ABSTRACT
Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.
Subject(s)
C9orf72 Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Dipeptides/metabolism , Proteolysis , Small Molecule Libraries/pharmacology , Animals , Cell Line , Codon, Initiator/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA Repeat Expansion/genetics , Disease Models, Animal , Drosophila/drug effects , Frontotemporal Dementia/pathology , HEK293 Cells , High-Throughput Screening Assays , Humans , Induced Pluripotent Stem Cells/pathology , Isoquinolines/pharmacology , Longevity/drug effects , Motor Neurons/drug effects , Motor Neurons/pathology , Protein Biosynthesis/drug effects , Proteolysis/drug effects , RNA Interference , Sulfonamides/pharmacologyABSTRACT
Neuroblastoma (NB) is the most commonly diagnosed extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. Although the 5-year survival rate of patients with a high-risk disease has increased in recent decades, NB remains a challenge in pediatric oncology, and the identification of novel potential therapeutic targets and agents is an urgent clinical need. The RNA-binding protein LIN28B has been identified as an oncogene in NB and is associated with a poor prognosis. Given that LIN28B acts by negatively regulating the biogenesis of the tumor suppressor let-7 miRNAs, we reasoned that selective interference with the LIN28B/let-7 miRNA interaction would increase let-7 miRNA levels, ultimately leading to reduced NB aggressiveness. Here, we selected (-)-epigallocatechin 3-gallate (EGCG) out of 4959 molecules screened as the molecule with the best inhibitory activity on LIN28B/let-7 miRNA interaction and showed that treatment with PLC/PLGA-PEG nanoparticles containing EGCG (EGCG-NPs) led to an increase in mature let-7 miRNAs and a consequent inhibition of NB cell growth. In addition, EGCG-NP pretreatment reduced the tumorigenic potential of NB cells in vivo. These experiments suggest that the LIN28B/let-7 miRNA axis is a good therapeutic target in NB and that EGCG, which can interfere with this interaction, deserves further preclinical evaluation.
Subject(s)
Catechin , MicroRNAs , Neuroblastoma , RNA-Binding Proteins , Catechin/analogs & derivatives , Catechin/pharmacology , Neuroblastoma/genetics , Neuroblastoma/pathology , Neuroblastoma/metabolism , Neuroblastoma/drug therapy , MicroRNAs/genetics , MicroRNAs/metabolism , Humans , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Animals , Mice , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Cell Proliferation/drug effects , Xenograft Model Antitumor Assays , Mice, NudeABSTRACT
Modern societies produce ever-increasing amounts of waste, e.g. organic fraction of municipal solid waste (OFMSW). According to the best available techniques, OFMSW should be treated through anaerobic digestion to recover biogas and subsequently composted. An innovative scheme is under investigation, where anaerobic digestion is combined with hydrothermal carbonization (HTC) and composting. The final product, referred to as hydrochar co-compost (HCO), is under study to be used as an unconventional soil improver/fertilizer. Recent studies showed that HCO is not phytotoxic. However, nothing is known about the toxicity of HCO on cells as part and organisms as a whole. This study aims to investigate in vitro genotoxicity and cytotoxicity of the HCO and its precursors in the production process. In particular, we tested water and methanolic extracts of HCO (WEHCO and MEHCO) from one side and methanolic extracts of hydrochar (MEH) and OFMSW digestate (MED) as well as liquor produced downstream HTC (HTCL) from the other side. Genotoxicity was investigated using cytokinesis-block micronucleus assay in Chinese Hamster Ovarian K1 (CHO-K1) cells. Cytotoxicity was tested in vitro against a panel of human cells line. Zebrafish embryo toxicity upon MEH treatment was also investigated. Results show that incubation of CHO-K1 cells with all the tested samples at different concentrations did not cause any induction of micronucleus formation compared to the vehicle-treated control. Treatment of cells with MEH, MED, HTCL and MEHCO, but not WEHCO, induced some degree of cytotoxicity and MEH showed to be more cytotoxic against tested cells compared to the MEHCO. Toxicity effect at the highest tested concentrations of MEH on zebrafish embryos resulted in coagulation, induction of pericardial edema and death. In conclusion, the hydrochar co-compost cytotoxicity is similar to standard compost cytotoxicity. Hence composting the hydrochar from OFMSW digestate is a good step to eliminate the cytotoxicity of hydrochar.
Subject(s)
Composting , Refuse Disposal , Anaerobiosis , Animals , Biofuels , Humans , Refuse Disposal/methods , Soil , Solid Waste , ZebrafishABSTRACT
Therapeutic approaches for cutaneous melanoma are flourishing, but despite promising results, there is an increasing number of reported primary or secondary resistance to the growing sets of drugs approved for therapy in the clinics. Combinatorial approaches may overcome resistance, as they may tackle specific weaknesses of melanoma cells, not sufficient on their own, but effective in combination with other therapies. The transgenic zebrafish line kita:ras develops melanoma with high frequency. At 3 dpf, transgenic kita:ras larvae show a hyperpigmentation phenotype as earliest evidence of abnormal melanocyte growth. Using this model, we performed a chemical screen based on automated detection of a reduction of melanocyte number caused by any of 1280 FDA or EMA approved drugs of the library. The analysis showed that 55 molecules were able to reduce by 60% or more the number of melanocytes per embryo. We further tested two compounds for each of the 5 classes, and a farnesyltransferase inhibitor (Lonafarnib), that inhibits an essential post-translational modification of HRAS and suppresses the hyperpigmentation phenotype. Combinations of Clotrimazole and Lonafarnib showed the most promising results in zebrafish embryos, allowing a dose reduction of both drugs. We performed validation of these observations in the metastatic human melanoma cell line A375M, and in normal human epithelial melanocytes (NHEM) in order to investigate the mechanism of action of Clotrimazole in blocking the proliferation of transformed melanocytes. Viability assay and analysis of energy metabolism in Clotrimazole treated cells show that this drug specifically affects melanoma cells in vitro and transformed melanocytes in vivo, having no effects on NHEM or wild type larvae. Similar effects were observed with another hit of the same class, Miconazole. Furthermore, we show that the effects of Clotrimazole are mediated by the inhibition of hexokinase activity, which is lethal to the abnormal metabolic profile of melanoma cells in vitro and in vivo. Thus, our study shows that the zebrafish can provide a phenotype-rich assay for fully automated screening approaches to identify drugs for synthetic lethal treatment in melanoma and suggest further testing of Clotrimazole in combinatorial treatments.
Subject(s)
Antineoplastic Agents/pharmacology , Clotrimazole/pharmacology , Melanoma/drug therapy , Piperidines/pharmacology , Pyridines/pharmacology , Skin Neoplasms/drug therapy , Animals , Animals, Genetically Modified , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Disease Models, Animal , Drug Screening Assays, Antitumor/methods , Farnesyltranstransferase/antagonists & inhibitors , Humans , Melanocytes/metabolism , Melanoma/metabolism , Miconazole/pharmacology , Skin Neoplasms/metabolism , Zebrafish , Melanoma, Cutaneous MalignantABSTRACT
The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.
Subject(s)
Cell Membrane/chemistry , PrPC Proteins/analysis , Prions/antagonists & inhibitors , Animals , Casein Kinase II/antagonists & inhibitors , Cell Line, Tumor , Cell Survival/drug effects , Drug Evaluation, Preclinical/methods , Fluorescent Dyes , Gene Expression , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , HEK293 Cells , Harmaline/analogs & derivatives , Harmaline/pharmacology , Hematoxylin/analogs & derivatives , Hematoxylin/pharmacology , Humans , Mice , Neuroblastoma , PrPC Proteins/genetics , Prions/biosynthesis , Prions/toxicity , Quinacrine/pharmacology , Tacrolimus/pharmacologyABSTRACT
Iron is an essential cellular nutrient, being a critical cofactor of several proteins involved in cell growth and replication. Compared with normal cells, neoplastic cells have been shown to require a greater amount of iron, thus laying the basis for the promising anticancer activity of iron chelators. In this work, we evaluated the effects of molecules with iron chelation activity on neuroblastoma (NB) cell lines. Of the 17 iron chelators tested, six reduced cell viability of two NB cell lines with an inhibition of growth of 50% below 10 µM; four of the six molecules-ciclopirox olamine (CPX), piroctone, 8-hydroxyquinoline, and deferasirox-were also shown to efficiently chelate intracellular iron within minutes after addition. Effects on cell viability of one of the compounds, CPX, were indeed dependent on chelation of intracellular iron and mediated by both G0/G1 cell cycle block and induction of apoptosis. By combined transcriptome and translatome profiling we identified early translational downregulation of several members of the heat shock protein group as a specific effect of CPX treatment. We functionally confirmed iron-dependent depletion of HSP90 and its client proteins at pharmacologically achievable concentrations of CPX, and we extended this effect to piroctone, 8-hydroxyquinoline, and deferasirox. Given the documented sensitivity of NB cells to HSP90 inhibition, we propose CPX and other iron chelators as investigational antitumor agents in NB therapy.
Subject(s)
Down-Regulation/physiology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/biosynthesis , Iron Chelating Agents/pharmacology , Neuroblastoma/metabolism , Protein Biosynthesis/physiology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/physiology , Down-Regulation/drug effects , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Humans , MCF-7 Cells , Protein Biosynthesis/drug effectsABSTRACT
BACKGROUND: The nerve growth factor (NGF) receptor tyrosine-kinase TrkA is a well-known determinant of the melanocytic lineage, through modulation of the MAPK and AKT cascades. While TrkA gene is frequently rearranged in cancers, its involvement in malignant melanoma (MM) development is still unclear. METHODS: We analyzed a dataset of primary cutaneous MM (n = 31) by array comparative genomic hybridization (aCGH), to identify genomic amplifications associated with tumor progression. The analysis was validated by genomic quantitative PCR (qPCR) on an extended set of cases (n = 64) and the results were correlated with the clinical outcome. To investigate TrkA molecular pathways and cellular function, we generated inducible activation of the NGF-TrkA signaling in human MM cell lines. RESULTS: We identified amplification of 1q23.1, where the TrkA locus resides, as a candidate hotspot implicated in the progression of MM. Across 40 amplicons detected, segmental amplification of 1q23.1 showed the strongest association with tumor thickness. By validation of the analysis, TrkA gene amplification emerged as a frequent event in primary melanomas (50 % of patients), and correlated with worse clinical outcome. However, experiments in cell lines revealed that induction of the NGF-TrkA signaling produced a phenotype of dramatic suppression of cell proliferation through inhibition of cell division and pronounced intracellular vacuolization, in a way straightly dependent on NGF activation of TrkA. These events were triggered via MAPK activity but not via AKT, and involved p21(cip1) protein increase, compatibly with a mechanism of oncogene-induced growth arrest. CONCLUSIONS: Taken together, our findings point to TrkA as a candidate oncogene in MM and support a model in which the NGF-TrkA-MAPK pathway may mediate a trade-off between neoplastic transformation and adaptive anti-proliferative response.
Subject(s)
Melanoma/genetics , Receptor, trkA/genetics , Skin Neoplasms/genetics , Analysis of Variance , Cell Cycle/physiology , Cell Line, Tumor , Cell Proliferation/physiology , Comparative Genomic Hybridization/methods , Disease Progression , Gene Amplification , Gene Expression Profiling , Humans , Kaplan-Meier Estimate , Melanoma/pathology , Mitogen-Activated Protein Kinase Kinases/physiology , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Receptor, trkA/metabolism , Signal Transduction/physiology , Skin Neoplasms/pathology , Melanoma, Cutaneous MalignantABSTRACT
Identification of prognostic melanoma-associated copy number alterations (CNAs) is still an area of active research. Here, we investigated by high-resolution array comparative genomic hybridization (aCGH) a cohort of 31 paraffin-preserved primary malignant melanomas (MMs), whose prognosis was not predictable on the basis of conventional histopathological parameters. Although we identified a variety of highly recurrent sites of genomic lesions, the total number of CNAs per patient was not a discriminator of MM outcome. Furthermore, validation of aCGH by quantitative PCR on an extended population of 65 MM samples confirmed the absence of predictive value for the most recurrent CNA loci. Instead, our analysis revealed specific prognostic potential of the frequency of homozygous deletions (representing less than 3% of the total CNAs on average per sample), which was strongly associated with sentinel lymph node (SLN) invasion (P = 0.003), and distant metastasis (P = 0.003). Increased number of homozygous deletions was also indicative of poor patient survival (P = 0.01), both in our samples and in an independent validation of public dataset of primary and metastatic MMs. Moreover, we identified 77 hotspots of minimal common homozygous deletions, enriched in genes involved in cell adhesion processes and cell-communication functions, which preferentially accumulated in primary MMs showing the most severe outcome. Therefore, specific loss of gene loci in regions of minimal homozygous deletion may represent a pivotal type of genomic alteration accumulating during MM progression with potential prognostic implication.
Subject(s)
Gene Deletion , Gene Frequency , Homozygote , Melanoma/diagnosis , Skin Neoplasms/diagnosis , DNA Copy Number Variations , Genetic Loci , Humans , Melanoma/secondary , Paraffin Embedding , Prognosis , Skin Neoplasms/pathology , Melanoma, Cutaneous MalignantABSTRACT
The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.
Subject(s)
Chromosomes, Human, Pair 3 , Histone Deacetylase Inhibitors , Leukemia, Myeloid, Acute , MDS1 and EVI1 Complex Locus Protein , Proteogenomics , Animals , Female , Humans , Mice , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Chromosomes, Human, Pair 3/genetics , Gene Expression Regulation, Leukemic/drug effects , Histone Deacetylase Inhibitors/pharmacology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , MDS1 and EVI1 Complex Locus Protein/metabolism , MDS1 and EVI1 Complex Locus Protein/genetics , Proteogenomics/methods , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins c-myc/genetics , Xenograft Model Antitumor AssaysABSTRACT
Integration of genome-wide profiles of DNA copy number alterations (CNAs) and gene expression variations (GEVs) could provide combined power to the identification of driver genes and gene networks in tumors. Here we merge matched genome and transcriptome microarray analyses from neuroblastoma samples to derive correlation patterns of CNAs and GEVs, irrespective of their genomic location. Neuroblastoma correlation patterns are strongly asymmetrical, being on average 10 CNAs linked to 1 GEV, and show the widespread prevalence of long range covariance. Functional enrichment and network analysis of the genes covarying with CNAs consistently point to a major cell function, the regulation of mitotic spindle assembly. Moreover, elevated expression of 14 key genes promoting this function is strongly associated to high-risk neuroblastomas with 1p loss and MYCN amplification in a set of 410 tumor samples (P < 0.00001). Independent CNA/GEV profiling on neuroblastoma cell lines shows that increased levels of expression of these genes are linked to 1p loss. By this approach, we reveal a convergence of clustered neuroblastoma CNAs toward increased expression of a group of prognostic and functionally cooperating genes. We therefore propose gain of function of the spindle assembly machinery as a lesion potentially offering new targets for therapy of high-risk neuroblastoma.
Subject(s)
Chromosome Aberrations , Neuroblastoma/genetics , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Spindle Apparatus/genetics , Cluster Analysis , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Humans , N-Myc Proto-Oncogene Protein , Neuroblastoma/metabolism , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Oncogene Proteins/metabolism , Prognosis , Spindle Apparatus/metabolismABSTRACT
Nucleic acid therapeutics have demonstrated an impressive acceleration in recent years. They work through multiple mechanisms of action, including the downregulation of gene expression and the modulation of RNA splicing. While several drugs based on the former mechanism have been approved, few target the latter, despite the promise of RNA splicing modulation. To improve our ability to discover novel RNA splicing-modulating therapies, we developed HCS-Splice, a robust cell-based High-Content Screening (HCS) assay. By implementing the use of a two-colour (GFP/RFP) fluorescent splicing reporter plasmid, we developed a versatile, effective, rapid, and robust high-throughput strategy for the identification of potent splicing-modulating molecules. The HCS-Splice strategy can also be used to functionally confirm splicing mutations in human genetic disorders or to screen drug candidates. As a proof-of-concept, we introduced a dementia-related splice-switching mutation in the Microtubule-Associated Protein Tau (MAPT) exon 10 splicing reporter. We applied HCS-Splice to the wild-type and mutant reporters and measured the functional change in exon 10 inclusion. To demonstrate the applicability of the method in cell-based drug discovery, HCS-Splice was used to evaluate the efficacy of an exon 10-targeting siRNA, which was able to restore the correct alternative splicing balance.
Subject(s)
Alternative Splicing , RNA Splicing , Humans , RNA Splicing/genetics , Alternative Splicing/genetics , Mutation/geneticsABSTRACT
Neuroblastoma (NB) is an aggressive childhood tumor, with high-risk cases having a 5-year overall survival probability of approximately 50%. The multimodal therapeutic approach for NB includes treatment with the retinoid isotretinoin (13-cis retinoic acid; 13cRA), which is used in the post-consolidation phase as an antiproliferation and prodifferentiation agent to minimize residual disease and prevent relapse. Through small-molecule screening, we identified isorhamnetin (ISR) as a synergistic compound with 13cRA in inhibiting up to 80% of NB cell viability. The synergistic effect was accompanied by a marked increase in the expression of the adrenergic receptor α1B (ADRA1B) gene. Genetic knockout of ADRA1B or its specific blockade using α1/α1B adrenergic antagonists led to selective sensitization of MYCN-amplified NB cells to cell viability reduction and neural differentiation induced by 13cRA, thus mimicking ISR activity. Administration of doxazosin, a safe α1-antagonist used in pediatric patients, in combination with 13cRA in NB xenografted mice exerted marked control of tumor growth, whereas each drug alone was ineffective. Overall, this study identified the α1B adrenergic receptor as a pharmacologic target in NB, supporting the evaluation of adding α1-antagonists to the post-consolidation therapy of NB to more efficiently control residual disease. SIGNIFICANCE: Targeting α-adrenergic receptors synergizes with isotretinoin to suppress growth and to promote differentiation of neuroblastoma, revealing a combinatorial approach for more effective management of the disease and prevention of relapse.
Subject(s)
Isotretinoin , Neuroblastoma , Humans , Mice , Child , Animals , Isotretinoin/pharmacology , Isotretinoin/therapeutic use , Adrenergic alpha-1 Receptor Antagonists/therapeutic use , Cell Line, Tumor , Neoplasm Recurrence, Local , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Neuroblastoma/metabolism , Cell Differentiation , Receptors, Adrenergic/therapeutic use , Recurrence , N-Myc Proto-Oncogene ProteinABSTRACT
Herein, we report a method for the isotopic labelling of hydantoins directly from CO2 by means of trimethyl-λ5-phosphine diiodide mediated carbonyl insertion. The method is suitable for 13C-labelling of diverse substrates and was implementated for 11C-labelling in PET-imaging facilities for the synthesis of radiotracers. Isolated yields of 90% and radiochemical yields of 89% were achieved for hydantoin containing drug candidates in formulation within 30 min with high molar activity (>400 MBq nmol-1).
Subject(s)
Hydantoins , Iodine , Carbon Dioxide/chemistry , Phosphines , RadiopharmaceuticalsABSTRACT
Efficient, wide-scale testing for SARS-CoV-2 is crucial for monitoring the incidence of the infection in the community. The gold standard for COVID-19 diagnosis is the molecular analysis of epithelial secretions from the upper respiratory system captured by nasopharyngeal (NP) or oropharyngeal swabs. Given the ease of collection, saliva has been proposed as a possible substitute to support testing at the population level. Here, we used a novel saliva collection device designed to favour the safe and correct acquisition of the sample, as well as the processivity of the downstream molecular analysis. We tested 1003 nasopharyngeal swabs and paired saliva samples self-collected by individuals recruited at a public drive-through testing facility. An overall moderate concordance (68%) between the two tests was found, with evidence that neither system can diagnose the infection in 100% of the cases. While the two methods performed equally well in symptomatic individuals, their discordance was mainly restricted to samples from convalescent subjects. The saliva test was at least as effective as NP swabs in asymptomatic individuals recruited for contact tracing. Our study describes a testing strategy of self-collected saliva samples, which is reliable for wide-scale COVID-19 screening in the community and is particularly effective for contact tracing.
Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19 Nucleic Acid Testing/standards , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Saliva/virology , COVID-19/diagnosis , COVID-19/virology , Female , Humans , Male , Mass Screening , Nasopharynx/virology , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Specimen Handling/methodsABSTRACT
Core facilities (CFs) provide a centralised access to costly equipment, scientific expertise, experimental design, day-to-day technical support and training of users. CFs have a tremendous impact on research outputs, skills and educational agendas, increasing the competencies of staff, researchers and students. However, the rapid development of new technologies and methodologies for the life sciences requires fast adaptation and development of existing core facilities and their technical and scientific staff. Given the scarcity of well-defined CF career paths, CF staff positions are typically filled by people having followed either academic or technical tracks. Each academic institution follows different policies and often fails to adequately recognize the merits of CF personnel and to support their training efficiently. Thus, the Core Technologies for Life Science association (CTLS), through the Training working group, has conducted an anonymous online survey to assess the training needs of CF personnel, as well as to identify common characteristics and challenges in this relatively new and dynamic career type. 275 individuals, including core managers and directors, technicians, technologists and administrators, participated in the survey. The survey was divided into 2 sections; the first, applied to all respondents, and the second, specifically targeted core management issues. Training needs in technological areas, financial and soft skills, management and administrative issues were surveyed as well. The lack of clarity and consistency regarding established career paths for CF professionals was evident from the second part of the survey, highlighting geographical or cultural differences. Gender balance was achieved and the distribution was always taken into account. The results of this survey highlight a need to develop better training resources for CF staff, to improve their recognition within academic institutions, and to establish a recognized career pathway.
Subject(s)
Curriculum , Universities , Humans , Surveys and QuestionnairesABSTRACT
Glioblastoma stem cells (GSCs) resist current glioblastoma (GBM) therapies. GSCs rely highly on oxidative phosphorylation (OXPHOS), whose function requires mitochondrial translation. Here we explore the therapeutic potential of targeting mitochondrial translation and report the results of high-content screening with putative blockers of mitochondrial ribosomes. We identify the bacterial antibiotic quinupristin/dalfopristin (Q/D) as an effective suppressor of GSC growth. Q/D also decreases the clonogenicity of GSCs in vitro, consequently dysregulating the cell cycle and inducing apoptosis. Cryoelectron microscopy (cryo-EM) reveals that Q/D binds to the large mitoribosomal subunit, inhibiting mitochondrial protein synthesis and functionally dysregulating OXPHOS complexes. These data suggest that targeting mitochondrial translation could be explored to therapeutically suppress GSC growth in GBM and that Q/D could potentially be repurposed for cancer treatment.
Subject(s)
Glioblastoma/genetics , Mitochondria/metabolism , Neoplastic Stem Cells/metabolism , Cell Line, Tumor , Cell Proliferation , HumansABSTRACT
Core facilities (CFs) provide a centralised access to costly equipment, scientific expertise, experimental design, day-to-day technical support and training of users. CFs have a tremendous impact on research outputs, skills and educational agendas, increasing the competencies of staff, researchers and students. However, the rapid development of new technologies and methodologies for the life sciences requires fast adaptation and development of existing core facilities and their technical and scientific staff. Given the scarcity of well-defined CF career paths, CF staff positions are typically filled by people having followed either academic or technical tracks. Each academic institution follows different policies and often fails to adequately recognize the merits of CF personnel and to support their training efficiently. Thus, the Core Technologies for Life Science association (CTLS), through the Training working group, has conducted an anonymous online survey to assess the training needs of CF personnel, as well as to identify common characteristics and challenges in this relatively new and dynamic career type. 275 individuals, including core managers and directors, technicians, technologists and administrators, participated in the survey. The survey was divided into 2 sections; the first, applied to all respondents, and the second, specifically targeted core management issues. Training needs in technological areas, financial and soft skills, management and administrative issues were surveyed as well. The lack of clarity and consistency regarding established career paths for CF professionals was evident from the second part of the survey, highlighting geographical or cultural differences. Gender balance was achieved and the distribution was always taken into account. The results of this survey highlight a need to develop better training resources for CF staff, to improve their recognition within academic institutions, and to establish a recognized career pathway.
ABSTRACT
RNA-binding proteins (RBPs) are pleiotropic factors that control the processing and functional compartmentalization of transcripts by binding primarily to mRNA untranslated regions (UTRs). The competitive and/or cooperative interplay between RBPs and an array of coding and noncoding RNAs (ncRNAs) determines the posttranscriptional control of gene expression, influencing protein production. Recently, a variety of well-recognized and noncanonical RBP domains have been revealed by modern system-wide analyses, underlying an evolving classification of ribonucleoproteins (RNPs) and their importance in governing physiological RNA metabolism. The possibility of targeting selected RNA-protein interactions with small molecules is now expanding the concept of protein "druggability," with new implications for medicinal chemistry and for a deeper characterization of the mechanism of action of bioactive compounds. Here, taking SF3B1, HuR, LIN28, and Musashi proteins as paradigmatic case studies, we review the strategies applied for targeting RBPs, with emphasis on the technological advancements to study protein-RNA interactions and on the requirements of appropriate validation strategies to parallel high-throughput screening (HTS) efforts.
Subject(s)
Drug Discovery/methods , High-Throughput Screening Assays/methods , Ribonucleoproteins/drug effects , Drug Delivery Systems , Protein Binding , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Untranslated RegionsABSTRACT
Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4-64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling.
Subject(s)
Mesenchymal Stem Cells/cytology , Quantum Theory , Adult , Electromagnetic Fields , Female , Humans , Immunophenotyping , Male , Mesenchymal Stem Cells/immunology , Middle Aged , Models, Biological , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain Reaction , Young AdultABSTRACT
Novel druggable targets have been discovered in neuroblastoma (NB), paving the way for more effective treatments. However, children with high-risk NB still show high mortality rates prompting for a search of novel therapeutic options. Here, we aimed at repurposing FDA-approved drugs for NB treatment by performing a high-content screening of a 349 anticancer compounds library. In the primary screening, we employed three NB cell lines, grown as three-dimensional (3D) multicellular spheroids, which were treated with 10 µmol/L of the library compounds for 72 hours. The viability of 3D spheroids was evaluated using a high-content imaging approach, resulting in a primary hit list of 193 compounds. We selected 60 FDA-approved molecules and prioritized drugs with multi-target activity, discarding those already in use for NB treatment or enrolled in NB clinical trials. Hence, 20 drugs were further tested for their efficacy in inhibiting NB cell viability, both in two-dimensional and 3D models. Dose-response curves were then supplemented with the data on side effects, therapeutic index, and molecular targets, suggesting two multiple tyrosine kinase inhibitors, ponatinib and axitinib, as promising candidates for repositioning in NB. Indeed, both drugs showed induction of cell-cycle block and apoptosis, as well as inhibition of colony formation. However, only ponatinib consistently affected migration and inhibited invasion of NB cells. Finally, ponatinib also proved effective inhibition of tumor growth in orthotopic NB mice, providing the rationale for its repurposing in NB therapy. Mol Cancer Ther; 17(7); 1405-15. ©2018 AACR.