Association of JAK/STAT genetic variants with cutaneous melanoma

Cutaneous melanoma; Genetic variant; PrognosisMelanoma cutáneo; Variante genética; PronósticoMelanoma cutani; Variant genètica; PronòsticBackground: The Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway regulates cutaneous melanoma (CM) development and progression. The JAK1, JAK2, and STAT3 proteins are encoded by polymorphic genes. This study aimed to verify whether single-nucleotide variants (SNVs) in JAK1 (c.1648+1272G>A, c.991-27C>T), JAK2 (c.-1132G>T, c.-139G>A), and STAT3 (c.*1671T>C, c.-1937C>G) altered the risk, clinicopathological aspects, and survival of CM patients as well as protein activity. Methods: CM patients (N = 248) and controls (N = 274) were enrolled in this study. Genotyping was performed by real-time polymerase chain reaction (PCR), and JAK1, JAK2, and STAT3 expression was assessed by quantitative PCR (qPCR). STAT3 c.-1937C>G SNV was investigated by luciferase, qPCR, western blot, apoptosis, and cell cycle assays in SKMEL-28 cells with CC or GG genotype. Results: Individuals with STAT3 c.*1671TT and c.-1937CC genotypes and TC haplotype of both SNVs were under about 2.0-fold increased risk of CM. Specific JAK1, JAK2, and STAT3 combined genotypes were associated with up to 4.0-fold increased risk of CM. Higher luciferase activity [4,013.34 vs. 2,463.32 arbitrary units (AU); p = 0.004], STAT3 expression by qPCR (649.20 vs. 0.03 AU; p = 0.003) and western blot (1.69 vs. 1.16 AU; p = 0.01), and percentage of cells in the S phase of the cell cycle (57.54 vs. 30.73%; p = 0.04) were more frequent in SKMEL-28 with STAT3 c.-1937CC than with GG genotype. CM cell line with CC genotype presented higher STAT3 protein levels than the one with GG genotype (1.93 versus 1.27 AU, p = 0.0027). Conclusion: Our data present preliminary evidence that inherited abnormalities in the JAK/STAT pathway can be used to identify individuals at a high risk of CM, who deserve additional attention for tumor prevention and early detection.This work was supported by grants from the FAPESP (2016/25407-4 and 2019/16776-4) and FAPESP 2019/09168-8

Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli

Bacterial transcription factors (TFs) are key devices for the engineering of complex circuits in many biotechnological applications, yet there are few well-characterized inducer-responsive TFs that could be used in the context of an animal or human host. We have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e., aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of BenR and XylS. By means of site-directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. Whereas this modification in BenR abolishes protein activity, in XylS, it increases the response to several inducers, including acetyl salicylic acid, to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with an enhanced response to benzoate, 3-methylbenzoate, 2-methylbenzoate, 4-methylbenzoate, salicylic acid, aspirin, and acetylsalicylic acid molecules for eliciting gene expression in E. coli.Fil: Monteiro, Lummy Maria Oliveira. Universidade de Sao Paulo; BrasilFil: Arruda, Leticia Magalhães. Universidade de Sao Paulo; BrasilFil: Sanches Medeiros, Ananda. Universidade de Sao Paulo; BrasilFil: Martins Santana, Leonardo. Universidade de Sao Paulo; BrasilFil: Alves, Luana de Fátima. Universidade de Sao Paulo; BrasilFil: Defelipe, Lucas Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Turjanski, Adrian Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Guazzaroni, Mara Eugenia. Universidade de Sao Paulo; BrasilFil: de Lorenzo, Victor. Consejo Superior de Investigaciones Científicas. Centro Nacional de Biotecnología; EspañaFil: Silva Rocha, Rafael. Universidade de Sao Paulo; Brasi

Desvendando as relações arquitetura/função de promotores bacterianos complexos utilizando abordagens de biologia sintética

Gene regulation has been studied extensively, however the complexity of the regulatory mechanisms still remains unknown. Understanding how gene regulation occurs is important not only to better understand the complexity of an organism but to postulate new rules, characterize new biological parts and then allow new design of biological circuits, for example. A possible strategy to unravel the mechanisms of action and complexity of bacterial promoters would be to combine the knowledge of gene regulation with the use of approaches from synthetic biology and bioinformatics, which, in turn, allow to design and build new functions in biological systems. Progress in synthetic biology is often made possible by powerful bioinformatics tools that allow the integration of the design, construction and testing stages of the biological engineering cycle. Consequently, the development of new bioinformatics tools is useful and important for scientists working on the design, development and testing of parts to extend or modify the behavior of organisms and design them to perform new tasks. In this context, the present thesis described (i) the existence of emergent properties in complex synthetic promoters in Escherichia coli, which could be extrapolated to naturally occurring regulatory systems and would significantly impact the engineering of synthetic biological circuits in bacteria. Taken together, these data demonstrate how small changes in the architecture of bacterial promoters could result in drastic changes in the final regulatory logic of the system, with important implications for the understanding of natural complex promoters in bacteria and their engineering for novel applications; (ii) the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e. Aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of these two proteins. As a result, a collection of engineered transcription factors (TFs) was generated with enhanced response to a well characterized and largely innocuous molecule with a potential for eliciting heterologous expression of bacterial genes in animal carriers; (iii) the complexity of transcription factors in environmental communities. We created one bacterial transcription factor database (BacTFDB) that was used to train a deep learning model to predict novel TFs and their families in metagenomics and metranscriptomics samples (PredicTF). PredicTF provides the first tool to profile TFs in yet-to be cultured bacteria and it opens the potential to evaluate regulatory networks in complex microbial communities. PredicTF is a flexible, open source pipeline able to predict and annotate TFs in genomes and metagenomes. PredicTF is avaliable at https://github.com/mdsufz/PredicTF.A regulação gênica tem sido estudada extensivamente, no entanto, a complexidade dos mecanismos regulatórios ainda permanece desconhecida. Entender os mecanismos da regulação gênica é importante não apenas para desvendar a complexidade de um organismo, mas para postular novas regras, caracterizar novas partes biológicas e então permitir novos designs de circuitos biológicos, por exemplo. Uma possível estratégia para desvendar os mecanismos de ação e complexidade dos promotores bacterianos seria combinar o conhecimento da regulação gênica com o uso de abordagens da biologia sintética e da bioinformática, que, por sua vez, permitem projetar e construir novas funções em sistemas biológicos. O progresso na biologia sintética é frequentemente possibilitado por poderosas ferramentas de bioinformática que permitem a integração das fases de design, construção e teste do ciclo de engenharia biológica. Consequentemente, o desenvolvimento de novas ferramentas de bioinformática é útil e importante para os cientistas que trabalham para estender ou modificar o comportamento dos organismos e projetá-los para realizar novas tarefas. Nesse contexto, a presente tese descreveu (i) a existência de propriedades emergentes em promotores sintéticos complexos em Escherichia coli, que poderiam ser extrapoladas para sistemas regulatórios de ocorrência natural e impactariam significativamente a engenharia de circuitos biológicos sintéticos em bactérias. Em resumo, esses dados demonstram como pequenas mudanças na arquitetura dos promotores bacterianos podem resultar em mudanças drásticas na lógica regulatória final do sistema, com implicações importantes na compreensão de promotores complexos naturais em bactérias e sua engenharia para novas aplicações; (ii) o mecanismo de reconhecimento do indutor de dois reguladores AraC/XylS de Pseudomonas putida (BenR e XylS) para a criação de um novo sistema de expressão responsivo ao ácido acetil salicílico (aspirina). Usando homologia de proteínas e docking molecular com o indutor benzoato e um conjunto de análogos químicos, identificamos o sítio de ligação conservado dessas duas proteínas. Como resultado, uma coleção de fatores de transcrição (TFs) engenheirados foram gerados com respostas aprimoradas a uma molécula bem caracterizada e amplamente inócua com um potencial para induzir a expressão heteróloga de genes bacterianos em animais; (iii) a complexidade dos fatores de transcrição em comunidades microbianas ambientais. Criamos um banco de dados de fatores de transcrição bacteriano (BacTFDB) que foi usado para treinar um modelo de Machine Learning para prever novos TFs e suas famílias em amostras metagenômicas e metranscriptômicas (PredicTF). PredicTF fornece a primeira ferramenta para traçar o perfil de TFs em bactérias ainda a serem cultivadas e abre o potencial para avaliar redes regulatórias em comunidades microbianas complexas. PredicTF é um pipeline de código aberto flexível capaz de prever e anotar TFs em genomas e metagenomas. PredicTF está disponível em https://github.com/mdsufz/PredicTF

Biochemical study of ?-glucosidases from Malbranchea pulchella and applications in agroindustrial residues and anthocyanins hydrolysis

?-glucosidases são enzimas que catalisam a hidrólise de ligações glucosídicas ?-1,4, ?-1,3 e ?-1,6 a partir da extremidade não redutora de oligossacarídeos de cadeias pequenas, alquil e aril ?-D-glucosídeos e dissacarídeos. Além de serem enzimas chave do complexo celulolítico, apresentam funções importantes como o melhoramento de aromas de vinhos e a hidrólise de antocianinas. Malbranchea pulchella usualmente é encontrado em fragmentos vegetais em decomposição ou material rico em celulose, podendo ser considerado promissor à produção de enzimas de interesse biotecnológico. Neste contexto, o objetivo desse projeto foi a caracterização funcional de uma ?-glucosidase de M. pulchella e sua aplicação na hidrólise de resíduos agroindustriais e de antocianinas. Uma BGL da família GH3 foi purificada com um fator de purificação 6,32 e recuperação de aproximadamente 35 %. A sua massa molecular aproximado foi de 100 kDa, e o Km, Vmáx e Kcat foram calculadas em 0,33 mM, 13,67 U/mg, 26,5 s-1 respectivamente. O dicroísmo circular revelou uma estrutura composta por aproximadamente 25% de ?-hélices e 20% de ?-folhas. A BGL apresentou pH e temperatura ótimos igual a 6,0 e 50 °C; e foi estável a 40 °C e apresentou boa estabilidade nos pH 5,0 a 8,0, por 24 horas. Nenhum dos sais de íons metálicos ativou a enzima e apenas o HgCl2 inibiu a atividade em 90%. A enzima não apresentou inibição em presença de glucose (0,1-1M) por até 24 horas. Além disso, a GH3 mostrou-se glicosilada e a proporção de açúcar corresponde a 15% massa da enzima. O efeito da celobiose (C) e do bagaço de cana-de-açúcar in natura (BCAN) na produção das BGLs foram avaliados em um DCCR, que indicou um modelo reduzido com influência das duas variáveis. A melhor condição de cultivo para a produção de BGLs foi 0,6% de C (p/v) e 4% de BCAN (p/v). Por meio de um planejamento de mistura, os resíduos BCAN, a casca de soja moída (CS) e o bagaço de cevada (BCev), foram avaliados quanto ao potencial de hidrólise a partir das enzimas presentes no extrato enzimático, resultando no maior potencial de hidrólise sobre o BCev, com a produção de aproximadamente 2 mg/mL de açúcares redutores em 48 horas. As BGLs presentes no extrato enzimático otimizado foram imobilizadas em suporte MANAE-agarose, Concanavalina A-Sepharose e BrCN-Sepharose. Os derivados BGL-MANAE e BGL-ConA foram ativados aproximadamente 10 e 3 vezes, respectivamente. BGL-MANAE e BGL-ConA foram mais estáveis que o controle BGLBrCN em todos os pH testados em 24 horas e, além disso, BGL-ConA permaneceu com 100% de sua atividade em temperaturas de 40 °C, 50 °C e 60 °C, já o BGL-MANAE mostrou-se estável a 40 °C permanecendo com 83% de sua atividade, ambos em 24 horas. BGL-MANAE e BGL-ConA apresentaram menor efeito inibitório em presença de diferentes concentrações de glucose e etanol quando comparados ao BGL-BrCN, e esses resultados indicaram que a imobilização de alguma forma colaborou para uma maior estabilidade ao pH e à temperatura, bem como ao aumento da tolerância por glucose e etanol. Os derivados puderam ser reutilizados por até 20 vezes e quando avaliados quanto à capacidade de clarificar vinhos e sucos de uva (hidrólise de antocianinas), BGL-MANAE clarificou 52% o vinho, 71% o vinho diluído, 77% o suco de uva e 56% o suco de uva diluído, e BGL-ConA em contrapartida clarificou 41% o vinho, 46% o vinho diluído, 63% o suco de uva e 23% o suco de uva diluído. BGL-MANAE foi mais eficiente que BGL-ConA na clarificação de vinhos e sucos de uva podendo ser considerado um biocatalisador promissor na hidrólise de antocianinas e, consequentemente, para a produção de vinhos brancos e rose a partir de diferentes variedades de uvas. Este trabalho pelo que sabemos, é o primeiro a usar BGLs imobilizadas com aplicação na clarificação de sucos de uva e vinhos, podendo ser considerado um trabalho inovador, e de grande importância para a indústria de alimentos e bebidas.?-glucosidases are enzymes that catalyze the hydrolysis of ?-1,4, ?-1,3 and ?-1,6 glucosidic linkages, from the non-reducing end of short chain oligosaccharides, alkyl and aryl ?-D-glucosides and disaccharides. Besides being complex key cellulolytic enzymes, they have important functions such as the improvement of wine flavors and anthocyanins hydrolysis. Malbranchea pulchella is usually found in decaying plant debris or in material rich in cellulose, for this reason it can be considered promising for the production of enzymes of biotechnological interest. In this context, the aim of this project was the functional characterization of a ?-glucosidase from M. pulchella and its application in the organic residues and anthocyanins hydrolysis. A BGL GH3 family produced by M. pulchella was purified with a purification factor and recovery of about 6.32 and 35 times. Its approximate molecular mass was 100 kDa, and Km, Vmax and kcat were 0.33 mM, 13.67 U/mg, 26.5 s-1, respectively. The circular dichroism revealed a structure composed of approximately 25% of ?-helix and 20% of ?-sheets. BGL presented optimum pH and temperature at 6.0 and 50 °C; and it was stable at 40 °C. It also showed good stability at pH 5.0 to 8.0, for 24 hours. None of the metal ions salts activated the enzyme and HgCl2 inhibited the activity by 90%. The enzyme showed no inhibition in the presence of glucose (0,1-1M) for 24 hours. Furthermore, it is glycosylated and the sugar proportion correspondsto 15% of the enzyme mass. The effect of cellobiose (C) and sugarcane bagasse in natura (BCAN) in the production of BGLs were evaluated in a CCRD, which indicated a reduced model of influence of the two variables. The best culture condition for BGLs production was 0.6% of C (w/v) and 4% (w/v) of BCAN. Through a mixture design, using the BCAN, ground soybean hulls (CS) and barley bagasse (BCev) were used to evaluate the potential of hydrolysis of these residues in the presence of enzymes present in the enzymatic extract, resulting in a greater efficiency of (BCev) hydrolysis, producing approximately 2 mg/mL of reducing sugars in 48 hours. The BGLs present in the optimized enzyme extract were also used in the immobilization on ionic support MANAE-agarose and affinity support Concanavalin A-Sepharose (ConA-Sepharose). The BGL-MANAE and BGL-ConA derivatives were activated approximately 10 and 3 times, respectively. BGL-MANAE and BGL-ConA were more stable than BGL-BrCN control in all pH tested within 24 hours. In addition, BGL-ConA remained 100% of its activity at 40 °C, 50 °C and 60 °C , and BGL-MANAE was stable at 40 °C and remained 83% of its activity, both in 24 hours. BGL-MANAE and BGL-ConA showed lower inhibitory effect in the presence of different glucose and ethanol concentrations when compared to BGL-BrCN and these results indicate that the immobilization, somehow, cooperated to a greater pH and temperature stability, as well as to increased tolerance by glucose and ethanol. The derivatives could be reused up to 20 times and when they were tested for their capacity to clarify wine and grape juice (anthocyanins hydrolysis), BGL-MANAE clarified 52% wine, 71% diluted wine, 77% grape juice and 56% diluted grape juice. On the other hand, BGL-ConA clarified 41% wine, 46% diluted wine, 63% grape juice and 23% diluted grape juice. BGL-MANAE was more efficient than BGL-ConA in clarifying wines and grape juices and it may be considered a promising biocatalyst in the anthocyanins hydrolysis, and consequently in the production of white and rose wines from different varieties of grapes. This work as we know, is the first to use immobilized BGLs applied in the clarification of grape juice and wine, for this reason, it can be considered an innovative work, and of great importance to the food and beverage industr

The Chromobacterium violaceum ArsR arsenite repressor exerts tighter control on its cognate promoter than the Escherichia coli system

Environmental bacteria are endowed with several regulatory systems that have potential applications in biotechnology. In this report, we characterize the arsenic biosensing features of the ars response system from Chromobacterium violaceum in the heterologous host Escherichia coli. We show that the native Pars/arsR system of C. violaceum outperforms the chromosomal ars copy of E. coli when exposed to micromolar concentrations of arsenite. To understand the molecular basis of this phenomenon, we analyzed the interaction between ArsR regulators and their promoter target sites as well as induction of the system at saturating concentrations of the regulators. In vivo titration experiments indicate that ArsR from C. violaceum has stronger binding affinity for its target promoter than the regulator from E. coli does. Additionally, arsenite induction experiments at saturating regulator concentration demonstrates that although the Pars/arsR system from E. coli displays a gradual response to increasing concentration of the inducer, the system from C. violaceum has a steeper response with a stronger promoter induction after a given arsenite threshold. Taken together, these data demonstrate the characterization of a novel arsenic response element from an environmental bacterium with potentially enhanced performance that could be further explored for the construction of an arsenic biosensor

Calibrating Transcriptional Activity Using Constitutive Synthetic Promoters in Mutants for Global Regulators in Escherichia coli

The engineering of synthetic circuits in cells relies on the use of well-characterized biological parts that would perform predicted functions under the situation considered, and many efforts have been taken to set biological standards that could define the basic features of these parts. However, since most synthetic biology projects usually require a particular cellular chassis and set of growth conditions, defining standards in the field is not a simple task as gene expression measurements could be affected severely by genetic background and culture conditions. In this study, we addressed promoter parameterization in bacteria in different genetic backgrounds and growth conditions. We found that a small set of constitutive promoters of different strengths controlling a short-lived GFP reporter placed in a low-copy number plasmid produces remarkably reproducible results that allow for the calibration of promoter activity over different genetic backgrounds and physiological conditions, thus providing a simple way to set standards of promoter activity in bacteria. Based on these results, we proposed the utilization of synthetic constitutive promoters as tools for calibration for the standardization of biological parts, in a way similar to the use of DNA and protein ladders in molecular biology as references for comparison with samples of interest

Neosartorya glabra polygalacturonase produced from fruit peels as inducers has the potential for application in passion fruit and apple juices

Summary Polygalacturonases are enzymes with the biotechnological potential for use in fruit juice clarification and for the enhancement of filtration efficiency. The aim of this work was to assess the production of polygalacturonase by the fungus Neosartorya glabra by means of solid-state and submerged fermentation using fruit peel residues as the carbon source, and also apply the enzyme in the clarification and decrease in viscosity of passion fruit and apple juices. The highest polygalacturonase (4.52 U/g/h) production was obtained by means of submerged fermentation in Vogel´s medium (1964) containing orange peel – Bahia variety (Citrus sinensis), at a concentration of 1.5% (w/v, dried mass) at 30-35°C for 72 h. The polygalacturonase of the crude extract presented optimal activity at 60°C and pH 5.5. The enzyme retained around 90% of the initial activity after 180 minutes at 40°C, and 50% of the initial activity after 150 minutes at 50°C. The enzyme was shown to be stable at acid pH values (3.0-6.5) after 120 minutes at 25oC. All these favourable enzymatic properties make the polygalacturonase attractive for potential uses in the industry of pectin-rich fruit juices, since the application of the crude extract to passion fruit (Passiflora edulis) juice caused an 80% reduction in viscosity and 75% decrease in light absorbance. In the processing of apple pulp juice (Malus domestica), there was a 50% reduction in viscosity and 78% decrease in light absorbance

Increased Malbranchea pulchella β-glucosidase production and its application in agroindustrial residue hydrolysis: A research based on experimental designs

β-Glucosidases are a limiting factor in the conversion of cellulose to glucose for the subsequent ethanol production. Here, β-glucosidase production by Malbranchea pulchella was optimized using Composite Central Designs and Response Surface Methodologies from a medium designed. The coefficient of determination (R2) was 0.9960, F-value was very high, and the lack of fit was found to be non-significant. This indicates a statistic valid and predictive result. M. pulchella enzymatic extract was successfully tested as an enzymatic cocktail in a mixture design using sugarcane bagasse, soybean hull and barley bagasse. We proved that the optimization of the β-glucosidase production and the application in hydrolysis using unexpansive biomass and agricultural wastes can be accomplished by means of statistical methodologies. The strategy presented here can be useful for the improvement of enzyme production and the hydrolysis process, arising as an alternative for bioeconomy

Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli

Bacterial transcription factors (TFs) are key devices for the engineering of complex circuits in many biotechnological applications, yet there are few well-characterized inducer-responsive TFs that could be used in the context of an animal or human host. We have deciphered the inducer recognition mechanism of two AraC/XylS regulators from Pseudomonas putida (BenR and XylS) for creating a novel expression system responsive to acetyl salicylate (i.e., aspirin). Using protein homology modeling and molecular docking with the cognate inducer benzoate and a suite of chemical analogues, we identified the conserved binding pocket of BenR and XylS. By means of site-directed mutagenesis, we identified a single amino acid position required for efficient inducer recognition and transcriptional activation. Whereas this modification in BenR abolishes protein activity, in XylS, it increases the response to several inducers, including acetyl salicylic acid, to levels close to those achieved by the canonical inducer. Moreover, by constructing chimeric proteins with swapped N-terminal domains, we created novel regulators with mixed promoter and inducer recognition profiles. As a result, a collection of engineered TFs was generated with an enhanced response to benzoate, 3-methylbenzoate, 2-methylbenzoate, 4-methylbenzoate, salicylic acid, aspirin, and acetylsalicylic acid molecules for eliciting gene expression in E. coli.R.S.-R. and M.-E.G. were supported by Young Research Awards, grant nos. 2012/22921-8 and 2015/04309-1, São Paulo Research Foundation (FAPESP). L.M.O.M., A.S.-M., L.M.-S., and L.F.A. were supported by FAPESP Ph.D. Fellowships (grant nos. 2016/19179-9, 2016/06323-4, 2017/17924-1, and 2018/04810-0)