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Spheroid image analysis, 3D invasion image analysis and antibodies list.

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In vivo imaging of SK-N-BE(2) tumor growth in NB orthotopic mice.

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.

Scope: Epidemiological evidence suggests that a reduced risk of colorectal cancer (CRC) is correlated with high consumption of fruits and vegetables, which are major sources of fiber and phytochemicals, such as flavan-3-ols. However, it remains unknown how these phytochemicals and their specific gut-related metabolites may alter cancer cell behavior. Methods and results: A focused screening using native (poly)phenols and gut microbial metabolites (GMMs) on 3D HCT116 spheroids is carried out using a high-throughput imaging approach. Dose–responses, IC50, and long-term exposure are calculated for the most promising native (poly)phenols and GMMs. As a result, this research shows that (poly)phenol catabolites may play a key role in preventing cancer propagation. Indeed, µM concentration levels of (4R)-5-(3ʹ,4ʹ-dihydroxyphenyl)-γ-valerolactone significantly decrease spheroid size at early stages of spheroid aggregation and gene expression of matrix metalloproteinases. Conclusion: A chronic exposure to (4R)-5-(3ʹ,4ʹ-dihydroxyphenyl)-γ-valerolactone may lead to a reduced CRC risk. Daily intake of monomeric, oligomeric, and polymeric flavan-3-ols may increase the colonic concentrations of this metabolite, and, in turn, this compound may act locally interacting with intestinal epithelial cells, precancerous and cancer cells.

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., Screening small‐molecule agonists for the ability to modulate accumulation of toxic polypeptides suggests PKA signaling, proteasomal and autophagy pathways as potential therapeutic targets for C9orf72‐associated ALS/FTD.

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) swabs, which requires the intervention of trained personnel. Given the ease of collection, saliva has been proposed as a possible substitute to support testing at the population level. Here we describe the set-up of a laboratory, in an academic context, for the high-throughput screening of SARS-CoV-2 in the saliva from the community. A novel saliva collection device was designed to favour the safe and correct acquisition of the sample as well as the processivity of the downstream molecular analysis. To test the performance of the system,1025 paired saliva and nasopharyngeal samples were collected from individuals recruited at a public drive through testing facility and analysed in parallel. An overall moderate concordance (68%) between the two tests was found, with evidence that neither test can diagnose the infection in 100% of the cases. While the two tests performed equally well in symptomatic individuals, their discordance was mainly restricted to samples from convalescent individuals. The saliva test was at least as effective as NP swabs in asymptomatic individuals recruited for contact tracing. Our study, therefore, indicates that saliva testing can be a reliable tool for wide-scale COVID-19 screening in the community.

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.

The pro-apoptotic property of n-BuOH extract of Limonium duriusculum (BEL) and its major isolated components [apigenin (APG1) and apigenin7-O-β-D-(6''-methylglucuronide) (APG2)] were tested. The anti-proliferative IC50 of BEL and APG1 was quantified as 7.60 µg/mL and 25.74 µM respectively, while APG2 did not affect cell proliferation in HCT116 p53 wild type cells. Growth inhibition by BEL treatment was associated with reduced signaling from the MAP kinase, activation of the p53 response pathway and PARP cleavage. The multi-effect of Limonium duriusculum could be due through their major apigenin compounds and the other bioactive constituents that may possibly act in synergy to exercise the most favorable anti-tumor activities.

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.

The pro-apoptotic property of

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrP

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.

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.

Prion diseases are neurodegenerative conditions characterized by the conformational conversion of the cellular prion protein (PrPC), an endogenous membrane glycoprotein of uncertain function, into PrPSc, a pathological isoform that replicates by imposing its abnormal folding onto PrPC molecules. A great deal of evidence supports the notion that PrPC plays at least two roles in prion diseases, by acting as a substrate for PrPSc replication, and as a mediator of its toxicity. This conclusion was recently supported by data suggesting that PrPC may transduce neurotoxic signals elicited by other disease-associated protein aggregates. Thus, PrPC may represent a convenient pharmacological target for prion diseases, and possibly other neurodegenerative conditions. Here, we sought to characterize the activity of chlorpromazine (CPZ), an antipsychotic previously shown to inhibit prion replication by directly binding to PrPC. By employing biochemical and biophysical techniques, we provide direct experimental evidence indicating that CPZ does not bind PrPC at biologically relevant concentrations. Instead, the compound exerts anti-prion effects by inducing the relocalization of PrPC from the plasma membrane. Consistent with these findings, CPZ also inhibits the cytotoxic effects delivered by a PrP mutant. Interestingly, we found that the different pharmacological effects of CPZ could be mimicked by two inhibitors of the GTPase activity of dynamins, a class of proteins involved in the scission of newly formed membrane vesicles, and recently reported as potential pharmacological targets of CPZ. Collectively, our results redefine the mechanism by which CPZ exerts anti-prion effects, and support a primary role for dynamins in the membrane recycling of PrPC, as well as in the propagation of infectious prions.

The phyllosphere is colonized by complex microbial communities, which are adapted to the harsh habitat. Although the role and ecology of nonpathogenic microorganisms of the phyllosphere are only partially understood, leaf microbiota could have a beneficial role in plant growth and health. Pesticides and biocontrol agents are frequently applied to grapevines, but the impact on nontarget microorganisms of the phyllosphere has been marginally considered. In this study, we investigated the effect of a chemical fungicide (penconazole) and a biological control agent ( Lysobacter capsici AZ78) on the leaf microbiota of the grapevine at three locations. Amplicons of the 16S rRNA gene and of the internal transcribed spacer were sequenced for bacterial and fungal identification, respectively. Pyrosequencing analysis revealed that the richness and diversity of bacterial and fungal populations were only minimally affected by the chemical and biological treatments tested, and they mainly differed according to grapevine locations. Indigenous microbial communities of the phyllosphere are adapted to environmental and biotic factors in the areas where the grapevines are grown, and they are resilient to the treatments tested. The biocontrol properties of phyllosphere communities against downy mildew differed among grapevine locations and were not affected by treatments, suggesting that biocontrol communities could be improved with agronomic practices to enrich beneficial populations in vineyards.

Here, we report the surprising and, to our knowledge, unique example of horizontal interkingdom transfer of a human opportunistic pathogen (Propionibacterium acnes) to a crop plant (the domesticated grapevine Vitis vinifera L.). Humans, like most organisms, have established a long-lasting cohabitation with a variety of microbes, including pathogens and gut-associated bacteria. Studies which have investigated the dynamics of such associations revealed numerous cases of bacterial host switches from domestic animals to humans. Much less is, however, known about the exchange of microbial symbionts between humans and plants. Fluorescent in situ hybridization localized P. acnes in the bark, in xylem fibers, and, more interestingly, inside pith tissues. Phylogenetic and population genetic analyses suggest that the establishment of the grapevine-associated P. acnes as obligate endophyte is compatible with a recent transfer event, likely during the Neolithic, when grapevine was domesticated.

Summary Endophytic microorganisms asymptomatically colonise plant tissues. Exploring the assembly dynamics of bacterial endophytic communities is essential to understand the functioning of the plant holobiont and to optimise their possible use as biopesticides or plant biostimulants. We studied the variation in endophytic communities in above and below-ground organs in Vitis vinifera in the field. To understand the specific effect of temperature on endophytic communities, a separate experiment was set up where grapevine cuttings were grown under controlled conditions at three different temperatures. Our findings reveal the succession of endophytic communities over the year. Endophytic communities of roots and stems differ in terms of composition and dynamic response to temperature. Noticeably, compositional differences during the seasons affected bacterial taxa more in stems than in roots, suggesting that roots offer a more stable and less easily perturbed environment. Correlation abundance networks showed that the presence of several taxa (including Bradyrhizobium, Burkholderia, Dyella, Mesorhizobium, Propionibacterium, and Ralstonia) is linked in both the field and the greenhouse. This article is protected by copyright. All rights reserved.

Neurodegenerative diseases (NDs) are often associated with the presence of misfolded protein inclusions. The chaperone HSPB8 is upregulated in mice, the human brain and muscle structures affected during NDs progression. HSPB8 exerts a potent pro-degradative activity on several misfolded proteins responsible for familial NDs forms. Here, we demonstrated that HSPB8 also counteracts accumulation of aberrantly localized misfolded forms of TDP-43 and its 25 KDa fragment involved in most sporadic cases of Amyotrophic Lateral Sclerosis (sALS) and of Fronto Lateral Temporal Dementia (FLTD). HSPB8 acts with BAG3 and the HSP70/HSC70-CHIP complex enhancing the autophagic removal of misfolded proteins. We performed a high-through put screening (HTS) to find small molecules capable of inducing HSPB8 in neurons for therapeutic purposes. We identified two compounds, colchicine and doxorubicin, that robustly up-regulated HSPB8 expression. Both colchicine and doxorubicin increased the expression of the master regulator of autophagy TFEB, the autophagy linker p62/SQSTM1 and the autophagosome component LC3. In line, both drugs counteracted the accumulation of TDP-43 and TDP-25 misfolded species responsible for motoneuronal death in sALS. Thus, analogs of colchicine and doxorubicin able to induce HSPB8 and with better safety and tolerability may result beneficial in NDs models.

We studied the distribution of fungal endophytes of grapevine ( Vitis vinifera L.) plants in a subalpine area of northern Italy, where viticulture is of high economic relevance. We adopted both cultivation-based and cultivation-independent approaches to address how various anthropic and nonanthropic factors shape microbial communities. Grapevine stems were harvested from several locations considering organic and integrated pest management (IPM) and from the cultivars Merlot and Chardonnay. Cultivable fungi were isolated and identified by internal-transcribed-spacer sequence analysis, using a novel colony-PCR method, to amplify DNA from fungal specimens. The composition of fungal communities was assessed using a cultivation-independent approach, automated ribosomal intergenic spacer analysis (ARISA). Multivariate statistical analysis of both culture-dependent and culture-independent data sets was convergent and indicated that fungal endophytic communities in grapevines from organically managed farms were different from those from farms utilizing IPM. Fungal communities in plants of cv. Merlot and cv. Chardonnay overlapped when analyzed using culture-dependent approaches but could be partially resolved using ARISA fingerprinting.

Plant pathogens and endophytes co-exist and often interact with the host plant and within its microbial community. The outcome of these interactions may lead to healthy plants through beneficial interactions, or to disease through the inducible production of molecules known as virulence factors. Unravelling the role of virulence in endophytes may crucially improve our understanding of host-associated microbial communities and their correlation with host health. Virulence is the outcome of a complex network of interactions, and drawing the line between pathogens and endophytes has proven to be conflictive, as strain-level differences in niche overlapping, ecological interactions, state of the host’s immune system and environmental factors are seldom taken into account. Defining genomic differences between endophytes and plant pathogens is decisive for understanding the boundaries between these two groups. Here we describe the major differences at the genomic level between seven grapevine endophytic test bacteria, and twelve reference strains. We describe the virulence factors detected in the genomes of the test group, as compared to endophytic and non-endophytic references, to better understand the distribution of these traits in endophytic genomes. To do this, we adopted a comparative whole-genome approach, encompassing BLAST-based searches through the GUI-based tools Mauve and BRIG as well as calculating the core and accessory genomes of three genera of enterobacteria. We outline divergences in metabolic pathways of these endophytes and reference strains, with the aid of the online platform RAST. We present a summary of the major differences that help in the drawing of the boundaries between harmless and harmful bacteria, in the spirit of contributing to a microbiological definition of endophyte.

Microbial plant endophytes are receiving ever-increasing attention as a result of compelling evidence regarding functional interaction with the host plant. Microbial communities in plants were recently reported to be influenced by numerous environmental and anthropogenic factors, including soil and pest management. In this study we used automated ribosomal intergenic spacer analysis (ARISA) fingerprinting and pyrosequencing of 16S rDNA to assess the effect of organic production and integrated pest management (IPM) on bacterial endophytic communities in two widespread grapevines cultivars (Merlot and Chardonnay). High levels of the dominant Ralstonia, Burkholderia and Pseudomonas genera were detected in all the samples We found differences in the composition of endophytic communities in grapevines cultivated using organic production and IPM. Operational taxonomic units (OTUs) assigned to the Mesorhizobium, Caulobacter and Staphylococcus genera were relatively more abundant in plants from organic vineyards, while Ralstonia, Burkholderia and Stenotrophomonas were more abundant in grapevines from IPM vineyards. Minor differences in bacterial endophytic communities were also found in the grapevines of the two cultivars.