Three new troglobitic species of Pseudochthonius Balzan, 1892 (Pseudoscorpiones, Chthoniidae) from northeastern Brazil.

Three new pseudoscorpion species, Pseudochthonius pali sp. nov., Pseudochthonius diamachi sp. nov., and Pseudochthonius koinopoliteia sp. nov., are herein described from caves of Bahia state, Brazil. They can be distinguished from congeners mainly by the absence of eyes or eyespots, chelal and cheliceral dentition, coxal chaetotaxy and body size. Some ecological remarks on each species, possible threats, recommendations for future research and a key to the Pseudochthonius species from Brazil are provided.

Paradigma manicomial e proibicionismo como operadores da guerra de raças no Brasil / Asylum paradigm and prohibitionism operating the racial war in Brazil / Paradigma asilar y prohibicionismo como operadores de la guerra racial en Brasil

Este artigo relaciona o paradigma manicomial, relativo à assistência psiquiátrica, à compreensão e ao manejo do campo da saúde mental, ao paradigma proibicionista, referente ao porte, uso e à circulação de drogas, como duas séries de políticas e práticas sociais que operam a guerra de raças que está na base do Estado brasileiro. Com isso, propomos uma investigação arqueogenealógica acerca do emaranhado de condições de emergência das práticas e objetos de saber-poder mobilizados por esses dois paradigmas, atentando ao caráter político das verdades que as sustentam. Dedicamo-nos especialmente ao período entre o final do século XIX e o começo do XX ao interrogar as dinâmicas de forças que constituem as práticas sociais e seus efeitos de subjetivação, produzidos pela sujeição de corpos por meio de uma diversidade de mecanismos morais, disciplinares, eugênicos, higienistas e biopolíticos que articulam os anseios de modernização e produtividade do Estado brasileiro à gestão dos problemas de saúde e segurança do país, colocando a pobreza, o vício e a doença como desdobramento da sua constituição racial. Concluímos, por fim, que o conflito de raças aparece como fundo intrínseco que se atualiza no cerne e a partir dos campos problemáticos da saúde mental e das drogas, colocando como saída dos impasses sociais e políticos eliminar ou pelo menos diluir, via miscigenação ou submissão para integração, o elemento físico e cultural do negro do Brasil.(AU)

This article puts in relation the asylum paradigm, associated to psychiatric care, to the understanding and management of the mental health field, to the prohibitionist paradigm, that refers to the possession, use and circulation of drugs, as two series of social policies and practices that operate racial war that is in the base of the Brazilian State. So on, we propose an archeogenealogical investigation about the emergency conditions of the practices and objects of knowledge-power organized by these two paradigms, paying attention to the political character of the truths that support them. Looking especially at the period between the end of the 19th century and the beginning of the 20th, we questioned the dynamics of forces that constitute social practices and their effects of subjectivation, produced by the subjection of bodies through moral, disciplinary, eugenic, hygienist and biopolitics mechanisms that articulate the modernization and productivity aspirations of the Brazilian State to the management of the country's health and safety problems, understanding poverty, addiction and disease as consequences of its racial constitution. We conclude that the conflict of races is an intrinsic background that is updated at the heart of the problematic fields of mental health and drugs. Considering this, the solution for social and political impasses is the elimination or at least dilution, through miscegenation or submission for integration, of the physical and cultural element of black people in Brazil.(AU)

Este artículo relaciona el paradigma asilar de atención psiquiátrica, comprensión y manejo del campo de la salud mental, con el paradigma prohibicionista, referente a al uso y circulación de drogas, como dos series de políticas y prácticas sociales que operan la guerra racial que está en el fundamento del Estado brasileño. Así, proponemos una investigación arqueogenealógica sobre las condiciones de emergencia de prácticas y objetos de saber-poder movilizados por estos dos paradigmas, prestando atención al carácter político de las verdades que los sustentan. Nos dedicamos especialmente al período entre finales del siglo XIX y principios del XX buscando la dinámica de fuerzas que constituyen a las prácticas sociales y sus efectos de subjetivación, producidos por la sujeción de los cuerpos a través de una diversidad de mecanismos morales, disciplinarios, eugenésicos, higienistas y biopolíticos que articulan las aspiraciones de modernización y productividad del Estado brasileño a la gestión de los problemas de salud y seguridad del país, comprendiendo la pobreza, la adicción y la enfermedad como resultado de su constitución racial. Finalmente, concluimos que el conflicto racial aparece como un trasfondo intrínseco que se actualiza en el cerne y desde los campos problemáticos de la salud mental y de las drogas, tomando como soluciones a los impasses sociales y políticos nacionales, la eliminación o al menos la dilución, a través del mestizaje o de la sumisión para fines de integración, del elemento físico y cultural del negro en Brasil.(AU)

CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane.

Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on in vitro tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars.

Direitos Sexuais e Reprodutivos nos Protocolos Profissionais sobre HIV/Aids / Sexual Rights and Reproductive Rights in Professional Protocols on HIV/AIDS / Derechos Sexuales y Reproductivos en Protocolos Profesionales sobre VIH/SIDA

O trabalho em saúde no campo do HIV/Aids requer uma abordagem interdisciplinar e tem como desafios fundamentais a adesão da população aos métodos e medidas de prevenção e tratamento, assim como a efetivação do acesso e da integralidade na assistência, o enfrentamento à discriminação e à estigmatização das populações-chave e das pessoas que vivem com HIV. Além disso, ele mobiliza questões complexas relacionados à sexualidade e ao exercício da autonomia cidadã. Tendo isso em vista, analisamos neste trabalho dezoito protocolos clínicos que instruem a atuação profissional na assistência ao HIV/Aids, pautando temas a serem abordados e procedimentos para qualificar o atendimento. Realizamos uma revisão integrativa buscando elucidar o entendimento dos diretos sexuais e reprodutivos presente nestes documentos. Com a síntese dos resultados obtidos, organizamos três categorias a partir das quais são desenvolvidos a temática dos direitos sexuais e reprodutivos: vulnerabilidade; autonomia; gênero e diversidade sexual. Concluímos que os tópicos da vulnerabilidade, reiteradamente abordado, e da sexualidade, que coloca progressivamente em questão a desnaturalização dos papéis sociais de gênero, buscam convergir aspectos sociais, culturais e individuais, indicando um aprimoramento das práticas de promoção de autonomia e a efetivação dos direitos sexuais e reprodutivos na assistência ao HIV/Aids.

Health work in the field of HIV/AIDS requires an interdisciplinary approach and has as fundamental challenges the population's adherence to prevention and treatment methods and measures, as well as the effective access and integrality in assistance, and the struggle against discrimination and stigmatization of key-populations and people living with HIV. In addition, it mobilizes complex issues related to sexuality and the exercise of citizenship autonomy. Considering this, we analyzed in this work eighteen clinical protocols that guide professional performance in HIV/AIDS care, organizing themes and procedures to qualify the care. We performed an integrative review to elucidate the understanding of sexual and reproductive rights present in these documents. With the synthesis of the results obtained, we organized three categories from which the theme of sexual and reproductive rights is developed: vulnerability; autonomy; gender and sexual diversity. We conclude that the topics of vulnerability, repeatedly addressed, and sexuality, which progressively questions the naturalization of social gender roles, try to converge social, cultural and individual aspects, indicating an improvement of practices that promotes sexual and reproductive autonomy and enforcement of rights in HIV/AIDS care.

El trabajo en salud en el campo del VIH/SIDA requiere un abordaje interdisciplinario y tiene como desafíos fundamentales la adhesión de la población a los métodos y medidas de prevención y tratamiento, así como el acceso efectivo y la atención integral, combatiendo la discriminación y estigmatización de las poblaciones-clave y de las personas que viven con el VIH. Además, moviliza temas complejos relacionados con la sexualidad y el ejercicio de la autonomía ciudadana. Considerando eso, analizamos dieciocho protocolos clínicos que orientan la actuación profesional en la atención al VIH/SIDA, orientando temas a ser abordados y procedimientos para calificar la atención. Realizamos una revisión integrativa buscando elucidar la comprensión de los derechos sexuales y reproductivos presente en estos documentos. Con la síntesis de los resultados obtenidos, organizamos tres categorías con las cuales se desarrolla la temática de los derechos sexuales y reproductivos: vulnerabilidad; autonomía; género y diversidad sexual. Concluimos que los temas vulnerabilidad, repetidamente abordado, y sexualidad, que progresivamente cuestiona la naturalización de los roles sociales de género, buscan converger aspectos sociales, culturales e individuales, indicando una mejora de las prácticas de promoción de la autonomía y la efectuación de los derechos sexuales y reproductivos en la atención del VIH/SIDA.

Nanoparticles in association with antimicrobial peptides (NanoAMPs) as a promising combination for agriculture development.

Antimicrobial peptides are small molecules, up to 10 kDa, present in all kingdoms of life, including in plants. Several studies report that these molecules have a broad spectrum of activity, including antibacterial, antifungal, antiviral, and insecticidal activity. Thus, they can be employed in agriculture as alternative tools for phytopathogen and pest control. However, the application of peptides in agriculture can present challenges, such as loss of activity due to degradation of these molecules, off-target effects, and others. In this context, nanotechnology can offer versatile structures, including metallic nanoparticles, liposomes, polymeric nanoparticles, nanofibers, and others, which might act both in protection and in release of AMPs. Several polymers and biomaterials can be employed for the development of nanostructures, such as inorganic metals, natural or synthetic lipids, synthetic and hybrid polymers, and others. This review addresses the versatility of NanoAMPs (Nanoparticles in association with antimicrobial peptides), and their potential applications in agribusiness, as an alternative for the control of phytopathogens in crops.

The effects of ageing, BMI and physical activity on blood IL-15 levels: A systematic review and meta-analyses.

AIM: The purpose of the study was to test the effect of ageing, BMI, physical activity and chronic exercise on IL-15 blood concentration by meta-analyses of the literature. METHODS: The search was performed on PubMed/MEDLINE, Web of Science, ProQuest, Embase and Cochrane databases. First meta-analysis compared blood IL-15 of healthy adults across three age groups (<35 years, 35-65 years, and >65 years), considering BMI as confounding factor; the second compared IL-15 levels between physically active and non-physically active individuals (cross-sectional studies); and the third tested the effect of chronic exercise interventions on blood IL-15 levels on participants of any age, sex, and health condition. RESULTS: From 2582 studies retrieved, 67 were selected for the three meta-analyses (age effect: 59; physical activity cross-sectional effect: 5; chronic exercise effect: 14). Older adults had lower blood IL-15 than young and middle-aged adults (5.30 pg/ml [4.76; 5.83]; 7.11 pg/ml [6.33; 7.88]; 7.10 pg/ml [5.55; 8.65], respectively). However, the subgroup of overweight older adults had higher IL-15 than young and middle aged overweight adults; Habitual physical activity did not affect blood IL-15 (standardized mean difference [SMD] 0.61 [-0.65; 1.88], p = 0.34); Chronic exercise reduced blood IL-15 in short-term interventions (<16 weeks) (SMD -0.14 [-0.27; -0.01], p = 0.04), but not studies of >16 weeks of intervention (SMD 0.44 [-0.26; 1.15], p = 0.22). CONCLUSION: The present meta-analyses highlight the complex interaction of age, BMI and physical activity on blood IL-15 and emphasize the need to take these factors into account when considering the role of this myokine in health throughout life.

Do the New Protocols of Platelet-Rich Fibrin Centrifugation Allow Better Control of Postoperative Complications and Healing After Surgery of Impacted Lower Third Molar? A Systematic Review and Meta-Analysis.

PURPOSE: Platelet concentrate generation protocols have undergone several modifications in recent years; in light of these new developments, this study review aims to evaluate the effects of platelet-rich fibrin (PRF) and the new centrifugation protocols, advanced platelet-rich fibrin (A-PRF), and leukocyte platelet-rich fibrin (L-PRF), after extraction of impacted mandibular third molar. Specifically, we assessed pain control, edema, trismus, and soft tissue healing, and also measured the degree of periodontal regeneration adjacent to the second molar. METHODS: PubMed, MEDLINE, EMBASE, Web of Science, Virtual health library (BVS), and Cochrane Library were searched up to Julye 2021; randomized controlled studies were included. This report followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and PICO (population, intervention, comparison, outcome) questions. This review has been registered at the International Prospective Register of Ongoing Systematic Reviews (PROSPERO) under the number CRD42019136701. The risk of bias screening and data extraction was performed according to the guidelines recommended by Cochrane. The quantitative analysis was performed using RevMan version 5.4. RESULTS: Of 17 studies included in the systematic review, 11 were eligible for the meta-analysis. The use of L-PRF was not associated with better soft tissue healing at day 7. (standard mean difference = -0.70; 95% confidence interval, -3.50 to 2.10; Z = 0.49; P = .62; heterogeneity = 0.00001; I2 = 97%). With L-PRF, qualitative analysis revealed better pocket depth and insertion level, and also better pain control at 1 and 3 days. With A-PRF, a lower consumption of analgesics was observed than with L-PRF. With both A-PRF and L-PRF, better control of edema (but not trismus) was observed. CONCLUSIONS: The use of L-PRF and A-PRF allows better control of pain and edema compared with the use of standard PRF protocols, but neither has an effect on trismus. The PRF and L-PRF protocols improve soft tissue healing, although not to a statistically significant degree; however, they could improve probing depth at the third month after third molar surgery.

O coronavírus como analisador das práticas de poder na atualidade / The coronavirus as an analyst of practices of power today / El coronavírus como analisador de las prácticas de poder en la actualidad

A pandemia de Covid-19 revolve e faz emergir diversos problemas novos e antigos transformando-se num potente analisador da atualidade. O presente ensaio aborda as estratégias de exercício do poder sobre a vida e a morte mobilizadas pelas ações de enfrentamento da crise sanitária e seus desdobramentos políticos que pautam os vários modos de gestão da sociedade. Nosso objetivo é problematizar a situação atual usando dados epidemiológicos e estatísticas estatais e de organizações internacionais, notícias da grande imprensa, colocando em cena medidas de governos, ações e políticas públicas adotadas no período de pandemia no Brasil, contrastando-o com o cenário internacional. Como resultados de uma abordagem crítica pelo prisma da genealogia dos poderes e da esquizoanálise acerca da constituição ético-política das práticas de saber-poder, expomos seus efeitos disciplinares, biopolíticos e necropolíticos em suas relações com o autoritarismo brasileiro.

The Covid-19 pandemic raises several new and old problems, transforming itself into a powerful analyzer of our present time. This essay addresses the different strategies of power over life and death mobilized by actions to face the health crisis and its unfolding in politics and different manners to manage the society. Our objective is to problematize the current situation using epidemiological data and statistics from state and international organizations and news from the mainstream media to look over government measures, public actions adopted during the pandemic period in Brazil and in the international scenario. As results of a critical approach that uses genealogy of power and schizoanalysis to focus the ethical-political constitution of knowledge-power practices, we expose its disciplinary, biopolitical and necropolitical effects in its relations with Brazilian authoritarianism.

La pandemia de Covid-19 plantea varios problemas nuevos o no, transformándose en un poderoso analizador de la actualidad. Este ensayo aborda las diferentes estrategias de poder sobre la vida y la muerte movilizadas por el enfrentamiento de la crisis sanitaria y sus desarrollos políticos que guían los modos de gestión de la sociedad. Nuestro objetivo es problematizar la situación actual utilizando datos y estadísticas epidemiológicas de organizaciones estatales e internacionales y noticias evidenciando las medidas gubernamentales, acciones y políticas públicas adoptadas durante la pandemia en Brasil y en el mundo. Como resultado de una análisis genealogíca y esquizoanálitica sobre la constitución ético-política de las prácticas de conocimiento-poder, exponemos sus efectos disciplinarios, biopolíticos y necropolíticos en sus relaciones con el autoritarismo brasileño.

Um estudo sobre o paradigma proibicionista e a (des)criminalização da maconha no Brasil / A study of the prohibitionist paradigm and the (de)criminalization of marihuana in Brasil / Un estudio sobre el paradigma prohibicionista y la (des)criminalización de la marihuana em Brasil

A maconha atualmente figura entre as substâncias consideradas ilícitas no Brasil. No entanto, nem sempre foi assim. O processo histórico que culminou com a proibição da maconha no Brasil é datado do início do século XX, perpassado por concepções eugenistas e higienistas que consolidaria o paradigma proibicionista.Todavia, ainda que paulatinamente, o proibicionismo vem sendo questionado em âmbito internacional e já há registros de outros modelos de relação de Estados com as drogas e dos indivíduos com as substâncias psicoativas. Sendo assim, o presente artigo, através da realização de entrevistas semiestruturadas, buscou identificar as diferentes maneiras pelas quais a proibição da maconha exerce seus efeitos nas subjetividades de indivíduos militantes, usuários ou não da planta. Dessa maneira, pretende-se demostrar a possibilidade de se pensar novas formas de se relacionar com a maconha, tanto em âmbito individual quanto em âmbito social, e ampliar o conhecimento da população acerca dos efeitos da proibição. (AU)

Marijuana currently ranks among the substances considered illegal in Brazil. However, it was not always like this. The historical process that culminated inthe prohibition of marijuana in Brazil is dated to the beginning of the 20th century, when eugenicist and hygienist ideas helped to consolidated the prohibitionist paradigm. However, even if gradually, prohibitionism has been questioned at the international level and there are already records of other models of relationship between States and drugs and between individuals and psychoactive substances. Therefore, this article, through semi-structured interviews, sought to identify the different ways in which the prohibition of marijuana exerts its effects on the subjectivities of individuals, users or not users of the plant. In this way, it is intended to demonstrate the possibility of thinking about new ways of relating to marijuana, both individually and socially and to broaden the population's knowledge about the effects of the ban. (AU)

La marihuana, en la actualidad, se encuentra como las substancias ilegales en Brasil. Pero no siempre fue así. El proceso histórico que culminó con la prohibición de marihuana en Brasil data del principio del siglo XX, influenciado por concepciones eugénicas e higienistas que consolidarían el paradigma prohibicionista. Aunque gradualmente, el prohibicionismo ha sido cuestionado y ya existen otros modos de relación entre los Estados con las drogas y también de las personas con las substancias. Por ello, este artículo, a través de entrevistas, buscó identificar las diferentes formas que la prohibición de la marihuana ejerce sus efectos sobre la subjetividad. Por lo tanto, tiene la intención de demonstrar la posibilidad de pensar en diferentes modos de relacionarse con la marihuana, tanto individual como socialmente, así como aumentar el conocimiento acerca de los efectos de la criminalización. (AU)

CRISPR Genome Editing Technology: A Powerful Tool Applied to Developing Agribusiness.

The natural increase of the world's population implies boosting agricultural demand. In the current non-optimistic global scenario, where adverse climate changes come associated with substantial population growth, the main challenge in agribusiness is food security. Recently, the CRISPR/Cas system has emerged as a friendly gene editing biotechnological tool, enabling a precise manipulation of genomes and enhancement of desirable traits in several organisms. This review highlights the CRISPR/Cas system as a paramount tool for the improvement of agribusiness products and brings up-to-date findings showing its potential applications in improving agricultural-related traits in major plant crops and farm animals, all representing economic-relevant commodities responsible for feeding the world. Several applied pieces of research have successfully demonstrated the CRISPR/Cas ability in boosting interesting traits in agribusiness products, including animal productivity and welfare, crop yield growth, and seed quality, reflecting positive impacts in both socioeconomics and human health aspects. Hence, the CRISPR/Cas system has revolutionized bioscience and biotechnology, and its concrete application in agribusiness goods is on the horizon.

Coloniality and perspectivism in psychology: from damnation to ecosophical care relations.

Inspired by Amerindian peoples' philosophy, this article aims to problematise the modern philosophical anthropology that underlies psychology as a social practice, and to use the Amerindian perspectivist philosophy proposed by Viveiros de Castro as a critique of Western values. Thus, we approach some of coloniality's epistemological implications in the institution of subject-object and nature-culture separations that grounds psychology. On the one hand, there is a totalising unification, which operates alongside a transcendental subject that subjugates its object. Beginning from nature's homogeneity, the differentiation and hierarchy of each being depends on its representations or its soul, expressed as a people's culture or as an individual psychology. This spiritual-representational aspect is the very foundation of the colonialism and racism of damnation, and their purpose in perpetuating the miserable ways of life of those considered to be scum. On the other hand, in Amerindian cosmogony, humanity is a pronominal mode, a non-exclusive perspectivist position of mankind, built in each context of relations. It is based on this idea that we posit other bases and a new agenda for psychology as a social practice which can enable us to broaden our ways of living towards ecosophical care, as a way of resistance to coloniality.

Author Correction: Enzymatic activity of a recombinant ß-1,4-endoglucanase from the Cotton Boll Weevil (Anthonomus grandis) aiming second generation ethanol production.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Corrigendum: Proteomic Analysis and Functional Validation of a Brassica oleracea Endochitinase Involved in Resistance to Xanthomonas campestris.

[This corrects the article DOI: 10.3389/fpls.2019.00414.].

Mosses: Versatile plants for biotechnological applications.

Mosses have long been recognized as powerful experimental tools for the elucidation of complex processes in plant biology. Recent increases in the availability of sequenced genomes and mutant collections, the establishment of novel technologies for targeted mutagenesis, and the development of viable protocols for large-scale production in bioreactors are now transforming mosses into one of the most versatile tools for biotechnological applications. In the present review, we highlight the astonishing biotechnological potential of mosses and how these plants are being exploited for industrial, pharmaceutical, and environmental applications. We focus on the biological features that support their use as model organisms for basic and applied research, and how these are being leveraged to explore the biotechnological potential in an increasing number of species. Finally, we also provide an overview of the available moss cultivation protocols from an industrial perspective, offering insights into batch operations that are not yet well established or do not even exist in the literature. Our goal is to bolster the use of mosses as factories for the biosynthesis of molecules of interest and to show how these species can be harnessed for the generation of novel and commercially useful bioproducts.

Enzymatic activity of a recombinant ß-1,4-endoglucanase from the Cotton Boll Weevil (Anthonomus grandis) aiming second generation ethanol production.

In the last years, the production of ethanol fuel has started to change with the introduction of second-generation ethanol (2 G Ethanol) in the energy sector. However, in Brazil, the process of obtaining 2 G ethanol did not reach a basic standard to achieve relevant and economically viable results. Several studies have currently been addressed to solve these issues. A critical stage in the bioethanol production is the deployment of efficient and stable enzymes to catalyze the saccharification step into the process of biomass conversion. The present study comprises a screening for genes coding for plant biomass degradation enzymes, followed by cloning a selected gene, addressing its heterologous expression, and characterizing enzymatic activity towards cellulose derived substrates, with a view to second-generation ethanol production. A cDNA database of the Cotton Boll Weevil, Anthonomus grandis (Coleoptera: Curculionidae), an insect that feeds on cotton plant biomass, was used as a source of plant biomass degradation enzyme genes. A larva and adult midgut-specific ß-1,4-Endoglucanase-coding gene (AgraGH45-1) was cloned and expressed in the yeast Pichia pastoris. Its amino acid sequence, including the two catalytic domains, shares high identity with other Coleoptera Glycosyl Hydrolases from family 45 (GH45). AgraGH45-1 activity was detected in a Carboxymethylcellulose (CMC) and Hydroxyethylcellulose (HEC) degradation assay and the optimal conditions for enzymatic activity was pH 5.0 at 50 °C. When compared to commercial cellulase from Aspergillus niger, Agra GH45-1 was 1.3-fold more efficient to degrade HEC substrate. Together, these results show that AgraGH45-1 is a valid candidate to be engineered and be tested for 2 G ethanol production.

Proteomic Analysis and Functional Validation of a Brassica oleracea Endochitinase Involved in Resistance to Xanthomonas campestris.

Black rot is a severe disease caused by the bacterium Xanthomonas campestris pv. campestris (Xcc), which can lead to substantial losses in cruciferous vegetable production worldwide. Although the use of resistant cultivars is the main strategy to control this disease, there are limited sources of resistance. In this study, we used the LC-MS/MS technique to analyze young cabbage leaves and chloroplast-enriched samples at 24 h after infection by Xcc, using both susceptible (Veloce) and resistant (Astrus) cultivars. A comparison between susceptible Xcc-inoculated plants and the control condition, as well as between resistant Xcc-inoculated plants with the control was performed and more than 300 differentially abundant proteins were identified in each comparison. The chloroplast enriched samples contributed with the identification of 600 additional protein species in the resistant interaction and 900 in the susceptible one, which were not detected in total leaf sample. We further determined the expression levels for 30 genes encoding the identified differential proteins by qRT-PCR. CHI-B4 like gene, encoding an endochitinase showing a high increased abundance in resistant Xcc-inoculated leaves, was selected for functional validation by overexpression in Arabidopsis thaliana. Compared to the wild type (Col-0), transgenic plants were highly resistant to Xcc indicating that CHI-B4 like gene could be an interesting candidate to be used in genetic breeding programs aiming at black rot resistance.

Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil α-amylase.

BACKGROUND: Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis α-amylase (AGA). RESULTS: We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. CONCLUSIONS: We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.

Gene-silencing suppressors for high-level production of the HIV-1 entry inhibitor griffithsin in Nicotiana benthamiana.

The exploration of emerging host organisms for the economic and efficient production of protein microbicides against HIV is urgently needed in resource-poor areas worldwide. In this study, the production of the novel HIV entry inhibitor candidate, griffithsin (GRFT), was investigated using Nicotiana benthamiana as the expression platform based on a non-viral vector. To increase the yield of recombinant GRFT, the RNA silencing defense mechanism of N. benthamiana was abolished by using three gene silencing suppressors. A transient expression system was used by transferring the GRFT gene, which encodes 122 amino acids, under the control of the enhanced CaMV 35S promoter. The presence of correctly assembled GRFT in transgenic leaves was confirmed using immunoglobulin-specific sandwich ELISA. The data demonstrated that the use of three gene silencing suppressors allowed the highest accumulation of GRFT, with a yield of 400 µg g-1 fresh weight, and this amount was reduced to 287 µg g-1 after purification, representing a recovery of 71.75%. The analysis also showed that the ability of GRFT expressed in N. benthamiana to bind to glycoprotein 120 is close to that of the GRFT protein purified from E. coli. Whole-cell assays using purified GRFT showed that our purified GRFT was potently active against HIV. This study provides the first high-level production of the HIV-1 entry inhibitor griffithsin with a non-viral expression system and illustrates the robustness of the co-agroinfiltration expression system improved through the use of three gene silencing suppressors.