Use of BODIPY-Labeled ATP Analogues in the Development and Validation of a Fluorescence Polarization-Based Assay for Screening of Kinase Inhibitors

Copyright © 2020 American Chemical Society. The screening of compound libraries to identify small-molecule modulators of specific biological targets is crucial in the process for the discovery of novel therapeutics and molecular probes. Considering the need for simple single-tool assay technologies with which one could monitor "all" kinases, we developed a fluorescence polarization (FP)-based assay to monitor the binding capabilities of protein kinases to ATP. We used BODIPY ATP-y-S as a probe to measure the shift in the polarization of a light beam when passed through the sample. We were able to optimize the assay using commercial Protein Kinase A (PKA) and H7 efficiently inhibited the binding of the probe when added to the reaction. Furthermore, we were able to employ the assay in a high-throughput fashion and validate the screening of a set of small molecules predicted to dock into the ATP-binding site of PKA. This will be useful to screen larger libraries of compounds that may target protein kinases by blocking ATP binding

Multicaloric effect in a multiferroic composite of Gd-5(Si,Ge)(4) microparticles embedded into a ferroelectric PVDF matrix

CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOThe coupling between electric, magnetic and elastic features in multiferroic materials is an emerging field in materials science, with important applications on alternative solid-state cooling technologies, energy harvesting and sensors/actuators. In this direction, we developed a thorough investigation of a multiferroic composite, comprising magnetocaloric/magnetostrictive Gd5Si2.4Ge1.6 microparticles blended into a piezo- and pyroelectric poly(vinylidene) fluoride (PVDF) matrix. Using a simple solvent casting technique, the formation and stabilization of PVDF electroactive phases are improved when the filler content increases from 2 to 12 weight fraction (wt.%). This effect greatly contributes to the magnetoelectric (ME) coupling, with the ME coefficient alpha(ME) increasing from 0.3 V/cm.Oe to 2.2 V/cm.Oe, by increasing the amount of magnetic material. In addition, magnetic measurements revealed that the ME-coupling has influenced the magnetocaloric effect via a contribution from the electroactive polymer and hence leading to a multicaloric effect. These results contribute to the development of multifunctional systems for novel technologies.9CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO203180/2014-3This work is funded by FEDER funds through the COMPETE 2020 Programme and National Funds throught FCT -Portuguese Foundation for Science and Technology under the projects POCI-01-0145-FEDER-029454, POCI-01-0145-FEDER-032527 and UID/FIS/04564/2016. This work was also supported by NECL with the project NORTE-01-0145-FEDER-022096 and by the European Union Horizon -2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 734801. The authors acknowledge K. Pirota and F. Beron for the FTIR measurements performed at Unicamp in Brazil. Special thanks to Dr. A. Aliev for help in automatizing the ME experiments at Amirkhanov Institute of Physics and the helpful discussions. Federal Fluminense University, Brazil, permanent address for MSR; Aveiro University, Portugal, temporary address during this work for MSR. VMA thanks the CNPq for the Grant No. 203180/2014-3. A.A. acknowledges Russian Science Foundation for support magnetoelectric studies (grant No. 18-79-10176). J.H. Belo thanks FCT for Grant No. SFRH/BD/88440/2012, Project PTDC/FISMA/31302/2017, and his contract No. DL57/2016 reference SFRH-BPD-87430/2012. Finally, the discussion of results has been made possible through the mobility grant provided by the 5 top 100 Russian Academic Excellence Project at the Immanuel Kant Baltic Federal University

Tumour risks and genotype-phenotype correlations associated with germline variants in succinate dehydrogenase subunit genes SDHB, SDHC and SDHD.

BACKGROUND: Germline pathogenic variants in SDHB/SDHC/SDHD are the most frequent causes of inherited phaeochromocytomas/paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. We estimate penetrance for symptomatic tumours and elucidate genotype-phenotype correlations in a large cohort of SDHB/SDHC/SDHD mutation carriers. METHODS: A retrospective survey of 1832 individuals referred for genetic testing due to a personal or family history of phaeochromocytoma/paraganglioma. 876 patients (401 previously reported) had a germline mutation in SDHB/SDHC/SDHD (n=673/43/160). Tumour risks were correlated with in silico structural prediction analyses. RESULTS: Tumour risks analysis provided novel penetrance estimates and genotype-phenotype correlations. In addition to tumour type susceptibility differences for individual genes, we confirmed that the SDHD:p.Pro81Leu mutation has a distinct phenotype and identified increased age-related tumour risks with highly destabilising SDHB missense mutations. By Kaplan-Meier analysis, the penetrance (cumulative risk of clinically apparent tumours) in SDHB and (paternally inherited) SDHD mutation-positive non-probands (n=371/67 with detailed clinical information) by age 60 years was 21.8% (95% CI 15.2% to 27.9%) and 43.2% (95% CI 25.4% to 56.7%), respectively. Risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4.2%(95% CI 1.1% to 7.2%). With retrospective cohort analysis to adjust for ascertainment, cumulative tumour risks for SDHB mutation carriers at ages 60 years and 80 years were 23.9% (95% CI 20.9% to 27.4%) and 30.6% (95% CI 26.8% to 34.7%). CONCLUSIONS: Overall risks of clinically apparent tumours for SDHB mutation carriers are substantially lower than initially estimated and will improve counselling of affected families. Specific genotype-tumour risk associations provides a basis for novel investigative strategies into succinate dehydrogenase-related mechanisms of tumourigenesis and the development of personalised management for SDHB/SDHC/SDHD mutation carriers

A Ligand Peptide Motif Selected from a Cancer Patient Is a Receptor-Interacting Site within Human Interleukin-11

Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11Rα is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11Rα has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs

Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations

​Leucine-rich repeat kinase 2 (​LRRK2) mutations are the most common genetic cause of Parkinson’s disease. ​LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson’s disease, but whether ​LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that ​LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase ​αTAT1 prevents association of mutant ​LRRK2 with microtubules, and the deacetylase inhibitor ​trichostatin A (​TSA) restores axonal transport. In vivo knockdown of the deacetylases ​HDAC6 and ​Sirt2, or administration of ​TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson’s disease

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio