Isolation and Identification of Pigment-Producing Endophytic Fungi from the Amazonian Species Fridericia chica.

Pigments of fungal origin have aroused increasing interest in the food dye and cosmetic industries since the global demand for natural dyes has grown. Endophytic microorganisms are a source of bioactive compounds, and Amazonian plant species can harbor fungi with a wide range of biotechnological applications. Popularly known in Brazil as crajiru, Fridericia chica is a medicinal plant that produces a red pigment. In this study, a total of 121 fungi were isolated in potato dextrose agar from three plants. We identified nine pigment-producing endophytic fungi isolated from branches and leaves of F. chica. The isolates that showed pigment production in solid media were molecularly identified via multilocus analysis as Aspergillus welwitschiae, A. sydowii, Curvularia sp., Diaporthe cerradensis (two strains), Hypoxylon investiens, Neoscytalidium sp. (two strains) and Penicillium rubens. These isolates were subjected to submerged fermentation in two culture media to obtain metabolic extracts. The extracts obtained were analyzed in terms of their absorbance between 400 and 700 nm. The pigmented extract produced by H. investiens in medium containing yeast extract showed maximum absorbance in the red absorption range (UA700 = 0.550) and significant antioxidant and antimicrobial activity. This isolate can thus be considered a new source of extracellular pigment.

Development of cassava starch-based films incorporated with phenolic compounds produced by an Amazonian fungus.

Drug-release systems have attracted attention over the last few years since they can be used as a substitute for traditional methods of drug delivery. These have the advantage of being directly administered at the treatment site and can maintain the drug at adequate levels for a longer period, thus increasing their efficacy. Starch-based films are interesting candidates for use as matrices for drug release, especially due to starch's non-toxic properties and its biocompatibility. Endophytic fungi are an important source of bioactive molecules, including secondary metabolites such as phenolic compounds with antioxidant activity. In the present study, cassava starch-based films were developed to act as release systems of phenolic compounds with antioxidant activity. The Amazonian endophytic fungus Aspergillus niger MgF2 was cultivated in liquid media, and the fungal extract was obtained by liquid-liquid partition with ethyl acetate. The starch-based films incorporated with the fungal extract were characterized in regards to their physicochemical properties. The release kinetics of the extract from the film and its antioxidant and cytotoxic properties were also evaluated. The films incorporated with the extract presented maximum release after 25 min at 37 °C and pH 6.8. In addition, it was observed that the antioxidant compounds of the fungal extract maintain their activity after being released from the film, and were non-toxic. Therefore, considering the promising physicochemical properties of the extract-incorporated films, and their considerable antioxidant capacity, the films demonstrate great biotechnological potential with diverse applications in the pharmacological and cosmetic industries.

Dectin-2 is critical for phagocyte function and resistance to Paracoccidioides brasiliensis in mice.

Germline-encoded pattern recognition receptors, particularly C-type lectin receptors (CLRs), are essential for phagocytes to sense invading fungal cells. Among CLRs, Dectin-2 (encoded by Clec4n) plays a critical role in the antifungal immune response as it recognizes high-mannose polysaccharides on the fungal cell wall, triggering phagocyte functional activities and ultimately determining adaptive responses. Here, we assessed the role of Dectin-2 on the course of primary Paracoccidioides brasiliensis systemic infection in mice with Dectin-2-targeted deletion. Paracoccidioides brasiliensis constitutes the principal etiologic agent of paracoccidioidomycosis, the most prominent invasive mycosis in Latin American countries. The deficiency of Dectin-2 resulted in shortened survival rates, high lung fungal burden, and increased lung pathology in mice infected with P. brasiliensis. Consistently, dendritic cells (DCs) from mice lacking Dectin-2 infected ex vivo with P. brasiliensis showed impaired secretion of several proinflammatory and regulatory cytokines, including TNF-α, IL-1ß, IL-6, and IL-10. Additionally, when cocultured with splenic lymphocytes, DCs were less efficient in promoting a type 1 cytokine pattern secretion (i.e., IFN-γ). In macrophages, Dectin-2-mediated signaling was required to ensure phagocytosis and fungicidal activity associated with nitric oxide production. Overall, Dectin-2-mediated signaling is critical to promote host protection against P. brasiliensis infection, and its exploitation might lead to the development of new vaccines and immunotherapeutic approaches.

We report a critical role of the innate immune receptor Dectin-2 during Paracoccidioides brasiliensis infection. Fungal sensing by Dectin-2 improved the survival of mice and lowered fungal burden. Further, Dectin-2 was required for cytokine production, phagocytosis, and fungal killing by phagocytes.

Genetic diversity and population structure of two Euglossini bee species in a host-parasite relationship.

In the current study, two euglossine species, Exaerete smaragdina and Eulaema nigrita, a cleptoparasite bee and its host, respectively, were used as models to: (i) access the genetic diversity and population structure of both species, sampled along a wide latitudinal range of Atlantic Forest, where the distribution of El. nigrita and Ex. smaragdina co-occurs; (ii) investigate the evolutionary history of these species through the Atlantic Forest, and in a wider scenario, to examine the evolutionary history of these species across others forest domains. Analyses involved males of El. nigrita and Ex. smaragdina sampled through Brazilian territory, including 19 sites in the Atlantic Forest. Bayesian Skyline Plot (BSP) was used to infer possible climate oscillations on population of both species over time. The BSP revealed stability in effective population size for both species in most of the Plio-Pleistocene period. However, BSP results aligned to the starlike configuration in the haplotype network, neutrality test, and population diversity patterns indicated population expansion of the two species during the late Pleistocene. Our findings suggest areas of potential refugia to the climatic oscillations of the Pleistocene in the Atlantic Forest in the Brazilian states of Espírito Santo for El. nigrita and Pernambuco for Ex. smaragdina.

Antimicrobial and Antioxidant Activities of Endophytic Fungi Associated with Arrabidaea chica (Bignoniaceae).

The endophytic fungal community of the Amazonian medicinal plant Arrabidaea chica (Bignoniaceae) was evaluated based on the hypothesis that microbial communities associated with plant species in the Amazon region may produce metabolites with interesting bioactive properties. Therefore, the antimicrobial and antioxidant activities of the fungal extracts were investigated. A total of 107 endophytic fungi were grown in liquid medium and the metabolites were extracted with ethyl acetate. In the screening of fungal extracts for antimicrobial activity, the fungus identified as Botryosphaeria mamane CF2-13 was the most promising, with activity against E. coli, S. epidermidis, P. mirabilis, B. subtilis, S. marcescens, K. pneumoniae, S. enterica, A. brasiliensis, C. albicans, C. tropicalis and, especially, against S. aureus and C. parapsilosis (MIC = 0.312 mg/mL). Screening for antioxidant potential using the DPPH elimination assay showed that the Colletotrichum sp. CG1-7 endophyte extract exhibited potential activity with an EC50 of 11 µg/mL, which is equivalent to quercetin (8 µg/mL). The FRAP method confirmed the antioxidant potential of the fungal extracts. The presence of phenolic compounds and flavonoids in the active extracts was confirmed using TLC. These results indicate that two of the fungi isolated from A. chica exhibit significant antimicrobial and antioxidant potential.

Antifungal activity of sustainable histone deacetylase inhibitors against planktonic cells and biofilms of Candida spp. and Cryptococcusneoformans.

The limited therapeutic options for fungal infections and the increased incidence of fungal strains resistant to antifungal drugs, especially Candida spp., require the development of new antifungal drugs and strategies. Histone deacetylase inhibitors (HDACi), like vorinostat, have been studied in cancer treatment and have antifungal effects, acting alone or synergistically with classical antifungals. Here we investigated the antifungal activity of two novel sustainable HDACi (LDT compounds) based on vorinostat structure. Molecular docking simulation studies reveal that LDT compounds can bind to Class-I HDACs of Candida albicans, C. tropicalis, and Cryptococcus neoformans, which showed similar binding mode to vorinostat. LDT compounds showed moderate activity when tested alone against fungi but act synergistically with antifungal azoles against Candida spp. They reduced biofilm formation by more than 50% in C. albicans (4 µg/mL), with the main action in fungal filamentation. Cytotoxicity of the LDT compounds against RAW264.7 cells was evaluated and LDT536 demonstrated cytotoxicity only at the concentration of 200 µmol/L, while LDT537 showed IC50 values of 29.12 µmol/L. Our data indicated that these sustainable and inexpensive HDACi have potential antifungal and antibiofilm activities, with better results than vorinostat, although further studies are necessary to better understand the mechanism against fungal cells.

Fungal infections are neglected diseases that affect more than a billion people worldwide. Some histone deacetylase inhibitors can act against fungal cells. Our data reveal that HDACi LDT536 and LDT537 have potential antibiofilm and antifungal activities.

Extracellular vesicle-based delivery of silencing sequences for the treatment of Machado-Joseph disease/spinocerebellar ataxia type 3.

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominantly inherited ataxia worldwide. It is caused by an over-repetition of the trinucleotide CAG within the ATXN3 gene, which confers toxic properties to ataxin-3 (ATXN3) species. RNA interference technology has shown promising therapeutic outcomes but still lacks a non-invasive delivery method to the brain. Extracellular vesicles (EVs) emerged as promising delivery vehicles due to their capacity to deliver small nucleic acids, such as microRNAs (miRNAs). miRNAs were found to be enriched into EVs due to specific signal motifs designated as ExoMotifs. In this study, we aimed at investigating whether ExoMotifs would promote the packaging of artificial miRNAs into EVs to be used as non-invasive therapeutic delivery vehicles to treat MJD/SCA3. We found that miRNA-based silencing sequences, associated with ExoMotif GGAG and ribonucleoprotein A2B1 (hnRNPA2B1), retained the capacity to silence mutant ATXN3 (mutATXN3) and were 3-fold enriched into EVs. Bioengineered EVs containing the neuronal targeting peptide RVG on the surface significantly decreased mutATXN3 mRNA in primary cerebellar neurons from MJD YAC 84.2 and in a novel dual-luciferase MJD mouse model upon daily intranasal administration. Altogether, these findings indicate that bioengineered EVs carrying miRNA-based silencing sequences are a promising delivery vehicle for brain therapy.

Lemon gum: Non-toxic arabinogalactan isolated from Citrus × latifolia with antiproliferative property against human prostate adenocarcinoma cells.

Lemon gum (LG) obtained from Citrus × latifolia in Brazil was isolated and characterized. In addition, gum biocompatibility was evaluated in vitro and in vivo by Galleria mellonella and mice model. The cytotoxicity against tumor cells was also evaluated. The ratio of arabinose:galactose: rhamnose:4-OMe-glucuronic acid was 1:0.65:0.06:0.15. Small traces of protein were detected, emphasizing the isolate purity. Molar mass was 8.08 × 105 g/mol, with three different degradation events. LG showed antiproliferative activity against human prostate adenocarcinoma cancer cells, with percentage superior to 50 %. In vivo toxicity models demonstrated that LG is biocompatible polymer, with little difference in the parameters compared to control group. These results demonstrate advance in the study of LG composition and toxicity, indicating a potential for several biomedical and biotechnological future applications.

Biotechnological Applications of Nanoencapsulated Essential Oils: A Review.

Essential oils (EOs) are complex mixtures of volatile and semi-volatile organic compounds that originate from different plant tissues, including flowers, buds, leaves and bark. According to their chemical composition, EOs have a characteristic aroma and present a wide spectrum of applications, namely in the food, agricultural, environmental, cosmetic and pharmaceutical sectors. These applications are mainly due to their biological properties. However, EOs are unstable and easily degradable if not protected from external factors such as oxidation, heat and light. Therefore, there is growing interest in the encapsulation of EOs, since polymeric nanocarriers serve as a barrier between the oil and the environment. In this context, nanoencapsulation seems to be an interesting approach as it not only prevents the exposure and degradation of EOs and their bioactive constituents by creating a physical barrier, but it also facilitates their controlled release, thus resulting in greater bioavailability and efficiency. In this review, we focused on selecting recent articles whose objective concerned the nanoencapsulation of essential oils from different plant species and highlighted their chemical constituents and their potential biotechnological applications. We also present the fundamentals of the most commonly used encapsulation methods, and the biopolymer carriers that are suitable for encapsulating EOs.

Unbalanced networks and disturbed kinetics of serum soluble mediators associated with distinct disease outcomes in severe COVID-19 patients.

The present study applied distinct models of descriptive analysis to explore the integrative networks and the kinetic timeline of serum soluble mediators to select a set of systemic biomarkers applicable for the clinical management of COVID-19 patients. For this purpose, a total of 246 participants (82 COVID-19 and 164 healthy controls - HC) were enrolled in a prospective observational study. Serum soluble mediators were quantified by high-throughput microbeads array on hospital admission (D0) and at consecutive timepoints (D1-6 and D7-20). The results reinforce that the COVID-19 group exhibited a massive storm of serum soluble mediators. While increased levels of CCL3 and G-CSF were associated with the favorable prognosis of non-mechanical ventilation (nMV) or discharge, high levels of CXCL10 and IL-6 were observed in patients progressing to mechanical ventilation (MV) or death. At the time of admission, COVID-19 patients presented a complex and robust serum soluble mediator network, with a higher number of strong correlations involving IFN-γ, IL-1Ra and IL-9 observed in patients progressing to MV or death. Multivariate regression analysis demonstrates the ability of serum soluble mediators to cluster COVID-19 from HC. Ascendant fold change signatures and the kinetic timeline analysis further confirmed that the pairs "CCL3 and G-CSF" and "CXCL10 and IL-6" were associated with favorable or poor prognosis, respectively. A selected set of systemic mediators (IL-6, IFN-γ, IL-1Ra, IL-13, PDGF and IL-7) were identified as putative laboratory markers, applicable as complementary records for the clinical management of patients with severe COVID-19.

Amazonian Melastomataceae blueberries: Determination of phenolic content, nutritional composition, and antioxidant and anti-glycation activities.

Berries come from hundreds of different species of plants spread around the world. Blackberries, blueberries and raspberries, for instance, are popular berries that have attracted attention for providing several benefits to human health. Wild berries from the Melastomataceae family are commonly encountered in the Amazon, although these small blue fruits are poorly consumed. Although domesticated fruits give better monetary profits, the consumption of wild fruits is a desirable option to afford income and/or food to communities at the same time as keep the Amazon region preserved. Aiming the divulgation of the nutritional potential of these plants, this paper describes the study of six species of Amazonian blueberries, five of them from the Clidemia genus and one from the Tococa genus, in regard to their nutritional and chemical composition and antioxidant activity (AA). The levels of moisture, ash, protein, lipids, carbohydrates, and the total caloric values obtained for the Amazonian blueberries were comparable to other common edible berries. Although the six species are similar in terms of nutritional composition, their anthocyanin profiles and contents are quite peculiar. Two non-methylated anthocyanins, cyanidin and delphinidin, which bound to a variable number of sugars, characterized the berries of the genera Clidemia and Tococa. Clidemia japurensis, Clidemia hirta and Tococa bullifera were rich in tri-glycosylated anthocyanins, although differences are notable between them. Clidemia pustulata and Clidemia capitellata were characterized by the prevalence of mono-glycosylated anthocyanins, and Clidemia rubra showed a unique profile with mono- and di-glycosylated homologous as the main anthocyanins. In addition to their different chemical profiles, the concentrations of anthocyanins and other phenolic compounds varied a lot among the six species studied. The species C. rubra had the highest total concentration of phenolic acids and flavonoids. Therefore, this study showed that the blueberries analyzed have potential to be better explored, which we suggest doing in a sustainable way, aiming at the preservation of the Amazon's biodiversity.

Starch as a Matrix for Incorporation and Release of Bioactive Compounds: Fundamentals and Applications.

Due to its abundance in nature and low cost, starch is one of the most relevant raw materials for replacing synthetic polymers in a number of applications. It is generally regarded as non-toxic, biocompatible, and biodegradable and, therefore, a safe option for biomedical, food, and packaging applications. In this review, we focused on studies that report the use of starch as a matrix for stabilization, incorporation, or release of bioactive compounds, and explore a wide range of applications of starch-based materials. One of the key application areas for bioactive compounds incorporated in starch matrices is the pharmaceutical industry, especially in orally disintegrating films. The packaging industry has also shown great interest in using starch films, especially those with antioxidant activity. Regarding food technology, starch can be used as a stabilizer in nanoemulsions, thus allowing the incorporation of bioactive compounds in a variety of food types. Starch also presents potential in the cosmetic industry as a delivery system. However, there are still several types of industry that could benefit from the incorporation of starch matrices with bioactive compounds, which are described in this review. In addition, the use of microbial bioactive compounds in starch matrices represents an almost unexplored field still to be investigated.

Data standardization of plant-pollinator interactions.

BACKGROUND: Animal pollination is an important ecosystem function and service, ensuring both the integrity of natural systems and human well-being. Although many knowledge shortfalls remain, some high-quality data sets on biological interactions are now available. The development and adoption of standards for biodiversity data and metadata has promoted great advances in biological data sharing and aggregation, supporting large-scale studies and science-based public policies. However, these standards are currently not suitable to fully support interaction data sharing. RESULTS: Here we present a vocabulary of terms and a data model for sharing plant-pollinator interactions data based on the Darwin Core standard. The vocabulary introduces 48 new terms targeting several aspects of plant-pollinator interactions and can be used to capture information from different approaches and scales. Additionally, we provide solutions for data serialization using RDF, XML, and DwC-Archives and recommendations of existing controlled vocabularies for some of the terms. Our contribution supports open access to standardized data on plant-pollinator interactions. CONCLUSIONS: The adoption of the vocabulary would facilitate data sharing to support studies ranging from the spatial and temporal distribution of interactions to the taxonomic, phenological, functional, and phylogenetic aspects of plant-pollinator interactions. We expect to fill data and knowledge gaps, thus further enabling scientific research on the ecology and evolution of plant-pollinator communities, biodiversity conservation, ecosystem services, and the development of public policies. The proposed data model is flexible and can be adapted for sharing other types of interactions data by developing discipline-specific vocabularies of terms.

Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis.

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host-pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.

Neuroprotective effects on microglia and insights into the structure-activity relationship of an antioxidant peptide isolated from Pelophylax perezi.

Tryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian's skin secretions. Here, we report the structural characterization and antioxidant properties of a novel tryptophyllin-like peptide, named PpT-2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP-HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT-2 (FPWLLS-NH2 ) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT-2 shared physicochemical properties with other well-known antioxidants. To test PpT-2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical-based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT-2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT-2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA-stimulated BV-2 microglia cells. We further explored possible bioactivities of PpT-2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT-2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT-2. This novel peptide, PpT-2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.

Thromboelastometry demonstrates endogenous coagulation activation in nonsevere and severe COVID-19 patients and has applicability as a decision algorithm for intervention.

In patients with severe forms of COVID-19, thromboelastometry has been reported to display a hypercoagulant pattern. However, an algorithm to differentiate severe COVID-19 patients from nonsevere patients and healthy controls based on thromboelastometry parameters has not been developed. Forty-one patients over 18 years of age with positive qRT-PCR for SARS-CoV-2 were classified according to the severity of the disease: nonsevere (NS, n = 20) or severe (S, n = 21). A healthy control (HC, n = 9) group was also examined. Blood samples from all participants were tested by extrinsic (EXTEM), intrinsic (INTEM), non-activated (NATEM) and functional assessment of fibrinogen (FIBTEM) assays of thromboelastometry. The thrombodynamic potential index (TPI) was also calculated. Severe COVID-19 patients exhibited a thromboelastometry profile with clear hypercoagulability, which was significantly different from the NS and HC groups. Nonsevere COVID-19 cases showed a trend to thrombotic pole. The NATEM test suggested that nonsevere and severe COVID-19 patients presented endogenous coagulation activation (reduced clotting time and clot formation time). TPI data were significantly different between the NS and S groups. The maximum clot firmness profile obtained by FIBTEM showed moderate/elevated accuracy to differentiate severe patients from NS and HC. A decision tree algorithm based on the FIBTEM-MCF profile was proposed to differentiate S from HC and NS. Thromboelastometric parameters are a useful tool to differentiate the coagulation profile of nonsevere and severe COVID-19 patients for therapeutic intervention purposes.

Hydrolytic enzyme production from açai palm (Euterpe precatoria) endophytic fungi and characterization of the amylolytic and cellulolytic extracts.

Enzymes are biocatalysts that are widely used in different industries and generate billions of dollars annually. With the advancement of biotechnology, new enzymatic sources are being evaluated, especially microbial ones, in order to find efficient producers. Endophytic fungi are promising sources of biomolecules; however, Amazonian species are still poorly studied as to their enzymatic production potential. In this sense, the production of hydrolases (amylases, lipases, cellulases and pectinases) was evaluated in endophytic fungi isolated from the leaves, roots and stems of açai palms (Euterpe precatoria). A qualitative test was carried out to detect the enzymatic synthesis in each isolate, and the most promising ones were cultivated using submerged fermentation. The enzyme extracts were quantified to determine those with the greatest activity. Cellulolytic and amylolytic extracts showed the highest enzymatic activities and were partially characterized. Among 50 isolates, 82.9% produced pectinase, 58.5% produced cellulase, 31.7% produced amylase, and 12.2% produced lipase. Penicillium sp. L3 was the best producer of amylase and Colletotrichum sp. S1 was the best producer of cellulase in liquid medium cultivation. The amylolytic extract showed the highest enzymatic activity at pH 8.0 and 45 °C, and the cellulolytic extract at pH 5.0 and 35 °C. The cellulase and amylase produced by the endophytes had their molecular masses estimated between 38 and 76 kDa. These results indicate that endophytic fungi from the açai palm can be used as a new source of hydrolytic enzymes, which can be applied in numerous biotechnological processes.

Hinge influences in murine IgG binding to Cryptococcus neoformans capsule.

Decades of studies on antibody structure led to the tenet that the V region binds antigens while the C region interacts with immune effectors. In some antibodies, however, the C region affects affinity and/or specificity for the antigen. One example is the 3E5 monoclonal murine IgG family, in which the mIgG3 isotype has different fine specificity to the Cryptococcus neoformans capsule polysaccharide than the other mIgG isotypes despite their identical variable sequences. Our group serendipitously found another pair of mIgG1/mIgG3 antibodies based on the 2H1 hybridoma to the C. neoformans capsule that recapitulated the differences observed with 3E5. In this work, we report the molecular basis of the constant domain effects on antigen binding using recombinant antibodies. As with 3E5, immunofluorescence experiments show a punctate pattern for 2H1-mIgG3 and an annular pattern for 2H1-mIgG1; these binding patterns have been associated with protective efficacy in murine cryptococcosis. Also as observed with 3E5, 2H1-mIgG3 bound on ELISA to both acetylated and non-acetylated capsular polysaccharide, whereas 2H1-mIgG1 only bound well to the acetylated form, consistent with differences in fine specificity. In engineering hybrid mIgG1/mIgG3 antibodies, we found that switching the 2H1-mIgG3 hinge for its mIgG1 counterpart changed the immunofluorescence pattern to annular, but a 2H1-mIgG1 antibody with an mIgG3 hinge still had an annular pattern. The hinge is thus necessary but not sufficient for these changes in binding to the antigen. This important role for the constant region in antigen binding could affect antibody biology and engineering.

Improvement of endoglucanase production by Aspergillus brasiliensis in solid-state fermentation using cupuaçu (Theobroma grandiflorum) residue as substrate.

AIMS: Optimize the production of Aspergillus brasiliensis endoglucanase in a solid-phase bioprocess using cupuaçu shell as substrate. METHODS AND RESULTS: The shells were supplemented with nitrogen and phosphorous and used as a substrate. The centesimal and inorganic composition of the residue was determined, and found to be rich in fibres, and possessed essential elements for fungal growth. In the initial cultivation of A. brasiliensis, endoglucanase activity of 7.35 U g-1 was obtained. A factorial experimental design was used to determine the most significant variables for the bioprocess. The interactions between moisture, temperature and nitrogen source were noteworthy (p < 0.05). From the rotational central composite design, the optimization of temperature and nitrogen supplementation was obtained, and this reached 40.50 U g-1 , which is an increase of more than five times the value obtained initially. The enzymatic extract was applied as the biocatalyst in the hydrolysis of cupuaçu shells and, after 48 h, it was possible to observe the production of reducing sugars. CONCLUSIONS: Cupuaçu shell can be used as a substrate for endoglucanase production by A. brasiliensis. The process was optimized for the cultivation temperature and the nitrogen source. The enzymatic extract can be applied in the hydrolysis of lignocellulosic biomass. SIGNIFICANCE AND IMPACT OF THE STUDY: Cupuaçu shells can be used to produce cellulases, a product of high added value that can generate economic and environmental benefits for communities and companies producing derivatives of the cupuaçu fruit.

Acetylated cashew gum and fucan for incorporation of lycopene rich extract from red guava (Psidium guajava L.) in nanostructured systems: Antioxidant and antitumor capacity.

Industrial application of lycopene is limited due to its chemical instability and low bioavailability. This study proposes the development of fucan-coated acetylated cashew gum nanoparticles (NFGa) and acetylated cashew gum nanoparticles (NGa) for incorporation of the lycopene-rich extract from red guava (LEG). Size, polydispersity, zeta potential, nanoparticles concentration, encapsulation efficiency, transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to characterize nanoparticles. The antioxidant activity was determinated and cell viability was evaluated in the human breast cancer cells (MCF-7) and human keratinocytes (HaCaT) by MTT assay. The toxic effect was evaluated by hemolysis test and by Galleria mellonella model. NFGa showed higher stability than NGa, having a size of 162.10 ± 3.21 nm, polydispersity of 0.348 ± 0.019, zeta potential -30.70 ± 0.53 mV, concentration of 6.4 × 109 nanoparticles/mL and 60% LEG encapsulation. Microscopic analysis revealed a spherical and smooth shape of NFGa. NFGa showed antioxidant capacity by ABTS method and ORAC assay. The NFGa presented significant cytotoxicity against MCF-7 from the lowest concentration tested (6.25-200 µg/mL) and did not affect the cell viability of the HaCaT. NFGa showed non-toxic effect in the in vitro and in vivo models. Therefore, NFGa may have a promising application in LEG stabilization for antioxidant and antitumor purposes.