Corrected and republished from: "Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence".

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants expected to lack NOP16 expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing potentially altered sugar transport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in G. mellonella. These results connect EV biogenesis, cargo, and cryptococcal virulence.

Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence and Requires the NOP16 Gene.

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants lacking NOP16 expression, we observed that this gene was required for EV production. Analysis of the small molecule composition of EVs produced by wild-type cells and two independent nop16Δ mutants revealed that the deletion of NOP16 resulted not only in a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the nop16Δ mutants were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were coinjected with the nop16Δ cells in G. mellonella. These results reveal a role for NOP16 in EV biogenesis and cargo, and also indicate that the composition of EVs is determinant for cryptococcal virulence.

Mass Spectrometry Analysis Reveals Lipids Induced by Oxidative Stress in Candida albicans Extracellular Vesicles.

Candida albicans is a commensal fungus in healthy humans that causes infection in immunocompromised individuals through the secretion of several virulence factors. The successful establishment of infection is owing to elaborate strategies to cope with defensive molecules secreted by the host, including responses toward oxidative stress. Extracellular vesicle (EV) release is considered an alternative to the biomolecule secretory mechanism that favors fungal interactions with the host cells. During candidiasis establishment, the host environment becomes oxidative, and it impacts EV release and cargo. To simulate the host oxidative environment, we added menadione (an oxidative stress inducer) to the culture medium, and we explored C. albicans EV metabolites by metabolomics analysis. This study characterized lipidic molecules transported to an extracellular milieu by C. albicans after menadione exposure. Through Liquid Chromatography coupled with Mass Spectrometry (LC-MS) analyses, we identified biomolecules transported by EVs and supernatant. The identified molecules are related to several biological processes, such as glycerophospholipid and sphingolipid pathways, which may act at different levels by tuning compound production in accordance with cell requirements that favor a myriad of adaptive responses. Taken together, our results provide new insights into the role of EVs in fungal biology and host-pathogen interactions.

Metabolomic analysis reveals stress tolerance mechanisms in common bean (Phaseolus vulgaris L.) related to treatment with a biostimulant obtained from Corynebacterium glutamicum.

Microbial biostimulants have emerged as a sustainable alternative to increase the productivity and quality of important crops. Despite this, the effects of the treatment on plant metabolism are poorly understood. Thus, this study investigated the metabolic response of common bean (Phaseolus vulgaris) related to the treatment with a biostimulant obtained from the extract of Corynebacterium glutamicum that showed positive effects on the development, growth, and yield of crops previously. By untargeted metabolomic analysis using UHPLC-MS/MS, plants and seeds were subjected to treatment with the biostimulant. Under ideal growth conditions, the plants treated exhibited higher concentration levels of glutamic acid, nicotiflorin and glycosylated lipids derived from linolenic acid. The foliar application of the biostimulant under water stress conditions increased the chlorophyll content by 17% and induced the accumulation of flavonols, mainly quercetin derivatives. Also, germination seed assays exhibited longer radicle lengths for seeds treated compared to the untreated control even in the absence of light (13-18% increase, p-value <0.05). Metabolomic analysis of the seeds indicated changes in concentration levels of amino acids (tryptophan, phenylalanine, tyrosine, glutamine, and arginine) and their derivatives. The results point out the enhancement of abiotic stress tolerance and the metabolic processes triggered in this crop associated with the treatment with the biostimulant, giving the first insights into stress tolerance mechanisms in P. vulgaris.

Molecular networking as a tool to annotate the metabolites of Bacillus sp. and Serratia marcescens isolates and evaluate their fungicidal effects against Magnapothe oryzae and Bipolaris oryzae.

Rhizobacteria are valuable sources of compounds that can be used for the integrated management of diseases in rice. Here, we aimed to explore the metabolism and organize and annotate the metabolites of Bacillus sp. and Serratia marcescens isolates using molecular networking and evaluate their fungicidal effects against Magnaporthe oryzae and Bipolaris oryzae. We obtained bacterial extracts after 6 and 16-h incubation via liquid-liquid extraction using ethyl acetate as solvent. We performed UHPLC-MS analysis and data processing using molecular networking and conducted biological assays in rice plants. Using the Global Natural Product Social spectral libraries, we annotated the following compounds: austinoneol, Phe-Pro, N-acetyl-l-leucine, Leu-Gly, Ile-Leu, Phe-Pro, 2,5-piperazinedione, 3-(1H-indol-3-methyl)-6-methyl-cyclo(d-Trp-l-Pro), and cholic acid. Results of the biological assays showed that the bacterial extracts reduced the mycelial growth of both pathogens in all treatments compared to the control. In the greenhouse setup, 8 days after the challenge for leaf gray spot and leaf blast, all treatments affected up to 4.4% of the leaf area, with an area under disease progress curve of 13.24, showing significant difference compared to the control, which affected 23% of the leaf area, with an AUDPC of 44.65. Our study provides potential new sources of natural products to be applied in the integrated management of rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03547-6.

Biotransformation of white and black mustard grains through germination and enzymatic hydrolysis revealed important metabolites for antioxidant properties and cytotoxic activity against Caco-2 cells.

Germination and enzymatic hydrolysis are biological processes with well-recognized positive effects on phenolic composition and antioxidant potential. This study aimed to apply those processes to white (Sinapsis alba) and black (Brassica nigra) mustard grains and to analyze the influences on the total phenolic content (TPC); phenolic and peptide profile determined by ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS); antioxidant potential (DPPH, ABTS, and FRAP assays); and cytotoxicity against Caco-2, a human colorectal adenocarcinoma cell line. Enzyme combinations for hydrolysis were different for each mustard grain, but for both species, enzymatic hydrolysis and germination showed a positive effect on antioxidant properties. From UPLC-HRMS analysis and molecular network studies, 14 peptides and 17 phenolic compounds were identified as metabolites released from mustard after processes application, which were strongly correlated with increased antioxidant activity. In addition, enzymatic hydrolysis applied in germinated mustard grains for both mustards increased the cytotoxic activity against Caco-2 human colorectal adenocarcinoma cell line.

Musa spp. cultivars as a neutralising source against some toxic activities of Bothrops and Crotalus genus snake venoms.

Accidents involving snakes from Bothrops spp. and Crotalus spp. constitute the most important cause of envenomation in Brazil and Argentina. Musa spp. (banana) have been reported to be used in popular medicine against snakebite by the members of the Canudos Settlement, located in Goiás. In this way, the aim of this work was to evaluate the antivenom effect of the Ouro (AA), Prata (AAB), Prata-anã (AAB) and Figo (ABB) cultivars against in vitro (phospholipase, coagulation and proteolytic) and in vivo (lethality and toxicity) activities caused by the venoms and toxicity (Artemia salina nauplii and Danio rerio embryos) of Musa spp. as well as the annotation of chemical compounds possibly related to these activities. From the in vitro antiophidic tests with the sap, we observed 100% inhibition of the phospholipase and coagulant activities with the cultivars Prata-anã and Figo against the venoms of B. alternatus and C. d. collineatus, B. diporus and B. pauloensis, respectively, and neutralisation of the lethality against the B. diporus venom. It was observed that the cultivars of Musa spp. did not show toxicity against Artemia salina nauplii and Danio rerio embryos. The sap analysis via HPLC-MS/MS allowed the annotation of the 13 compounds: abscisic acid, shikimic acid, citric acid, quinic acid, afzelechin, Glp-hexose, glucose, sucrose, isorhamnetin-3-O-galactoside-6-raminoside, kaempferol-3-glucoside-3-raminoside, myricetin-3-O-rutinoside, procyanidin B1 and rutin. Therefore, it can be seen that Musa spp. is a potential therapeutic agent that can act to neutralise the effects caused by snakebites.

Dereplication of polar extracts from Croton antisyphiliticus Mart. roots and its anti-inflammatory and antinociceptive potential.

Croton antisyphiliticus Mart. is a plant popularly used in folk medicine by traditional communities from Brazilian savannah to treat general inflammation. According to ethnopharmacological data, this specie can be considered a source of biologically active molecules for the development of new drugs. Thereby, this study reports the results of the dereplication approach of C. antisyphiliticus roots extracts and the in vivo evaluation of its potential antinociceptive and anti-inflammatory in albino Swiss mice. Based on HPLC coupled to Q-Exactive Orbitrap Mass Spectrometer and using GNPS, a total of thirteen polyphenolic compounds were noticed, including four compounds that have been reported for the first time in the genus Croton. Ethanolic and aqueous roots extracts demonstrated a dose-dependent inhibition for the number of writes, reduced pain induced by formalin and hyperalgesia induced by carrageenan. These extracts also reduced paw edema, cell migration, and myeloperoxidase activity, with effects similar to indomethacin and dexamethasone drugs.

Traversing the Cell Wall: The Chitinolytic Activity of Histoplasma capsulatum Extracellular Vesicles Facilitates Their Release.

Histoplasma capsulatum is the causative agent of histoplasmosis. Treating this fungal infection conventionally has significant limitations, prompting the search for alternative therapies. In this context, fungal extracellular vesicles (EVs) hold relevant potential as both therapeutic agents and targets for the treatment of fungal infections. To explore this further, we conducted a study using pharmacological inhibitors of chitinase (methylxanthines) to investigate their potential to reduce EV release and its subsequent impact on fungal virulence in an in vivo invertebrate model. Our findings revealed that a subinhibitory concentration of the methylxanthine, caffeine, effectively reduces EV release, leading to a modulation of H. capsulatum virulence. To the best of our knowledge, this is the first reported instance of a pharmacological inhibitor that reduces fungal EV release without any observed fungicidal effects.

The Symbiotic Fungus Leucoagaricus gongylophorus (Möller) Singer (Agaricales, Agaricaceae) as a Target Organism to Control Leaf-Cutting Ants.

Atta and Acromyrmex are the main genera of leaf-cutting ants present in North and South America, causing extensive damage to agroforestry. Control of the ants requires high handling costs with few effective methods available to decrease the losses. The symbiosis between the leaf-cutting ants and the fungus Leucoagaricus gongylophorus is essential for ant nest survival. Therefore, L. gongylophorus may be a key target in controlling leaf-cutting ants, since its reduction may cause an imbalance in the symbiosis necessary to maintain the nest. Among the options for natural fungal control, plant species are considered important sources of compounds belonging to several classes of natural products that show potential as antifungal agents. This review also presents studies that establish that the antagonist fungi from the Escovopsis and Trichoderma genera effectively reduce the development of L. gongylophorus. The development of nanostructured delivery systems, which have shown advantages over conventional formulations, is suggested for ant control; no commercial nanotechnology-based product has yet been developed, and this appears to be a new approach for future studies.

Recovery and purification of bikaverin produced by Fusarium oxysporum CCT7620.

Microbial pigments have a distinguished potential for applications in food and pharmaceutical industries, stimulating the research in this field. The present study evaluated the ideal conditions for extracting bikaverin (red pigment) from the biomass of Fusarium oxysporum CCT7620. Among the solvents tested, ethyl acetate extraction resulted in the highest bikaverin concentration and the kinetic study revealed a saturation in bikaverin concentration from 256 min on. Based on a preliminary economic study, three sequential extractions with ethyl acetate was considered the ideal protocol to recover bikaverin. After extraction, chromatographic methods were tested to purify bikaverin. The use of silica gel or Sephadex (open column) could not successfully purify bikaverin, but the semi-preparative HPLC resulted in a bikaverin-enriched fraction with a purity degree equivalent to the commercial analytical standard. This work provides relevant information regarding the extraction and purification of bikaverin, which may be useful for other downstraming processes.

O copinho oferecido pelos cuidadores aos recém-nascidos prematuros hospitalizados / The cup offered by caregivers to premature newborns in hospital

Resumo:OBJETIVO:verificar se o conhecimento da técnica de oferta da dieta pelo copo, o recebimento de treinamento e o tempo de trabalho influenciam a postura do técnico de enfermagem, a postura do recém-nascido e o posicionamento do copo.MÉTODOS:trata-se de um estudo observacional, descritivo, transversal, do qual participaram 15 profissionais, técnicos de enfermagem responsáveis pela administração da dieta às crianças, no momento do oferecimento do leite pelo copo, na ausência da genitora. Foi definido nível de significância estatística de 0,05.RESULTADOS:apenas nove indivíduos foram treinados (p=0,273) e 11 referiram não ter conhecimento da técnica (p=0,011). O oferecimento da dieta ocorreu em pé (p=0,001), com o derramamento do leite na boca da criança (p=0,010), sendo posicionada com as regiões occipital e cervical apoiadas (p-valor = 0,001). Não houve associação entre o conhecimento da técnica, recebimento de treinamento, tempo de trabalho às variáveis postura do técnico de enfermagem, postura do recém-nascido e posicionamento do copo.CONCLUSÃO:a postura de oferta da alimentação é realizada em pé, há derramamento da dieta na cavidade oral do bebê e não há influência das posturas do neonato, do técnico ou do copo por conta do conhecimento da técnica, realização de treinamento e tempo de trabalho.

Abstract:PURPOSE: to verify if knowing the technique of offering the diet in a cup, the training and the working time have influence on the nursing technician posture, the newborn posture and the cup position.METHODS: it was an observational, descriptive, transversal study, in which participated 15 professionals - nursing technicians responsible for diet administration in children, offering milk in a cup, during mother's absence. The statistical significance level was 0.05.RESULTS: only nine individuals were trained (p = 0.273) and 11 technicians declared had no knowledge about the technique (p = 0.011). The diet was offered with technicians standing (p = 0.001), pouring milk into child's mouth (p = 0.010) positioned with the occipital and cervical regions supported (p = 0.001). There was no association between technique knowledge, receiving training and working time to the different nursing technician's posture, newborn's posture and cup position.CONCLUSION: the posture of offering the diet is standing, milk is poured into babies' oral cavity and there is no influence in newborn, technician or cup position, caused by technical knowledge, training and working time.

Abordagem psicológica na atenção básica em saúde: da fragmentação à integralidade / The psychological approach in primary healthcare: from fragmentation to comprehensiveness

A Estratégia Saúde da Família tem sido a principal resposta de organização do cuidado perante os atendimentos médicos de visão biologicista, hospitalocêntricos e voltados para ações curativas. Por essa razão, este estudo teve por objetivo analisar como os profissionais médicos da Atenção Básica em Saúde, na Estratégia Saúde da Família, realizam o cuidado integral individual, tendo como foco a dimensão psicológica no processo saúde-doença das pessoas. A abordagem qualitativa foi o método desta investigação. Os dados foram coletados por meio de entrevistas semiestruturadas e interpretados pela análise de conteúdo, modalidade temática, constituindo-se três temas. Identificou-se que a abordagem da dimensão psicológica pelos médicos se desenvolve na perspectiva da integralidade em um esforço de reconstrução das práticas fragmentadas em saúde. No entanto, percebe-se que há dificuldades e desafios a serem superados pelos profissionais médicos junto aos usuários, à equipe e à gestão do sistema. Dessa forma, a integralidade surge como a capacidade de os profissionais interagirem com os usuários, produzindo, assim, um território comum que possibilite o diálogo entre os sujeitos, gerando uma reorganização do cuidado.

The Family Health Strategy has been the principal response in the organization of healthcare to narrowly biological, hospital-centered, and mainly curative medical care. This study analyzed how physicians working in primary care under the Family Health Strategy in Brazil conduct comprehensive individual care, with a focus on the psychological dimension in the health-disease process. The study used a qualitative approach. The data were collected with semi-structured interviews and interpreted by means of content analysis (thematic modality), comprising three themes. We found that the approach to the psychological dimension by physicians is developed from the perspective of comprehensiveness in an effort to reconstruct fragmented health practices. However, we observed difficulties and challenges to be overcome by physicians with the clientele, the health team, and management. Comprehensiveness thus emerges as the capacity of professionals to interact with users, thus producing a common ground that allows dialogue between the stakeholders, thereby fostering reorganization of care.