyte differentiation using current microscopy techniques involves cell staining and washing, while using flow cytometry involves cell retrieval in suspension. These methods induce biases, are difficult to reproduce, and involve tedious optimizations. In this study, we present digital holographic microscopy (DHM) as a label-free, nonperturbing means to quantify lipid droplets in differentiating adipocytes in a robust medium- to high-throughput manner. Taking advantage of the high refractive index of lipid droplets, DHM can assess the production of intracellular lipid droplets by differences in phase shift in a quantitative manner. Adipocytic differentiation, combined with other morphological features including cell confluence and cell death, was tracked over 6 days in live OP9 mesenchymal stromal cells. We compared DHM with other currently available methods of lipid droplet quantification and demonstrated its robustness with modulators of adipocytic differentiation in a dose-responsive manner. This study suggests DHM as a novel marker-free nonperturbing method to study lipid droplet accumulation and may be envisioned for drug screens and mechanistic studies on adipocytic differentiation.

The ionotropic GABAA receptors represent the main target for different groups of widely used drugs having hypnotic and anxiolytic effects. So far, most approaches used to assess GABA activity involve invasive low -throughput electrophysiological techniques or rely on fluorescent dyes, preventing the ability to conduct noninvasive and thus nonperturbing screens. To address this limitation, we have developed an automated marker-free cell imaging method, based on digital holographic microscopy (DHM). This technology allows the automatically screening of compounds in multiple plates without having to label the cells or use special plates. This methodological approach was first validated by screening the GABAA receptor expressed in HEK cells using a selection of active compounds in agonist, antagonist, and modulator modes. Then, in a second blind screen of a library of 3041 compounds (mostly composed of natural products), 5 compounds having a specific agonist action on the GABAA receptor were identified. The hits validated from this unbiased screen were the natural products muscimol, neurosteroid alphaxalone, and three compounds belonging to the avermectin family, all known for having an agonistic effect on the GABAA receptor. The results obtained were exempt from false negatives (structurally similar unassigned hits), and false-positive hits were detected and discarded without the need for performing electrophysiological measurements. The outcome of the screen demonstrates the applicability of our screening by imaging method for the discovery of new chemical structures, particularly regarding chemicals interacting with the ionotropic GABAA receptor and more generally with any ligand-gated ion channels and transporters.

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.

Parkinson’s disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (α-syn) into intraneuronal inclusions named Lewy bodies (LB). Although it is widely believed that α-syn plays a central role in the pathogenesis of PD and synucleinopathies, the processes that govern α-syn fibrillization and LB formation in the brain remain poorly understood. In this work, we sought to reverse engineer LBs and dissect the spatiotemporal events involved in their biogenesis at the genetic, molecular, biochemical, structural, and cellular levels. Toward this goal, we took advantage of a seeding-based model of α-syn fibril formation in primary neurons and further developed this model to generate the first neuronal model that reproduces the key events leading to LB formation; including seeding, fibrillization, and the formation of LB-like inclusions that recapitulate many of the biochemical, structural, and organizational features of LBs found in post-mortem human PD brain tissues. Next, we applied an integrative approach combining confocal and correlative light-electron microscopy (CLEM) imaging methods with biochemical profiling of α-syn species and temporal proteomic and transcriptomic analyses to dissect the molecular events associated with LB formation and maturation and to elucidate their contributions to neuronal dysfunctions and neurodegeneration in PD and synucleinopathies. The results from these studies demonstrate that LB formation involves a complex interplay between α-syn fibrillization, post-translational modifications, and interactions between α-syn aggregates and membranous organelles, including mitochondria and the autophagosome and endolysosome. Furthermore, we demonstrate that the process of LB formation and maturation, rather than simply fibril formation, is the major driver of neurodegeneration through disruption of cellular functions and inducing mitochondria damage and deficits, as well as synaptic dysfunctions. Having a neuronal model that allows for unlinking of the key processes involved in LB formation is crucial for elucidating the molecular and cellular determinants of each process and their contributions to neuronal dysfunction and degeneration in PD and synucleinopathies. Such a model is essential to efforts to identify and investigate the mode of action and toxicity of drug candidates targeting α-syn aggregation and LB formation.

We automated a system for performing quantitative cellular assays using label-free Digital Holographic Microscopy (DHM) and validated its applicability for cell viability assays in a drug screening context 1-2. Our system allows performing fast and cost-effective screening assays for a wide range of chemical biology and drug discovery applications. In addition to classic phenotypes recordings related to morphological changes and cell movements, we have demonstrated that target-based cellular assays can also be addressed by DHM for therapeutically relevant chloride channel receptors 3. DH-imaging (DHI) technology, scalable for screening by imaging approaches in a high-throughput manner can also deliver highly informative data through long term experiments (time lapse). Examples of label free imaging screens we validated or currently under development will be described:Profiling cancer cell-line sensitivities with small moleculesMonitoring the activity of the GABA A chloride channel receptor ('an optical electrode')Assessing cardiotoxycity of compounds using human cardiomyocytes derived from induced pluripotent stem cells (iPSCs) 4.

In previous work, we showed that telomeres of normal cells are organized within the 3D space of the interphase nucleus in a nonoverlapping and cell cycle-dependent manner. This order is distorted in tumor cell nuclei where telomeres are found in close association forming aggregates of various numbers and sizes. Here we show that c-Myc overexpression induces telomeric aggregations in the interphase nucleus. Directly proportional to the duration of c-Myc deregulation, we observe three or five cycles of telomeric aggregate formation in interphase nuclei. These cycles reflect the onset and propagation of breakage-bridge-fusion cycles that are initiated by end-to-end telomeric fusions of chromosomes. Subsequent to initial chromosomal breakages, new fusions follow and the breakage-bridge-fusion cycles continue. During this time, nonreciprocal translocations are generated. c-Myc-dependent remodeling of the organization of telomeres thus precedes the onset of genomic instability and subsequently leads to chromosomal rearrangements. Our findings reveal that c-Myc possesses the ability to structurally modify chromosomes through telomeric fusions, thereby reorganizing the genetic information.

Cellular assays using label-free Digital Holographic Microscopy (DHM) have been previously validated for cell viability assays in a drug screening context. Our automated DHM system allows performing fast and cost-effective screening assays for a wide range of applications for monitoring cell morphological changes and cell movements upon interaction with interfering compounds. In addition to these classic phenotypic assays, it has been demonstrated that target-based cellular assays can also be addressed by DHM for therapeutically relevant chloride channel receptors. Our DH-imaging (DHI) technology, potentially scalable for screening by imaging approaches in a high-throughput manner can also deliver highly informative data through long term experiments. Three examples of phenotypic screens are detailed in the present chapter: a label-free profiling approach, a cell proliferation assay, and methods for monitoring the activity of the GABAA chloride channel receptor.

The canonical Wnt signaling pathway plays key roles in stem-cell maintenance, progenitor cell expansion, and lineage decisions. Transcriptional responses induced by Wnt depend on the association of either beta-catenin or gamma-catenin with lymphoid enhancer factor/T cell factor transcription factors. Here we show that hematopoiesis, including thymopoiesis, is normal in the combined absence of beta- and gamma-catenin. Double-deficient hematopoietic stem cells maintain long-term repopulation capacity and multilineage differentiation potential. Unexpectedly, 2 independent ex vivo reporter gene assays show that Wnt signal transmission is maintained in double-deficient hematopoietic stem cells, thymocytes, or peripheral T cells. In contrast, Wnt signaling is strongly reduced in thymocytes lacking TCF-1 or in nonhematopoietic cells devoid of beta-catenin. These data provide the first evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of beta- and gamma-catenin.

Extrachromosomal elements (EEs) were first discovered as minute chromatin bodies [Cox et al. Minute chromatin bodies in malignant tumors of childhood. Lancet 1965;62:55-8], and subsequently characterized as small circular DNA molecules physically separated from chromosomes. They include episomes, minichromosomes, small polydispersed DNAs or double minutes. This review focuses on eukaryotic EEs generated by genome rearrangements under physiological or pathological conditions. Some of those rearrangements occur randomly, but others are strictly non-random, highly regulated, and involve specific chromosomal locations (V(D)J-recombination, telomere maintenance mechanisms, c-myc deregulation). The multiple mechanisms of EEs formation are strongly interconnected and frequently linked to gene amplification. Identification of genes located on EEs will undoubtedly allow a better understanding of genome dynamics and oncogenic pathways.

The proto-oncogene c-myc has been the subject of intensive research since its discovery. It is already known that this oncogene targets multiple pathways for the initiation and promotion of tumor formation, and that deregulation of this protein is observed in numerous cancers. However, despite the plethora of information gathered, the exact role and mechanism of action of the protein still remains enigmatic. This review focuses on the role of the c-Myc protein in the induction of genomic instability and its link with the development of cancer. We briefly describe c-Myc protein, its binding partners and downstream targets as well as its role in inducing genomic instability and the c-myc-related diseases in humans and mice with regard to genomic instability. This review emphasizes the notions that c-Myc is a multifunctional protein which also affects the stability of the whole genome and triggers the initiation of a complex network of genomic instability and therefore acts beyond the characteristics of classical transcription factors that only regulate a limited number of downstream targets. We propose that c-Myc is a structural modifier of the genome that affects the nuclear organization and an important molecule in tumor cell progression through the induction of genomic instability.

Objective Some phenotypic and functional properties of lymphocytes from bone marrow or peripheral blood stem cell donors were compared in a randomized study. Materials and Methods Lymphocyte subsets were analyzed by immunocytometry in blood harvested from bone marrow donors (n = 27) and from peripheral blood stem cell donors before and after granulocyte colony-stimulating factor mobilization (n = 23) and in bone marrow and peripheral blood stem cell grafts. Results Granulocyte colony-stimulating factor mobilization increased the blood T and B, but not NK, lymphocyte counts. All lymphocyte counts were ∼10-fold higher in peripheral blood stem cell grafts than in bone marrow grafts. Analysis of CD25, CD95, HLA-DR, and CD45RA expression shows that T-cell activation level was lower after granulocyte colony-stimulating factor mobilization. Similarly, granulocyte colony-stimulating factor reduced by twofold to threefold the percentage of interferon-γ, interleukin-2, and tumor necrosis factor-α–secreting cells within the NK, NK-T, and T-cell subsets and severely impaired the potential for interferon-γ production at the single-cell level. mRNA levels of both type 1 (interferon-γ, interleukin-2) and type 2 (interleukin-4, interleukin-13) cytokines were ∼10-fold lower in peripheral blood stem cell grafts than in bone marrow grafts. This reduced potential of cytokine production was not associated with a preferential mobilization of so-called "suppressive" cells (CD3 + CD4 − CD8 − , CD3 + CD8 + CD56 + , or CD3 + TCRVA24 + CD161 + ), nor with a modulation of killer cell receptors CD161, NKB1, and CD94 expression by NK, NK-T, or T cells. Conclusion Our data demonstrate in a randomized setting that quantitative as well as qualitative differences exist between a bone marrow and a peripheral blood stem cell graft, whose ability to produce type 1 and type 2 cytokines is impaired.

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-β-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multiround infection, nelfinavir mesylate., Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-β-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multiround infection, nelfinavir mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of human immunodeficiency virus (HIV) aspartyl protease that is used to treat AIDS. It is not effective against single-round HAdV infections. Here, we show that nelfinavir inhibits lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighboring uninfected cells. HAdV lacking ADP was insensitive to nelfinavir but gave rise to comet-shaped foci, indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and -B14p1 lacking ADP were highly sensitive to nelfinavir, although HAdV-A31, -B3, -B7, -B11, -B16, -B21, -D8, -D30, and -D37 were less sensitive. Conspicuously, nelfinavir uncovered slow-growing round HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of nonlytic cell-to-cell transmission. Our study demonstrates the repurposing potential of nelfinavir with postexposure efficacy against different HAdVs and describes an alternative nonlytic cell-to-cell transmission mode of HAdV.