Comparative microbiome analysis in cystic fibrosis and non-cystic fibrosis bronchiectasis.

BACKGROUND: Bronchiectasis is a condition characterized by abnormal and irreversible bronchial dilation resulting from lung tissue damage and can be categorized into two main groups: cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). Both diseases are marked by recurrent infections, inflammatory exacerbations, and lung damage. Given that infections are the primary drivers of disease progression, characterization of the respiratory microbiome can shed light on compositional alterations and susceptibility to antimicrobial drugs in these cases compared to healthy individuals. METHODS: To assess the microbiota in the two studied diseases, 35 subjects were recruited, comprising 10 NCFB and 13 CF patients and 12 healthy individuals. Nasopharyngeal swabs and induced sputum were collected, and total DNA was extracted. The DNA was then sequenced by the shotgun method and evaluated using the SqueezeMeta pipeline and R. RESULTS: We observed reduced species diversity in both disease cohorts, along with distinct microbial compositions and profiles of antimicrobial resistance genes, compared to healthy individuals. The nasopharynx exhibited a consistent microbiota composition across all cohorts. Enrichment of members of the Burkholderiaceae family and an increased Firmicutes/Bacteroidetes ratio in the CF cohort emerged as key distinguishing factors compared to NCFB group. Staphylococcus aureus and Prevotella shahii also presented differential abundance in the CF and NCFB cohorts, respectively, in the lower respiratory tract. Considering antimicrobial resistance, a high number of genes related to antibiotic efflux were detected in both disease groups, which correlated with the patient's clinical data. CONCLUSIONS: Bronchiectasis is associated with reduced microbial diversity and a shift in microbial and resistome composition compared to healthy subjects. Despite some similarities, CF and NCFB present significant differences in microbiome composition and antimicrobial resistance profiles, suggesting the need for customized management strategies for each disease.

Role of partial dehydration in a naturally ventilated room on the mycobiota, ochratoxins, volatile profile and phenolic composition of Merlot grapes intended for wine production.

Dehydration of grapes has been used in various regions of the world to produce special wines, aiming to add value to oenological products. Post-harvest dehydration in rooms may be carried out regardless of weather conditions, without the additional cost of a specific infrastructure, in addition to the benefits of protecting the grapes from damages and environmental pollution. The objective of this study was to verify, for the first time, the impact of the dehydration in a naturally ventilated room on the quality of Merlot grapes. Physicochemical characteristics, mycobiota, occurrence of mycotoxins, volatile profile and phenolic composition of grapes were monitored on 7th, 14th and 21st days of dehydration (weight loss of 10, 20 and 27%, respectively). A decrease in aw (6%), pH (4%), and berry hardness (58%), along with an increase in total soluble solid content (15%) were observed during dehydration. The presence of Pestalotiopsis clavispora, Neopestalotiopsis clavispora, Colletotrichum siamense and Alternaria porri was favored during the dehydration process, while a decrease in the occurrence of Aspergillus niger and Phanerochaete sp. was verified. A. niger isolates showed no potential to produce forms of ochratoxins. These toxins were also not found in the grape samples. Regarding the volatile profile, 1-hexanal, 2-hexenal, and 1-octanal gave rise to the corresponding alcohols during dehydration, such as 1-hexanol, 2-hexen-1-ol, and 1-octanol. Acids (hexanoic, decanoic, and 3-hexenoic) resulted in the respective ethyl esters (hexanoate, decanoate, and ethyl 3-hexenoate) during dehydration. Terpenes as limonene, myrcene, and geraniol decreased throughout dehydration, while their biotransformation products (α-terpineol, 6-methyl-5-hepten-2-one, and linalool, respectively) had an increase in concentration. The phenolic content oscillated during dehydration, with an emphasis on increased levels of four hydroxybenzoic acids (ethyl gallate, p-hydroxybenzoic acid, gallic acid-hexose, and gallic acid), two hydroxycinnamic acids (caffeic acid and caftaric acid), two flavonols (kaempeferol galactoside and quercetin) and two anthocyanins (peonidin 3-O-hexoside and delphinidin 3-O-hexoside). Grapes of satisfactory quality were produced by dehydration in a naturally ventilated room. Even small wine producers can be encouraged to implement this procedure for the diversification of oenological products, as it has no costs related to the implementation of chambers/tunnels.

Structure-Function Insights of Jaburetox and Soyuretox: Novel Intrinsically Disordered Polypeptides Derived from Plant Ureases.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) do not have a stable 3D structure but still have important biological activities. Jaburetox is a recombinant peptide derived from the jack bean (Canavalia ensiformis) urease and presents entomotoxic and antimicrobial actions. The structure of Jaburetox was elucidated using nuclear magnetic resonance which reveals it is an IDP with small amounts of secondary structure. Different approaches have demonstrated that Jaburetox acquires certain folding upon interaction with lipid membranes, a characteristic commonly found in other IDPs and usually important for their biological functions. Soyuretox, a recombinant peptide derived from the soybean (Glycine max) ubiquitous urease and homologous to Jaburetox, was also characterized for its biological activities and structural properties. Soyuretox is also an IDP, presenting more secondary structure in comparison with Jaburetox and similar entomotoxic and fungitoxic effects. Moreover, Soyuretox was found to be nontoxic to zebra fish, while Jaburetox was innocuous to mice and rats. This profile of toxicity affecting detrimental species without damaging mammals or the environment qualified them to be used in biotechnological applications. Both peptides were employed to develop transgenic crops and these plants were active against insects and nematodes, unveiling their immense potentiality for field applications.

Risk assessment of the antifungal and insecticidal peptide Jaburetox and its parental protein the Jack bean (Canavalia ensiformis) urease.

Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.

Interaction of jack bean (Canavalia ensiformis) urease and a derived peptide with lipid vesicles.

Ureases are metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Jack bean (Canavalia ensiformis) produces three isoforms of urease (Canatoxin, JBU and JBURE-II). Canatoxin and JBU display several biological properties independent of their ureolytic activity, such as neurotoxicity, exocytosis-inducing and pro-inflammatory effects, blood platelets activation, insecticidal and antifungal activities. The Canatoxin entomotoxic activity is mostly due to an internal peptide, named pepcanatox, released upon the hydrolysis of the protein by insect cathepsin-like digestive enzymes. Based on pepcanatox sequence, Jaburetox-2Ec was produced in Escherichia coli. JBU and its peptides were shown to permeabilize membranes through an ion channel-based mechanism. Here we studied the JBU and Jaburetox-2Ec interaction with platelet-like multilamellar liposomes (PML) using Dynamic Light Scattering and Small Angle X-ray Scattering techniques. We also analyzed the interaction of JBU with giant unilamellar vesicles (GUVs) using Fluorescence Microscopy. The interaction of vesicles with JBU led to a slight reduction of hydrodynamic radius, and caused an increase in the lamellar repeat distance of PML, suggesting a membrane disordering effect. In contrast, Jaburetox-2Ec decreased the lamellar repeat distance of PML membranes, while also diminishing their hydrodynamic radius. Fluorescence microscopy showed that the interaction of GUVs with JBU caused membrane perturbation with formation of tethers. In conclusion, JBU can interact with PML, probably by inserting its Jaburetox "domain" into the PML external membrane. Additionally, the interaction of Jaburetox-2Ec affects the vesicle's internal bilayers and hence causes more drastic changes in the PML membrane organization in comparison with JBU.

Pigment production by filamentous fungi on agro-industrial byproducts: an eco-friendly alternative.

The search for new sources of natural pigments has increased, mainly because of the toxic effects caused by synthetic dyes used in food, pharmaceutical, textile, and cosmetic industries. Fungi provide a readily available alternative source of natural pigments. In this context, the fungi Penicillium chrysogenum IFL1 and IFL2, Fusarium graminearum IFL3, Monascus purpureus NRRL 1992, and Penicillium vasconiae IFL4 were selected as pigments producers. The fungal identification was performed using ITS and part of the ß-tubulin gene sequencing. Almost all fungi were able to grow and produce water-soluble pigments on agro-industrial residues, with the exception of P. vasconiae that produced pigments only on potato dextrose broth. The production of yellow pigments was predominant and the two strains of P. chrysogenum were the largest producers. In addition, the production of pigments and mycotoxins were evaluated in potato dextrose agar using TOF-MS and TOF-MS/MS. Metabolites as roquefortine C, chrysogine were found in both extracts of P. chrysogenum, as well fusarenone X, diacetoxyscirpenol, and neosolaniol in F. graminearum extract. In the M. purpureus extract, the pigments monascorubrin, rubropunctatin, and the mycotoxin citrinin were found. The crude filtrates have potential to be used in the textile industry; nevertheless, additional pigment purification is required for food and pharmaceutical applications.

Probiotic potential of Lactobacillus spp. isolated from Brazilian regional ovine cheese.

Twelve Lactobacillus isolates from Brazilian starter-free ovine cheeses were evaluated for their probiotic potential. The strains were identified by 16S rDNA sequencing as Lactobacillus plantarum (7), Lb. brevis (2), Lb. casei (2) and Lb. parabuchneri (1). All strains showed variable resistance to gastric juices and relative tolerance to pancreatin and bile salts. Only five strains of Lb. plantarum could not deconjugate the sodium salt of taurodeoxycholic acid. Autoaggregation ability after 24 h was above 50% and hydrophobicity was higher than 60% for most strains. All lactobacilli could inhibit linolenic acid oxidation, except Lb. parabuchneri strain, whereas none of them could scavenge DPPH radical. ß-Galactosidase activity ranged from 47·7 to 2503 Miller units. Inhibition of food pathogens Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Escherichia coli and Salmonella typhimurium was demonstrated and the production of organic acids could be associated with this effect. The Lactobacillus strains from Brazilian regional ovine cheese showed interesting functional characteristics, mainly the strains Lb. brevis SM-B and Lb. plantarum SM-I. Both presented high acid tolerance. In addition, Lb. brevis SM-B also displayed remarkable antioxidant activity and Lb. plantarum SM-I was the highest ß-galactosidase producer, exhibited high autoaggregation and hydrophobicity properties.

Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger.

A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.

Production of cellulolytic enzymes by Aspergillus phoenicis in grape waste using response surface methodology.

The production of cellulolytic enzymes by the fungus Aspergillus phoenicis was investigated. Grape waste from the winemaking industry was chosen as the growth substrate among several agro-industrial byproducts. A 2 x 2 central composite design was performed, utilizing the amount of grape waste and peptone as independent variables. The fungus was cultivated in submerged fermentation at 30 degrees C and 120 rpm for 120 h, and the activities of total cellulases, endoglucanases, and beta-glucosidases were measured. Total cellulases were positively influenced by the linear increase of peptone concentration and decrease at axial concentrations of grape waste and peptone. Maximum activity of endoglucanase was observed by a linear increase of both grape waste and peptone concentrations. Concentrations of grape waste between 5 and 15 g/L had a positive effect on the production of beta-glucosidase; peptone had no significant effects. The optimum production of the three cellulolytic activities was observed at values near the central point. A. phoenicis has the potential for the production of cellulases utilizing grape waste as the growth substrate.