EGCG Disrupts the LIN28B/Let-7 Interaction and Reduces Neuroblastoma Aggressiveness.

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.

Alpha-1 Adrenergic Antagonists Sensitize Neuroblastoma to Therapeutic Differentiation.

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.

Streptogramin A derivatives as mitochondrial translation inhibitors to suppress glioblastoma stem cell growth.

New therapeutic strategies for glioblastoma treatment, especially tackling the tumour's glioblastoma stem cell (GSC) component, are an urgent medical need. Recently, mitochondrial translation inhibition has been shown to affect GSC growth, clonogenicity, and self-renewal capability, therefore becoming an attractive therapeutic target. The combination of streptogramins B and A antibiotics quinupristin/dalfopristin (Q/D), which inhibits mitochondrial ribosome function, affects GSCs more effectively in vitro than the standard of care temozolomide. Here, docking calculations based on the cryo-EM structure of the Q/D-bound mitochondrial ribosome have been used to develop a series of streptogramin A derivatives. We obtained twenty-two new and known molecules starting from the dalfopristin and virginiamycin M1 scaffolds. A structure-activity relationship refinement was performed to evaluate the capability of these compounds to suppress GSC growth and inhibit mitochondrial translation, either alone or in combination with quinupristin. Finally, quantitative ultra HPLC-mass spectrometry allowed us to assess the cell penetration of some of these derivatives. Among all, the fluorine derivatives of dalfopristin and virginiamycin M1, (16R)-1e and (16R)-2e, respectively, and flopristin resulted in being more potent than the corresponding lead compounds and penetrating to a greater extent into the cells. We, therefore, propose these three compounds for further evaluation in vivo as antineoplastic agents.

Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency.

A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.

Different approaches to supplement polysaccharide-degrading enzymes in vinification: Effects on color extraction, phenolic composition, antioxidant activity and sensory profiles of Malbec wines.

Enzymes play an essential role in the winemaking process. Besides an improvement in the color of the wine it also enhances the quality, which makes it well worth to develop new strategies of application. The present study examined two approaches to apply microbial enzymes during different winemaking techniques using Malbec grapes from the San Rafael wine region, Mendoza, Argentina. i) Torulaspora delbrueckii was used in sequential inoculation with an autochthonous Saccharomyces cerevisiae strain. The former strain was first inoculated to produce in situ an enzyme complex during winemaking for four days, and subsequently the latter strain was added. The process was carried out under traditional winemaking conditions at 22 °C (Td-Sc). ii) A second approach was through inoculation of a multi-enzyme extract produced by Aureobasidium pullulans m11-2 during pre-fermentative cold maceration (PCM) for four days at 8 °C (PCM-E), and S. cerevisiae was inoculated at the end of the PCM. Controls were carried out without enzymatic treatments (Sc and PMC, respectively). T. delbrueckii started the fermentation and remained viable throughout the process. The fermentation kinetics were not affected in the mixed culture compared with a pure culture of S. cerevisiae. Monitoring of the pectinolytic activity of Td-Sc showed maximum activity (14.2 U mg-1) on day one of the inoculation, while in PCM-E the must activity increased during the PCM until day one of fermentation, reaching a maximum of 16.85 U mg-1. Both enzyme-treated wines maintained their red hue, regardless of the duration of the macerations, while the control wines were characterized by a faster rate of color loss and more yellow tones. In the sensory evaluation, enzymatically treated wines presented greater color intensity, redder and more violet tones, floral and fruit aromas, which are also the distinctive features of the Malbec variety. Anthocyanin values obtained in PCM and in PCM-E were higher than in vinifications without cold maceration (Td-Sc and Sc), which could be attributed to the effectiveness of the maceration technique. Similarly, the composition of stilbenes, especially trans-resveratrol, showed an increase in PCM techniques with or without enzymatic treatment, and both techniques were comparable to one other and statistically superior to traditional winemaking.

Yeast Multi-Enzymatic Systems for Improving Colour Extraction, Technological Parameters and Antioxidant Activity of Wine.

Research background: Wine yeasts are a heterogeneous microbial group with high enzymatic potential that makes them a useful tool in winemaking. With a better understanding of their oenological properties, selection procedures can be optimised to obtain more efficient strains. The present study aims to isolate and select yeasts from wine grape surface by studying their production of enzymes that hydrolyse plant cell wall polymers and by linking them to different technological parameters and antioxidant activity of wines. Experimental approach: Yeasts that are able to produce carbohydrolases and related enzymes of oenological importance were firstly selected on plates and subsequently identified. Then, a secondary selection of yeasts was carried out according to technological effects of their extracellular enzyme extracts on short macerations. In this way, the colour extraction, total polyphenol content, clarification, filterability and antioxidant activity were studied. This approach makes it possible to correlate the microorganism capacity to produce cell wall-depolymerizing enzymes with their technological effects. Results and conclusions: From 366 isolates, 96 strains (26.2%) showed at least one of the polysaccharidase activities and 55 strains (57.3%) of them exhibited activities of multiple enzymes that degrade plant cell wall polymers. Sixteen strains were selected and identified as Aureobasidium, Candida, Debaryomyces, Hanseniaspora, Metschnikowia, Pichia, Saccharomyces and Torulaspora. Pectinolytic enzymes had the highest hydrolytic activity. Aureobasidium pullulans had a broader enzyme blend and higher activity, dominated by pectinases and followed by xylanases and cellulases. Moreover, the Torulaspora delbrueckii m7-2 strain produced high amounts of polysaccharidase and this was strain-dependent. Strains that produced enzyme extracts with a wide range of activities that were also the highest, also had the best chromatic and technological properties. Cluster analysis confirmed that A. pullulans R-22, m11-2, m86-1 and m86-2 and T. delbrueckii m7-2 could be correlated with a better effect on filterability, clarification and extraction of bioactive compounds, encouraging future studies regarding their application in winemaking. Novelty and scientific contribution: The study of yeast multi-enzymatic systems impacting the grape maceration process enables a proper selection criterion for wine yeasts to improve colour extraction, technological parameters and antioxidant activity of Malbec wine. This work shows that A. pullulans and T. delbruekii have a high enzymatic potential for oenological purposes.

Inhibition of mitochondrial translation suppresses glioblastoma stem cell growth.

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.

Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila.

N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.

Interference with ethylene perception at receptor level sheds light on auxin and transcriptional circuits associated with the climacteric ripening of apple fruit (Malus x domestica Borkh.).

Apple (Malus x domestica Borkh.) is a model species for studying the metabolic changes that occur at the onset of ripening in fruit crops, and the physiological mechanisms that are governed by the hormone ethylene. In this study, to dissect the climacteric interplay in apple, a multidisciplinary approach was employed. To this end, a comprehensive analysis of gene expression together with the investigation of several physiological entities (texture, volatilome and content of polyphenolic compounds) was performed throughout fruit development and ripening. The transcriptomic profiling was conducted with two microarray platforms: a dedicated custom array (iRIPE) and a whole genome array specifically enriched with ripening-related genes for apple (WGAA). The transcriptomic and phenotypic changes following the application of 1-methylcyclopropene (1-MCP), an ethylene inhibitor leading to important modifications in overall fruit physiology, were also highlighted. The integrative comparative network analysis showed both negative and positive correlations between ripening-related transcripts and the accumulation of specific metabolites or texture components. The ripening distortion caused by the inhibition of ethylene perception, in addition to affecting the ethylene pathway, stimulated the de-repression of auxin-related genes, transcription factors and photosynthetic genes. Overall, the comprehensive repertoire of results obtained here advances the elucidation of the multi-layered climacteric mechanism of fruit ripening, thus suggesting a possible transcriptional circuit governed by hormones and transcription factors.