Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project. V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft

CONSTANS–FKBP12 interaction contributes to modulation of photoperiodic flowering in Arabidopsis

Flowering time is a key process in plant development. Photoperiodic signals play a crucial role in the floral transition in Arabidopsis thaliana, and the protein CONSTANS (CO) has a central regulatory function that is tightly regulated at the transcriptional and post-translational levels. The stability of CO protein depends on a light-driven proteasome process that optimizes its accumulation in the evening to promote the production of the florigen FLOWERING LOCUS T (FT) and induce seasonal flowering. To further investigate the post-translational regulation of CO protein we have dissected its interactome network employing in vivo and in vitro assays and molecular genetics approaches. The immunophilin FKBP12 has been identified in Arabidopsis as a CO interactor that regulates its accumulation and activity. FKBP12 and CO interact through the CCT domain, affecting the stability and function of CO. fkbp12 insertion mutants show a delay in flowering time, while FKBP12 overexpression accelerates flowering, and these phenotypes can be directly related to a change in accumulation of FT protein. The interaction is conserved between the Chlamydomonas algal orthologs CrCO–CrFKBP12, revealing an ancient regulatory step in photoperiod regulation of plant development.Ministerio de Ciencia BIO2014-52425-P, BIO2017-83629-RJunta de Andalucía P08-AGR-03582, BIO-281European Union GA83831

Synthesis and characterization of novel hydroxy- and aminobisphosphonates

Bisphosphonates (BPs) are a family of drugs that are successfully used in the treatment of various calcium-related disorders such as Pagetμs disease, osteoporosis and bone metastases. In addition, functional BPs have been also used in the treatment of metal intoxication and as novel ligands for well-defined radioactive metal complexes that can be used in imagiology, scintigraphy and radiotherapy applications [1,2]. The indazole derivatives are pharmacologically important compounds and the indazole ring system forms the basis of a number of drug molecules. Condensed pyrazoles are also known as pharmacophoric elements in numerous active compounds. However, in comparison with other heteroaromatic compounds, the chemistry of indazole and condensed pyrazoles remains less studied [3]. The present work is to extend the previous studies in indazolebisphosphonates [4] in order to obtain new BPs derived from indazole and condensed pyrazole with potential biological/therapeutical activities. Herein, we report the synthesis and characterization of a series of new 1-hydroxybisphosphonates (1) and aminobisphosphonates (2) (substituted at different C- or N-positions of the indazole ring - N-1, C-5, C-6, C-7

Phylogenetic analysis and geographical distribution of Theileria equi and Babesia caballi sequences from horses residing in Spain

The intraerythrocytic protozoans Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), one of the most important equine tick-borne diseases due to its significant impact on global international horse trade. Although EP is known to be endemic in Spain, previous phylogenetic studies have only been conducted for limited geographical regions. Therefore, the objective of this study was to evaluate the genetic diversity and distribution of these parasite species nationwide. This was performed by amplification of the 18S small subunit (SSU) rRNA gene from 100 EP positive equine blood samples using a nested PCR protocol, and sequencing the obtained amplicons. Seventy-seven T. equi and six B. caballi isolates were successfully sequenced and phylogenetic analysis revealed that the T. equi isolates grouped into the previously described clades A (n = 21/77), D (n = 1/77) and E (n = 55/77), while B. caballi isolates were placed into clades A (n = 5/6) and B (n = 1/6). Isolates from T. equi clade D and B. caballi clade B have not previously been reported in Spain. A greater intra-clade diversity (97.3–98.3 % identity) was observed between T. equi clade E isolates compared to those within clade A (99.7–100 % identity). Additionally, a multivariable logistic regression model was used to analyse associations between the clade of T. equi infection and available epidemiological data. Horses residing in Spanish northern regions were statistically more likely to be infected with T. equi clade E (p = 0.01). We conclude that while extensive sequence variation of equine piroplasms exists in Spanish infected horses, a requirement for increased equine movement controls between Spain and EP-endemic countries should be considered

The work–recovery cycle of kidney strain and inflammation in sugarcane workers following repeat heat exposure at work and at home

PurposeTo examine heat exposure at work and home and the work–recovery cycle and temporal variation of kidney strain, muscle injury and inflammation biomarkers in sugarcane workers.Methods20 male sugarcane workers (age: 33 ± 7 years) with a workplace Rest.Shade.Hydration (RSH) intervention were observed over 4 days, at the end (18 h post-shift recovery) and beginning of a work week (42 h post-shift recovery). Measures included work intensity (heart rate), gastro-intestinal temperature, estimated body core temperature (using heart rate), fluid consumption, pre- and post-work blood and urine samples, physical activity (accelerometery) away from work, plus ambient heat exposure at work and home.ResultsOn workdays, workers awakened at approx. 02:40 after 5 h sleep in ~ 30 °C. Across work shifts, daily average WBGT ranged from 26 to 29 °C (cooler than normal) and average workload intensity ranged from 55 to 58%HRmax. Workers reported consuming ~ 8 L of water and ~ 4 × 300 mL bags of electrolyte fluid each day. Serum creatinine, cystatin C and creatine phosphokinase markedly increased post-work and decreased during recovery; serum potassium did the opposite (all p &lt; 0.01). Biomarker concentration changes were similar between recovery periods (18 h vs. 42 h; all p &gt; 0.27). C-reactive protein was the highest at the end of the work week (p = 0.01).ConclusionDespite RSH intervention, cross-shift kidney strain was marked (recovering overnight) and systemic inflammation increased over the work week. Thus, biomonitoring of kidney function in occupational populations should be performed before a work shift at any point in the work week. This is essential knowledge for field studies and surveillance.<br/

Evaluation of Bio Briquettes made from Musa acuminata Colla, Musa acuminata and Musa balbisiana Silk, and Citrus reticulata and Citrus sinensis Peels

Accumulation of food waste and the burning of coal emit harmful chemicals which contribute to environmental problems such as climate change and global warming. These also risk the health of people, which causes deaths. Briquettes help improve and preserve the environment by lessening food waste and coal emissions. This study aims to determine the best treatment for briquettes to help disadvantaged communities and alleviate the adverse effects on the environment and health. A combination of banana (Musa acuminata Colla (AA Group) \u27Lakatan\u27 and Musa acuminata × M. balbisiana (AAB Group) \u27Silk\u27, and orange (Citrus × reticulata and Citrus × sinensis) peels were used as bases for the briquettes. Sawdust also served as a controlled treatment, and two different binder treatments were also used, namely paper pulp and cassava starch. The briquettes\u27 quality was tested based on their density, burning rate, ignition time, and efficiency (Water Boiling Test). One-way Multivariate Analysis of Variance (One-way MANOVA), Shapiro-Wilk Normality Test and Levene’s Homogeneity of Variances Test, One-way ANOVA, Post-Hoc Test, specifically Tukey’s LSD were then used to analyze the gathered results. Results revealed that the best briquettes are orange & cassava (density), banana & paper (burning rate), sawdust & cassava (ignition), and sawdust & cassava (efficiency). The findings indicate that the best briquettes were sawdust & cassava (most efficient in Water Boiling Test and fastest to ignite) and banana & paper (lowest burning rate) briquettes. Additionally, the findings suggest different production practices

Avaliação de políticas publicas na construção da cidade: o caso de Lençóis Paulista

Introdução: O trabalho analisa programas públicos que foram implantados em Lençóis Paulista - município de aproximadamente 60 mil habitantes, distante 300 km de São Paulo. A cidade inseriu-se em uma estratégia de modernização mais geral da administração, participando do Programa de Apoio à Administração Municipal (Progam). O Progam consolidou um conjunto de softwares aplicativos integrados em um sistema de gestão (Enterprise Resource Planning), um método educativo sobre técnicas de gestão para as equipes governativas locais e uma abordagem inovadora de coleta e sistematização de demandas produzidas por atores sociais organizados em processos participativos de construção de políticas territoriais. Objetivos: recuperar a história do Progam em Lençóis Paulista, particularmente no que se refere à fase de "leituras comunitárias" nos bairros da cidade e de introdução das novas tecnologias de gestão; buscar as relações entre as demandas expressas pela população e a elaboração do texto legal do Plano Diretor (LC 35/2006, alterada pela LC 43/2007) e de outras legislações pertinentes; pesquisar e criar um instrumento metodológico de avaliação de Políticas Públicas para medir os impactos do "programa" na cidade. Métodos: para atingir o objetivo, recuperou-se toda a documentação existente sobre as leituras comunitárias e o Plano Diretor Participativo de Lençóis; e foram realizadas entrevistas, filmadas, semi-dirigidas com gestores públicos municipais, com intenção de produção de um vídeo posteriormente. ; estabeleceu-se um roteiro e foi produzido um vídeo. Durante todo o processo os alunos estiveram várias vezes em visitas programadas em Lençóis Paulista conhecendo os gestores e debatendo a realidade da cidade com eles. Toda parte de filmagem foi feita pelos alunos. Resultados: O principal resultado foi colocar alunos de administração pública em contato com a gestão de um município concreto. Outros resultados significativos foram os efeitos de discutir e produzir o vídeo com os alunos, além da discussão de toda a avaliação de um projeto de modernização da administração municipal baseado em incorporação de softwares de gestão e de formação educacional de pessoas para o serviço público local

Canvass: a crowd-sourced, natural-product screening library for exploring biological space

NCATS thanks Dingyin Tao for assistance with compound characterization. This research was supported by the Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (NIH). R.B.A. acknowledges support from NSF (CHE-1665145) and NIH (GM126221). M.K.B. acknowledges support from NIH (5R01GM110131). N.Z.B. thanks support from NIGMS, NIH (R01GM114061). J.K.C. acknowledges support from NSF (CHE-1665331). J.C. acknowledges support from the Fogarty International Center, NIH (TW009872). P.A.C. acknowledges support from the National Cancer Institute (NCI), NIH (R01 CA158275), and the NIH/National Institute of Aging (P01 AG012411). N.K.G. acknowledges support from NSF (CHE-1464898). B.C.G. thanks the support of NSF (RUI: 213569), the Camille and Henry Dreyfus Foundation, and the Arnold and Mabel Beckman Foundation. C.C.H. thanks the start-up funds from the Scripps Institution of Oceanography for support. J.N.J. acknowledges support from NIH (GM 063557, GM 084333). A.D.K. thanks the support from NCI, NIH (P01CA125066). D.G.I.K. acknowledges support from the National Center for Complementary and Integrative Health (1 R01 AT008088) and the Fogarty International Center, NIH (U01 TW00313), and gratefully acknowledges courtesies extended by the Government of Madagascar (Ministere des Eaux et Forets). O.K. thanks NIH (R01GM071779) for financial support. T.J.M. acknowledges support from NIH (GM116952). S.M. acknowledges support from NIH (DA045884-01, DA046487-01, AA026949-01), the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program (W81XWH-17-1-0256), and NCI, NIH, through a Cancer Center Support Grant (P30 CA008748). K.N.M. thanks the California Department of Food and Agriculture Pierce's Disease and Glassy Winged Sharpshooter Board for support. B.T.M. thanks Michael Mullowney for his contribution in the isolation, elucidation, and submission of the compounds in this work. P.N. acknowledges support from NIH (R01 GM111476). L.E.O. acknowledges support from NIH (R01-HL25854, R01-GM30859, R0-1-NS-12389). L.E.B., J.K.S., and J.A.P. thank the NIH (R35 GM-118173, R24 GM-111625) for research support. F.R. thanks the American Lebanese Syrian Associated Charities (ALSAC) for financial support. I.S. thanks the University of Oklahoma Startup funds for support. J.T.S. acknowledges support from ACS PRF (53767-ND1) and NSF (CHE-1414298), and thanks Drs. Kellan N. Lamb and Michael J. Di Maso for their synthetic contribution. B.S. acknowledges support from NIH (CA78747, CA106150, GM114353, GM115575). W.S. acknowledges support from NIGMS, NIH (R15GM116032, P30 GM103450), and thanks the University of Arkansas for startup funds and the Arkansas Biosciences Institute (ABI) for seed money. C.R.J.S. acknowledges support from NIH (R01GM121656). D.S.T. thanks the support of NIH (T32 CA062948-Gudas) and PhRMA Foundation to A.L.V., NIH (P41 GM076267) to D.S.T., and CCSG NIH (P30 CA008748) to C.B. Thompson. R.E.T. acknowledges support from NIGMS, NIH (GM129465). R.J.T. thanks the American Cancer Society (RSG-12-253-01-CDD) and NSF (CHE1361173) for support. D.A.V. thanks the Camille and Henry Dreyfus Foundation, the National Science Foundation (CHE-0353662, CHE-1005253, and CHE-1725142), the Beckman Foundation, the Sherman Fairchild Foundation, the John Stauffer Charitable Trust, and the Christian Scholars Foundation for support. J.W. acknowledges support from the American Cancer Society through the Research Scholar Grant (RSG-13-011-01-CDD). W.M.W.acknowledges support from NIGMS, NIH (GM119426), and NSF (CHE1755698). A.Z. acknowledges support from NSF (CHE-1463819). (Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (NIH); CHE-1665145 - NSF; CHE-1665331 - NSF; CHE-1464898 - NSF; RUI: 213569 - NSF; CHE-1414298 - NSF; CHE1361173 - NSF; CHE1755698 - NSF; CHE-1463819 - NSF; GM126221 - NIH; 5R01GM110131 - NIH; GM 063557 - NIH; GM 084333 - NIH; R01GM071779 - NIH; GM116952 - NIH; DA045884-01 - NIH; DA046487-01 - NIH; AA026949-01 - NIH; R01 GM111476 - NIH; R01-HL25854 - NIH; R01-GM30859 - NIH; R0-1-NS-12389 - NIH; R35 GM-118173 - NIH; R24 GM-111625 - NIH; CA78747 - NIH; CA106150 - NIH; GM114353 - NIH; GM115575 - NIH; R01GM121656 - NIH; T32 CA062948-Gudas - NIH; P41 GM076267 - NIH; R01GM114061 - NIGMS, NIH; R15GM116032 - NIGMS, NIH; P30 GM103450 - NIGMS, NIH; GM129465 - NIGMS, NIH; GM119426 - NIGMS, NIH; TW009872 - Fogarty International Center, NIH; U01 TW00313 - Fogarty International Center, NIH; R01 CA158275 - National Cancer Institute (NCI), NIH; P01 AG012411 - NIH/National Institute of Aging; Camille and Henry Dreyfus Foundation; Arnold and Mabel Beckman Foundation; Scripps Institution of Oceanography; P01CA125066 - NCI, NIH; 1 R01 AT008088 - National Center for Complementary and Integrative Health; W81XWH-17-1-0256 - Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program; P30 CA008748 - NCI, NIH, through a Cancer Center Support Grant; California Department of Food and Agriculture Pierce's Disease and Glassy Winged Sharpshooter Board; American Lebanese Syrian Associated Charities (ALSAC); University of Oklahoma Startup funds; 53767-ND1 - ACS PRF; PhRMA Foundation; P30 CA008748 - CCSG NIH; RSG-12-253-01-CDD - American Cancer Society; RSG-13-011-01-CDD - American Cancer Society; CHE-0353662 - National Science Foundation; CHE-1005253 - National Science Foundation; CHE-1725142 - National Science Foundation; Beckman Foundation; Sherman Fairchild Foundation; John Stauffer Charitable Trust; Christian Scholars Foundation)Published versionSupporting documentatio

Avaliação de políticas publicas na construção da cidade: o caso de Lençóis Paulista

Introdução: O trabalho analisa programas públicos que foram implantados em Lençóis Paulista - município de aproximadamente 60 mil habitantes, distante 300 km de São Paulo. A cidade inseriu-se em uma estratégia de modernização mais geral da administração, participando do Programa de Apoio à Administração Municipal (Progam). O Progam consolidou um conjunto de softwares aplicativos integrados em um sistema de gestão (Enterprise Resource Planning), um método educativo sobre técnicas de gestão para as equipes governativas locais e uma abordagem inovadora de coleta e sistematização de demandas produzidas por atores sociais organizados em processos participativos de construção de políticas territoriais. Objetivos: recuperar a história do Progam em Lençóis Paulista, particularmente no que se refere à fase de "leituras comunitárias" nos bairros da cidade e de introdução das novas tecnologias de gestão; buscar as relações entre as demandas expressas pela população e a elaboração do texto legal do Plano Diretor (LC 35/2006, alterada pela LC 43/2007) e de outras legislações pertinentes; pesquisar e criar um instrumento metodológico de avaliação de Políticas Públicas para medir os impactos do "programa" na cidade. Métodos: para atingir o objetivo, recuperou-se toda a documentação existente sobre as leituras comunitárias e o Plano Diretor Participativo de Lençóis; e foram realizadas entrevistas, filmadas, semi-dirigidas com gestores públicos municipais, com intenção de produção de um vídeo posteriormente. ; estabeleceu-se um roteiro e foi produzido um vídeo. Durante todo o processo os alunos estiveram várias vezes em visitas programadas em Lençóis Paulista conhecendo os gestores e debatendo a realidade da cidade com eles. Toda parte de filmagem foi feita pelos alunos. Resultados: O principal resultado foi colocar alunos de administração pública em contato com a gestão de um município concreto. Outros resultados significativos foram os efeitos de discutir e produzir o vídeo com os alunos, além da discussão de toda a avaliação de um projeto de modernização da administração municipal baseado em incorporação de softwares de gestão e de formação educacional de pessoas para o serviço público local

Avaliação de políticas publicas na construção da cidade: o caso de Lençóis Paulista

Introdução: O trabalho analisa programas públicos que foram implantados em Lençóis Paulista - município de aproximadamente 60 mil habitantes, distante 300 km de São Paulo. A cidade inseriu-se em uma estratégia de modernização mais geral da administração, participando do Programa de Apoio à Administração Municipal (Progam). O Progam consolidou um conjunto de softwares aplicativos integrados em um sistema de gestão (Enterprise Resource Planning), um método educativo sobre técnicas de gestão para as equipes governativas locais e uma abordagem inovadora de coleta e sistematização de demandas produzidas por atores sociais organizados em processos participativos de construção de políticas territoriais. Objetivos: recuperar a história do Progam em Lençóis Paulista, particularmente no que se refere à fase de "leituras comunitárias" nos bairros da cidade e de introdução das novas tecnologias de gestão; buscar as relações entre as demandas expressas pela população e a elaboração do texto legal do Plano Diretor (LC 35/2006, alterada pela LC 43/2007) e de outras legislações pertinentes; pesquisar e criar um instrumento metodológico de avaliação de Políticas Públicas para medir os impactos do "programa" na cidade. Métodos: para atingir o objetivo, recuperou-se toda a documentação existente sobre as leituras comunitárias e o Plano Diretor Participativo de Lençóis; e foram realizadas entrevistas, filmadas, semi-dirigidas com gestores públicos municipais, com intenção de produção de um vídeo posteriormente. ; estabeleceu-se um roteiro e foi produzido um vídeo. Durante todo o processo os alunos estiveram várias vezes em visitas programadas em Lençóis Paulista conhecendo os gestores e debatendo a realidade da cidade com eles. Toda parte de filmagem foi feita pelos alunos. Resultados: O principal resultado foi colocar alunos de administração pública em contato com a gestão de um município concreto. Outros resultados significativos foram os efeitos de discutir e produzir o vídeo com os alunos, além da discussão de toda a avaliação de um projeto de modernização da administração municipal baseado em incorporação de softwares de gestão e de formação educacional de pessoas para o serviço público local.</p