Advances of development and application amino acid biosensors / 生物工程学报

Amino acids are the basic building blocks of protein that are very important to the nutrition and health of humans and animals, and widely used in feed, food, medicine and daily chemicals. At present, amino acids are mainly produced from renewable raw materials by microbial fermentation, forming one of the important pillar industries of biomanufacturing in China. Amino acid-producing strains are mostly developed through random mutagenesis- and metabolic engineering-enabled strain breeding combined with strain screening. One of the key limitations to further improvement of production level is the lack of efficient, rapid, and accurate strain screening methods. Therefore, the development of high-throughput screening methods for amino acid strains is very important for the mining of key functional elements and the creation and screening of hyper-producing strains. This paper reviews the design of amino acid biosensors and their applications in the high-throughput evolution and screening of functional elements and hyper-producing strains, and the dynamic regulation of metabolic pathways. The challenges of existing amino acid biosensors and strategies for biosensor optimization are discussed. Finally, the importance of developing biosensors for amino acid derivatives is prospected.

A disciplina psicologia do esporte nos cursos de psicologia e educação física / Sports psychology in psychology and physical education courses / La materia psicología del deporte en los cursos de psicología y educación física

Este discute a representatividade da disciplina Psicologia do Esporte nos cursos de Psicologia e Educação Física em instituições de ensino superior reconhecidas pelo MEC e situadas na região Sul do país. Foi realizado um estudo documental, com base nos currículos das Instituições. Os resultados revelaram que no Sul do Brasil 21,02% dos cursos de Psicologia, 41,96% dos cursos de bacharelado em Educação Física e apenas 14,83% dos cursos de licenciatura em Educação Física apresentam a disciplina Psicologia do Esporte em sua grade curricular. Observou-se que a disciplina é ofertada mais frequentemente em regime obrigatório nos cursos de bacharelado em Educação Física. Nos cursos de Psicologia, quando ofertada, costuma ser optativa. Os resultados evidenciam uma maior oferta da disciplina para os estudantes de Educação Física, em relação aos de Psicologia, o que pode estar relacionado ao próprio contexto de surgimento da disciplina e sua popularização no meio acadêmico. Para que esse panorama possa mudar e se possa oferecer uma formação adequada no curso de Psicologia para fomentar essa opção de carreira, há necessidade de se repensar o currículo e o próprio perfil do egresso, de forma a dar mais oportunidade aos estudantes para que conheçam as bases teóricas e os campos de aplicação da Psicologia do Esporte. Tal lacuna pode acarretar a fragilização da disseminação desse conhecimento aos estudantes de graduação e a consequente ocupação do mercado de trabalho.(AU)

This study discusses the representativeness of Sports Psychology in Psychology and Physical Education courses at higher education institutions from Southern Brazil. A documentary study was conducted based on the institutions' curricula. Results show that 21.02% of the Psychology major, 41.96% of the bachelor's in Physical Education, and only 14.83% of the license in Physical Education offer Sports Psychology in their curricula. Sports Psychology is most often offered as a compulsory subject in the bachelor's program in Physical Education, whereas Psychology courses offer it mainly as an elective. Physical Education students have greater contact with the discipline when compared with Psychology students, which may be explained by its context of development and popularization in the academic environment. To change this scenario and offer adequate education in the Psychology programs to foster this career option, institutions must rethink their curriculum and the graduate profile itself. This would give students better opportunity to get to know its theoretical bases and fields of application. Such a gap can hinder the dissemination of this knowledge to undergraduate students and the consequent labor market occupation.(AU)

El objetivo de este estudio es discutir la representatividad de la materia Psicología del Deporte en los cursos de Psicología y Educación Física en instituciones de educación superior de la región Sur de Brasil, reconocidas por el Ministerio de Educación (MEC). Se realizó un estudio documental, basado en los planes de estudio de las instituciones. Los resultados revelaron que, en el Sur de Brasil, el 21,02% de los cursos de Psicología, el 41,96% de los cursos de licenciatura en Educación Física y sólo el 14,83% de los cursos de profesorado en Educación tienen la materia Psicología del Deporte en sus planes de estudio. Se observó que la materia Psicología del Deporte se ofrece con mayor frecuencia como asignatura obligatoria en los cursos de licenciatura en Educación Física. Cuando se ofrece en los cursos de Psicología, es una materia optativa. Los resultados muestran una mayor oferta para los estudiantes de Educación Física en comparación con Psicología, lo que puede estar relacionado con el contexto del surgimiento de la Psicología del Deporte como materia y su popularización en el ámbito académico. Para que este escenario cambie y sea posible ofrecer una formación adecuada en el curso de Psicología con el fin de fomentar esta opción de carrera, es necesario repensar el plan de estudios y el perfil del egresado, así los estudiantes tendrán más oportunidades de conocer sus bases teóricas y sus campos de actuación. Tal brecha puede debilitar la difusión de este conocimiento a los estudiantes de grado y la consecuente ocupación en el mercado laboral.(AU)

As startups no contexto da organização capitalista financeira e as subjetividades empreendedoras / Startups in the context of financial capitalist organization and entrepreneurial subjectivities / Startups en el contexto de la organización capitalista financiera y subjetividades emprendedoras

As startups são empresas que apresentam modelos de negócios marcados pela inovação, rapidez, flexibilidade e alta capacidade de adaptação aos mercados. Atuando em diferentes setores socioeconômicos, elas prometem criar e transformar produtos e serviços. A emergência e disseminação dessas empresas ocorrem em um momento histórico de mudanças iniciadas a partir de 1970 e marcadas pelas crises geradas com o esgotamento do paradigma da sociedade urbano industrial. No Brasil, o número desse modelo de negócio apresentou uma expansão expressiva, alcançando a marca de 13.374 nos últimos cinco anos. Atento a esse cenário, o objetivo desta pesquisa consistiu em compreender como sujeitos, grupos e instituições atribuem sentidos à experiência de trabalho nas chamadas startups. Na parte teórica, as condições sociais e econômicas que possibilitaram a emergência e disseminação das startups são analisadas em uma perspectiva crítica. A parte empírica, por sua vez, apresenta depoimentos de empreendedores relatando o contexto geral de atuação nas startups. Ao final deste artigo, conclui-se que há uma instrumentalização capitalística de componentes subjetivos específicos selecionados e colocados em circulação para fortalecer o modo de produção capitalista financeirizado.(AU)

Startups are companies that have business models characterized by innovation, speed, flexibility, and a high capacity to adapt to markets. Operating in different socioeconomic sectors, they promise to create and transform products and services. The emergence and dissemination of these companies occur at a historical moment of changes that began from 1970 and are marked by the crises generated by the exhaustion of the paradigm of industrial urban society. In Brazil, the number of businesses in this model showed a significant expansion, reaching 13,374 companies in the last five years. Attentive to this scenario, the objective of this research was to understand how subjects, groups, and institutions attribute meanings to the work experience in so-called startups. In the theoretical part, the social and economic conditions that enabled the emergence and dissemination of startups are analyzed in a critical perspective. The empirical part presents entrepreneurs reporting the general context of action in startups. At the end of this article, it is concluded that there is a capitalistic instrumentalization of specific subjective components that are selected and put into circulation to strengthen the financed capitalist production.(AU)

Las startups son empresas que tienen modelos de negocio marcados por la innovación, la velocidad, la flexibilidad y una alta capacidad de adaptación a los mercados. Desde diferentes sectores socioeconómicos, las startups prometen crear y transformar productos y servicios. La aparición y difusión de estas empresas se produce en un momento histórico de cambios que comenzó a partir de 1970 y que está marcado por crisis generadas por el agotamiento del paradigma de la sociedad urbana industrial. En Brasil, estas empresas se expandieron significativamente alcanzando la marca de 13.374 empresas en los últimos cinco años. En este escenario, el objetivo de esta investigación fue entender cómo los sujetos, grupos e instituciones atribuyen significados a la experiencia laboral en las startups. En la parte teórica, se analizan las condiciones sociales y económicas que permitieron el surgimiento y la difusión de las startups en una perspectiva crítica. La parte empírica presenta testimonios de emprendedores que informan sobre el trabajo en startups. La investigación concluye que hay una instrumentalización capitalista de componentes subjetivos específicos que se seleccionan y ponen en circulación para fortalecer el modo de producción capitalista financiero.(AU)

High-throughput screening of SARS-CoV-2 main and papain-like protease inhibitors

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (Mpro) and papain like protease (PLpro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851Mpro inhibitors and 205 PLpro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both Mpro and PLpro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of Mpro inhibitors and over 20% of PLpro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 Mpro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.

Planejamento, síntese e avaliação biológica de candidatos a fármacos: busca de inibidores epigenéticos da Sirtuína 2 Sir2 / Design, synthesis and biological evaluation of drug candidates: search for Sirtuin 2 (Sir 2) epigenetic inhibitors

As doenças negligenciadas são causadas por agentes infecciosos e parasitários, como vírus, bactérias, protozoários e helmintos. Essas doenças são prevalentes em populações de baixa renda que vivem em países em desenvolvimento e são responsáveis por incapacitar e levar milhares de pessoas à morte. Este nome se dá pois, apesar de sua grande relevância médica, recebem pouca atenção dos governos e indústrias farmacêuticas. Dentre essas doenças podemos destacar a Doença de Chagas, doença infecciosa causada pelo parasita hemoflagelado Trypanosoma cruzi. Endêmica em 21 países, com 6 a 7 milhões de pessoas infectadas resultando em 7500 mortes por ano. A quimioterapia disponível contra essa parasitose é baseada em apenas dois medicamentos, o benznidazol e o nifurtimox, ativos principalmente na fase aguda da doença e com efeitos adversos graves que comprometem a adesão ao tratamento e, além disso, apesar dos enormes esforços na pesquisa de novos agentes antichagásicos em nível nacional e internacional, na maioria realizada academicamente, ainda não foram encontradas alternativas terapêuticas para a doença, persistindo, assim, a necessidade de descoberta e desenvolvimento de novos fármacos. O início de um planejamento de um novo fármaco se dá pela definição de um alvo bioquímico a ser utilizado na busca de moléculas que possam exercer a função de inibidores ou moduladores, conforme a atividade biológica desejada. Neste sentido, as sirtuínas 2 (Sir2) são enzimas que se mostraram essenciais para o crescimento in vitro do T. cruzi em suas formas amastigota e epimastigota. No caso de tripanossomatídeos, em geral, a superexpressão de Sir2 está relacionada à sobrevivência de formas amastigotas. Assim, essas evidências indicam que a Sir2 de tripanosomatídeos tem grande potencial como alvo biológico na busca e desenvolvimento de novos fármacos antichagásicos. O objetivo principal deste projeto foi identificar moléculas que apresentaram atividade inibitória para a sirtuína 2 de T. cruzi por meio da utilização da estratégia de Planejamento de Fármacos Baseada no Ligante - Ligand Based Drug Design (LBDD) e o desenvolvimento de análogos dos inibidores da Sir2. A modificação molecular está entre algumas das técnicas tradicionais usadas no desenvolvimento racional de um fármaco, e é usada principalmente no desenvolvimento de análogos, e busca melhorar as propriedades farmacocinéticas e/ou farmacodinâmicas de um protótipo, obter propriedades de interação semelhantes ao alvo e, em alguns casos, revelar uma atividade biológica. Com este intuito, análogos do sirtinol e da salermida foram sintetizados e uma nova rota sintética utilizando o microrreator em fluxo contínuo foi desenvolvida e apresentou rendimento superior quando comparado à síntese em bancada. A partir desta metodologia foram obtidos 20 compostos. Os ensaios in vitro contra formas amastigotas do T. cruzi indicaram que 8 compostos inibiram a atividade parasitária em mais de 50%, na dose de 10 µM, sendo que alguns destes apresentaram maior inibição parasitária quando comparados ao benznidazol, o fármaco de referência e único disponível no Brasil. Com estes resultados preliminares, novos ensaios estão sendo realizados para identificar potência e mecanismo de ação destes candidatos a agentes tripanomicidas

Neglected diseases are caused by infectious and parasitic agents such as viruses, bacteria, protozoa and helminths. These diseases are prevalent in low-income populations living in developing countries and are responsible for disabling and killing thousands of people. They get this name because, despite their great medical relevance, they end up receiving little attention from governments and pharmaceutical industries. Among these diseases, we can highlight Chagas disease, an infectious endemic disease caused by the hemoflagellate parasite Trypanosoma cruzi. This disease is endemic in 21 countries, with 6 to 7 million people infected resulting in 7,500 deaths per year. Chemotherapy is based on just two drugs, benznidazole and nifurtimox, which are mainly active in the acute phase of the disease. These drugs have adverse effects that compromise adherence, even more, considering that they are not effective from the point of view of the chronic phase of the disease. Despite the enormous efforts in researching new anti-chagasic agents at the national and international level, and mostly carried out academically, therapeutic alternatives for the disease have not yet been found, thus, the need for the discovery and development of new drugs persists. Sirtuins 2 (Sir2) are enzymes that have been shown to be essential for the in vitro growth of T. cruzi in its amastigote and epimastigote forms. In the case of trypanosomatids in general, Sir2 overexpression is related to the survival of amastigote forms. Sir2 inhibitors, such as sirtinol, have shown efficacy in leishmanicides. Thus, these evidences indicate that Sir2 from trypanosomatids can be considered as a biological target in the search and development of new anti-chagasic drugs. The beginning of a new drug planning study is the definition of a biochemical target to be used in the search for molecules that can play the role of inhibitors or modulators, according to the desired biological activity. The main objective of this project was to identify molecules that presented inhibitory activity to sirtuin 2 of T. cruzi using the Ligand Based Drug Design (LBDD) strategy of planning and the development of analogues of Sir2 inhibitors. Molecular modification is a traditional technique used in the rational development of a drug, as well as the use of natural products, combinatorial chemistry, high-throughput screening (HTS), among others. Mainly used in the development of analogues, molecular modification is applied for different purposes, among them, it seeks to improve the pharmacokinetic and/or pharmacodynamic properties of a prototype, obtain target-like interaction properties and, in some cases, reveal an activity biological. For this purpose, analogues of sirtinol and salermide were synthesized and a new synthetic route using the microreactor in continuous flow was developed and presented superior yield when compared to benchtop synthesis. From this methodology, 20 compounds were obtained. in vitro assays against amastigote forms of T. cruzi indicated that 8 compounds inhibited parasitic activity by more than 50% at a dose of 10 µM, and some of these showed greater parasitic inhibition when compared to benznidazole, the reference drug, and only available in Brazil. With these preliminary results, new assays are being carried out to identify the potency and mechanism of action of these candidate trypanocidal agents

Advances in high-content screening applications in toxicology research / 中华预防医学杂志

The toxicity data of chemicals and drugs increases rapidly, while the animal experimental-based tests method could not meet the current demand of health risk assessment. The high-throughput screening techniques based on in vitro alternative models, integrating with computational methods and information technology to establish toxicity tests strategy promises to address this problem. High-content screening (HCS) technology uses automated microscopy and quantitative image platforms to perform multi-parameter and high-throughput phenotypic analysis via a visualization and quantification manner, and to quickly and effectively assess toxicity and prioritization of chemicals, which promotes the development of in vitro toxicity tests and computational toxicology. HCS technology has been included as an important tool for Toxicity Testing in the 21st Century (Tox21) and chemical risk prioritization. Its applications have been widely utilized in the research field of toxicity tests and chemical toxicity mechanisms. In this review, we describe the development of HCS technology, technical points, toxicological applications, and the future directions and challenges of HCS, so as to provide references for the toxicity testing technology and risk assessment methodology.

Construction of a high-throughput screening model for mitochondrial function of Aconiti Lateralis Radix Praeparata by machine learning algorithm and mechanism analysis / 中国中药杂志

A high-throughput screening machine learning model for mitochondrial function was constructed, and compounds of Aco-niti Lateralis Radix Praeparata were predicted. Deoxyaconitine with the highest score and benzoylmesaconine with the lowest score among the compounds screened by the model were selected for mitochondrial mechanism analysis. Mitochondrial function data were collected from PubChem and Tox21 databases. Random forest and gradient boosted decision tree algorithms were separately used for mo-deling, and ECFP4(extended connectivity fingerprint, up to four bonds) and Mordred descriptors were employed for training, respectively. Cross-validation test was carried out, and balanced accuracy(BA) and overall accuracy were determined to evaluate the performance of different combinations of models and obtain the optimal algorithm and hyperparameters for modeling. The data of Aconiti Lateralis Radix Praeparata compounds in TCMSP database were collected, and after prediction and screening by the constructed high-throughput screening machine learning model, deoxyaconitine and benzoylmesaconine were selected to measure mitochondrial membrane potential, reactive oxygen species(ROS) level and protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax) and peroxisome proliferator-activated receptor-γ-coactivator 1α(PGC-1α). The results showed that the model constructed using gradient boosted decision tree+Mordred algorithm performed better, with a cross-validation BA of 0.825 and a test set accuracy of 0.811. Deoxyaconitine and benzoylmesaconine changed the ROS level(P<0.001), mitochondrial membrane potential(P<0.001), and protein expression of Bcl-2(P<0.001, P<0.01) and Bax(P<0.001), and deoxyaconitine increased the expression of PGC-1α protein(P<0.01). The high-throughput screening model for mitochondrial function constructed by gradient boosted decision tree+Mordred algorithm was more accurate than that by random forest+ECFP4 algorithm, which could be used to build an algorithm model for subsequent research. Deoxyaconitine and benzoylmesaconine affected mitochondrial function. However, deoxyaconitine with higher score also affected mitochondrial biosynthesis by regulating PGC-1α protein.

Compromiso e intención de abandono en adolescentes de selecciones nacionales argentinas: el papel de los agentes sociales y efectos del género / Engagement and intentions of drop-out in adolescents of Argentinean national teams: The role of social agents and gender effects

Resumen Introducción/objetivo. El objetivo de esta investigación es examinar el poder predictivo de la percepción de los estilos interpersonales de entrenadores/as, padres y madres en el compromiso e intención de abandono de adolescentes deportistas argentinos de alto rendimiento, considerando el papel del género en esta relación. Método. Participaron 234 adolescentes de entre 12 y 16 años (M = 14.48, DT = 1.09) seleccionados para los Juegos Olímpicos de la Juventud -Buenos Aires 2018-, quienes cumplimentaron cuestionarios para la evaluación de las variables de interés. Resultados. Los varones perciben mayores niveles de compromiso que las mujeres y estas perciben mayor apoyo a la autonomía de la madre. El apoyo a la autonomía del entrenador tuvo mayor capacidad predictiva en el compromiso de los varones y en la intención de abandono del deporte de las mujeres. En ambos géneros, el efecto del apoyo a la autonomía del entrenador fue mayor que el de los padres. Conclusiones. Estos resultados muestran la importancia de los otros significativos en la participación deportiva y contribuyen al conocimiento de los factores que pueden favorecer el desarrollo positivo en deportistas adolescentes de alto rendimiento.

Abstract Introduction/Objective. This research aims to examine the predictive power of perceived interpersonal styles of coaches, fathers and mothers on engagement and intentions to drop-out of Argentinean high-performance adolescent athletes considering the role of gender in this relationship. Method. A total of 234 adolescents aged 12-16 years (M = 14.48, SD = 1.09) selected for the Youth Olympic Games -Buenos Aires 2018- participated in the study, who answered questionnaires assessing the variables of interest. Results. Males perceived higher levels of engagement than females, and females perceived higher autonomy support from the mother. Coaches' autonomy support had greater predictive power on males' engagement and females' intentions to drop-out sport. In both genders, the effect of coach autonomy support was greater than parents. Conclusions. These results show the importance of significant others in sport participation and contribute to the knowledge of factors that may foster positive development in high-performance adolescence athletes.

In Silico Screening of DNA Gyrase B Potent Flavonoids for the Treatment of Clostridium difficile Infection from PhytoHub Database

Abstract Clostridium difficile infection (CDI) is the most common hospital acquired diarrheal disease with its increasing incidence and mortality rate globally. DNA Gyrase B (GyrB) is a key component of DNA replication process across all bacterial genera; thus, this offers a potential target for the treatment of CDI. In the present study, several virtual screening approaches were employed to identify a novel C. difficile GyrB inhibitor. The 139 known metabolites were screened out from the 480 flavonoids in PhytoHub database. Molinspiration and PROTOX II servers were used to calculate the ADME properties and oral toxicity of the metabolites, whereas mutagenicity, tumorigenicity, irritant, and reproductive effect were predicted using DataWarrior program. The binding mode and the binding efficiency of the screened flavonoids against the GyrB were studied using FlexX docking program. From virtual screening of 139 metabolites, we found 25 flavonoids with no mutagenicity, tumorigenicity, irritant, and reproductive effect. Docking study suggested that flavonoids 1030 ((-)-epicatechin 3'-O-sulfate), 1032 ((-)-epicatechin 4'-O-sulfate), 1049 (3'-O-methyl-(-)-epicatechin 4-O-sulfate), 1051 (3'-O-methyl-(-)-epicatechin 7-O-sulfate), 1055 (4'-O-methyl-(-)-epicatechin 7-O-sulfate) and 1317 (quercetin sulfate) have significantly higher binding affinity than the known GyrB inhibitor novobiocin. The results from molecular dynamics simulation and free energy calculations based on solvated interaction energy suggested that (-)-epicatechin 3'-O-sulfate could be a potential drug candidate in the management of CDI.

Determinantes do desempenho esportivo na ginástica de trampolim / Determinants of sport performance in trampoline gymnastics / Determinantes del rendimiento deportivo en la gimnasia de trampolín

Introdução: encontrar crianças com elevado potencial esportivo é uma importante etapa para o processo de formação esportiva, pois é o primeiro passo na descoberta de novos talentos visando o alto rendimento. O objetivo foi investigar os determinantes do desempenho na ginástica de trampolim e sua importância atribuída de acordo com os aparelhos da modalidade. Metodologia: participaram 40 experts(treinadores, gestores e árbitros) de ginástica de trampolim com experiência nacional e internacional. Os experts responderam sobre a importância atribuída aos fatores antropométricos, físico-motor, técnico, tático, psicológico e socioambiental. Avaliaram a importância dos fatores e indicadores de desempenho nos aparelhos trampolim acrobático, tumbling e duplo minitrampolim. Resultados e discussão: os determinantes do desempenho em ordem de importância decrescente foram: físico-motores, técnicos, psicológicos, táticos, antropométricos e socioambientais, com variação entre os aparelhos. Os indicadores de desempenho: massa muscular, velocidade, agilidade, flexibilidade, equilíbrio e força rápida de membros superiores apresentaram diferenças entre os aparelhos. Conclusão: revelou-se a opinião dos experts sobre quais os fatores e indicadores de desempenho são os mais importantes na ginástica de trampolim com implicações na detecção e seleção de talentos.

Introduction: Finding childrenwithhigh sports potential is an important stage in the sports training process, as it is the first step to discover new talents aiming to high performance. The objectivewas to investigate the determinants of performance in trampoline gymnastics, and Its attributed importanceaccording to discipline apparatuses. Methodology: Forty trampoline gymnastics experts (coaches, managersand referees) with national and international experience volunteered. The experts answered aboutthe importance given to the anthropometric, physicomotor, technical, tactical, psychological and social-environmental factors. They evaluated the importance of the factors and performance indicators on the acrobatic, tumbling, and double mini trampoline. Results and discussion:The determinants of performance in descent importance order were the physicomotor, technical, psychological, tactical anthropometric and social-environmental factors. Therefore the indicators of performance: muscle mass, speed, agility, flexibility, balance and upper limbs explosive strength have been differently distinguished among the apparatuses. Conclusion:The expert's opinions were revealedabout which determinants of performance have greater value in the detection and selection of talents.

Introducción: Encontrar niños conelevadopotencial deportivo es una etapa importante en el proceso de formación deportiva, ya que es el primer paso para encontrar nuevos talentos con el objetivo del alto rendimiento deportivo. El objetivofue investigar los determinantes del rendimiento en la gimnasia de trampolín, e la importancia atribuida de acuerdo con los aparatos de la disciplina. Metodología:Han participadocuarentaexpertos en gimnasia en trampolín (entrenadores, gestores y árbitros) conexperiencianacional y internacional. Los expertos respondieron sobre la importancia dada a los factores antropométricos, fisicomotores, técnicos, tácticos, psicológicos y socio-ambientales. Evaluaron la importancia de los factores y los indicadores de rendimiento en el trampolínacrobático, tumbling y el doble mini. Resultados e discusión:Los determinantes del desempeño en orden de importancia decreciente fueron: físico motores, técnicos, psicológicos, tácticos, antropométricos y socio ambientales. Los indicadores de rendimiento, masa muscular, velocidad, agilidad, flexibilidad, equilibrio y fuerza rápida de las extremidades superiores tienen diferencias entre los aparatos. Conclusión: se reveló laopinión de los expertos sobre cualeslos determinantes del desempeño son más importantes e de más valor en la detección y selección de talentos en la gimnasia de trampolín.

Optimizations of an ELISA-like high-throughput screening assay for the discovery of β-catenin/TCF4 interaction antagonists / 生物工程学报

In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.

Advances of high-throughput screening system in reengineering of biological entities / 生物工程学报

Enzymes and cell factories are the core of industrial biotechnology. They play important roles in various fields such as medicine, chemical industry, food, agriculture, and energy. Usually, natural enzymes and cells need to be engineered to improve the catalytic efficiency, stability and enantioselectivity. Directed evolution makes it possible to rapidly improve the properties of enzymes and cell factories. Sensitive and reliable high-throughput screening approaches are the key for successful and efficient engineering of enzymes and cell factories. In this review, we first summarize the advantages and disadvantages of different screening methods and signal generation strategies as well as their application scope; we then describe the latest advances of ultra-high throughput screening technology applied in the directed evolution of enzymes and cell factories in the past three years. On this basis, we discuss the limiting factors that need to be further improved for high-throughput screening systems and forecast the future development trends of high-throughput screening methods, hoping that researchers in various fields including biotechnology and instrument development can cooperate closely to enhance the reliability and applicability of the high-throughput screening techniques.

High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M

High-throughput screening of Saccharomyces cerevisiae efficiently producing tyrosine / 生物工程学报

Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.

Rapid generation of double-layer emulsion droplets based on microfluidic chip / 生物工程学报

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 μm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 μm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.

Clinical and biomedical applications of surface plasmon resonance systems

Abstract Surface plasmon resonance (SPR)-based biosensors offer superior analytical features such as simplicity, sensitivity, and specificity when compared to conventional methods in clinical analyses. In addition, they deliver real-time monitoring of label-free analytes with high-throughput approaches requiring little sample pretreatment that allows the analysis of virtually every clinical sample type to determine the amount and/or activity of any molecule of interest. Accordingly, SPR emerges as a novel, efficient, powerful, and relatively low-cost alternative tool for routine clinical analysis, opening also new horizons for developments in personalized medicine applied to diagnostics or therapeutics’ monitoring.

Development of an ELISA-based high throughput screening method for novel anticancer agents targeting β-catenin/Lef1 interaction / 生物工程学报

To develop an enzyme-linked immunosorbent assay (ELISA)-based high throughput screening (HTS) method for β-catenin/Lef1 interaction antagonists screening, Escherichia coli Rosetta (DE3) competent cells were transformed with β-catenin-pET-30a(+) plasmid. β-catenin protein was expressed after induction and purified using affinity chromatography. The biological activity of purified β-catenin was further analyzed by GST Pulldown assay. The β-catenin/GST-Lef1 binding model was established using ELISA principle, and the ELISA-based HTS method was further optimized through determination of an optimal coated concentration of GST-Lef1 and working concentration of β-catenin. The results showed that β-catenin protein was successfully expressed and purified. The GST Pulldown assay demonstrated a perfect biological activity for purified β-catenin. Subsequently, the ELISA-based HTS method was performed using 10 μg/mL GST-Lef1 and 6 μg/mL β-catenin, with the Z factor of 0.76. Taken together, we have successfully developed a simple, robust and reliable ELISA-based HTS method for screening of novel Wnt inhibitors targeting β-catenin/Lef1 interaction.

Protein engineering: from directed evolution to computational design / 生物工程学报

By constructing mutant libraries and utilizing high-throughput screening methods, directed evolution has emerged as the most popular strategy for protein design nowadays. In the past decade, taking advantages of computer performance and algorithms, computer-assisted protein design has rapidly developed and become a powerful method of protein engineering. Based on the simulation of protein structure and calculation of energy function, computational design can alter the substrate specificity and improve the thermostability of enzymes, as well as de novo design of artificial enzymes with expected functions. Recently, machine learning and other artificial intelligence technologies have also been applied to computational protein engineering, resulting in a series of remarkable applications. Along the lines of protein engineering, this paper reviews the progress and applications of computer-assisted protein design, and current trends and outlooks of the development.

High-throughput micro-phenotyping measurements applied to assess stalk lodging in maize ( Zea mays L. )

BACKGROUND: The biomechanical properties of maize stalks largely determine their lodging resistance, which affects crop yield per unit area. However, the quantitative and qualitative relationship between micro-phenotypes and the biomechanics of maize stalks is still under examined. In particular, the roles of the number, geometry, and distribution of vascular bundles of stalks in maize lodging resistance remain unclear. Research on these biomechanical properties will benefit from high-resolution micro-phenotypic image acquisition capabilities, which have been improved by modern X-ray imaging devices such as micro-CT and the development of micro-phenotyping analysis software. Hence, high-throughput image analysis and accurate quantification of anatomical phenotypes of stalks are necessary. RESULTS: We have updated VesselParser version 1.0 to version 2.0 and have improved its performance, accuracy, and computation strategies. Anatomical characteristics of the second and third stalk internodes of the cultivars 'Jingke968' and 'Jingdan38' were analyzed using VesselParser 2.0. The relationships between lodging resistance and anatomical phenotypes of stalks between the two different maize varieties were investigated. The total area of vascular bundles in the peripheral layer, auxiliary axis diameter, and total area of vascular bundles were revealed to have the highest correlation with mechanical properties, and anatomical phenotypes of maize stalk were better predictors of mechanical properties than macro features observed optically from direct measurement, such as diameter and perimeter. CONCLUSIONS: This study demonstrates the utility of VesselParser 2.0 in assessing stalk mechanical properties. The combination of anatomical phenotypes and mechanical behavior research provides unique insights into the problem of stalk lodging, showing that micro phenotypes of vascular bundles are good predictors of maize stalk mechanical properties that may be important indices for the evaluation and identification of the biomechanical properties to improve lodging resistance of future maize varieties.

Recent Advances in Function-based Metagenomic Screening / 基因组蛋白质组与生物信息学报·英文版

Metagenomes from uncultured microorganisms are rich resources for novel enzyme genes. The methods used to screen the metagenomic libraries fall into two categories, which are based on sequence or function of the enzymes. The sequence-based approaches rely on the known sequences of the target gene families. In contrast, the function-based approaches do not involve the incorporation of metagenomic sequencing data and, therefore, may lead to the discovery of novel gene sequences with desired functions. In this review, we discuss the function-based screening strategies that have been used in the identification of enzymes from metagenomes. Because of its simplicity, agar plate screening is most commonly used in the identification of novel enzymes with diverse functions. Other screening methods with higher sensitivity are also employed, such as microtiter plate screening. Furthermore, several ultra-high-throughput methods were developed to deal with large metagenomic libraries. Among these are the FACS-based screening, droplet-based screening, and the in vivo reporter-based screening methods. The application of these novel screening strategies has increased the chance for the discovery of novel enzyme genes.