Biodiversity and characterization of Staphylococcus species isolated from a small manufacturing dairy plant in Portugal

The level and the diversity of the staphylococcal community occurring in the environment and dairy products of a small manufacturing dairy plant were investigated. Species identification was performed using different molecular methods, viz. Multiplex-PCR, amplified ribosomal DNA restriction analysis (ARDRA), and sodA gene sequencing. The main species encountered corresponded to Staphylococcus equorum (41 isolates, 39.0%), S. saprophyticus (28 isolates, 26.7%) and S. epidermidis (15 isolates, 14.3%). Additionally, low incidence of enterotoxin genes was obtained, with only 9 strains (8.6%) being positive for one or more toxin genes. With regard to antimicrobial resistance, 57.1% of the isolates showed at least resistance against one antibiotic, and 28.6% were multi-resistant, which might accomplish resistance for up to 6 antibiotics simultaneously. These results provided evidence that the presence of Staphylococcus species in dairy environment are mostly represented by S. equorum and S. saprophyticus, and illustrate that carrying antimicrobial resistance genes has become reasonably widespread in cheese and dairy environment.info:eu-repo/semantics/acceptedVersio

An integrated in vitro approach unveils the biocompetence and glutathiolomic profile of a human hepatocyte-like cell 3d model

Funding: This work was supported by FCT (Portugal) through the research grant PTDC/MED-TOX/29183/2017. Acknowledgments: The authors thank ECBio S.A. for providing the hnMSCs and F.A. Beland (NCTR, Jefferson, AR, USA) for the kind donation of nevirapine. FCT (UID/DTP/04138/2019, UID/QUI/00100/2019, RECI/QEQ-MED/0330/2012, SFRH/BD/144130/2019 to J.S.R., SFRH/BD/110945/2015 to P.F.P. and CEECIND/02001/2017 to A.M.M.A) are also acknowledged.The need for competent in vitro liver models for toxicological assessment persists. The differentiation of stem cells into hepatocyte-like cells (HLC) has been adopted due to its human origin and availability. Our aim was to study the usefulness of an in vitro 3D model of mesenchymal stem cell-derived HLCs. 3D spheroids (3D-HLC) or monolayer (2D-HLC) cultures of HLCs were treated with the hepatotoxic drug nevirapine (NVP) for 3 and 10 days followed by analyses of Phase I and II metabolites, biotransformation enzymes and drug transporters involved in NVP disposition. To ascertain the toxic effects of NVP and its major metabolites, the changes in the glutathione net flux were also investigated. Phase I enzymes were induced in both systems yielding all known correspondent NVP metabolites. However, 3D-HLCs showed higher biocompetence in producing Phase II NVP metabolites and upregulating Phase II enzymes and MRP7. Accordingly, NVP-exposure led to decreased glutathione availability and alterations in the intracellular dynamics disfavoring free reduced glutathione and glutathionylated protein pools. Overall, these results demonstrate the adequacy of the 3D-HLC model for studying the bioactivation/metabolism of NVP representing a further step to unveil toxicity mechanisms associated with glutathione net flux changes.publishersversionpublishe

Identification of lignocellulose-degrading enzymes using metagenomic approaches

Composting units which handle lignocellulosic residues are suitable sources of novel and promising lignocellulose-degrading enzymes such as cellulases, xylanases and amylases. These enzymes have practical application in many industries where lignocellulose is converted into several added-value bioproducts. However, the effective conversion of lignocellulose by a sustainable process is currently incomplete. Therefore, there is a need to find novel and robust catalysts to overcome this fact. Function- and sequence-based metagenomic approaches were used to identify novel lignocellulose-degrading enzymes with interesting industrial applications.info:eu-repo/semantics/publishedVersio

Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes/lipidic 3 bilayer nanocomposite

An enzymatic biosensor based on nitric oxide reductase (NOR; purified from Marinobacter hydrocarbonoclasticus) was developed for nitric oxide (NO) detection. The biosensor was prepared by deposition onto a pyrolytic graphite electrode (PGE) of a nanocomposite constituted by carboxylated single-walled carbon nanotubes (SWCNTs), a lipidic bilayer [1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG)] and NOR. NOR direct electron transfer and NO bioelectrocatalysis were characterized by several electrochemical techniques. The biosensor development was also followed by scanning electron microscopy and Fourier transform infrared spectroscopy. Improved enzyme stability and electron transfer (1.96 × 10-4 cm.s-1 apparent rate constant) was obtained with the optimum SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR) ratio of 4/2.5/4 (v/v/v), which biomimicked the NOR environment. The PGE/[SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR] biosensor exhibited a low Michaelis-Menten constant (4.3 μM), wide linear range (0.44-9.09 μM), low detection limit (0.13 μM), high repeatability (4.1% RSD), reproducibility (7.0% RSD), and stability (ca. 5 weeks). Selectivity tests towards L-arginine, ascorbic acid, sodium nitrate, sodium nitrite and glucose showed that these compounds did not significantly interfere in NO biosensing (91.0 ± 9.3%-98.4 ± 5.3% recoveries). The proposed biosensor, by incorporating the benefits of biomimetic features of the phospholipid bilayer with SWCNT's inherent properties and NOR bioelectrocatalytic activity and selectivity, is a promising tool for NO.FG thanks to Fundacão para a Ciência e a Tecnologia, MCTES (FCT/MCTES) for the fellowship grant SFRH/BD/52502/2014, which is financed by national funds and co-financed by FSE. LBM thanks to FCT/MCTES for the CEEC-Individual 2017 Program Contract. This work was supported by the PTDC/BB-BQB/0129/2014 project (FCT/MCTES), by FCT/MEC with national funds and co-funded by FEDER, and also by the Associate Laboratory Research Unit for Green Chemistry - Technologies and Processes Clean – LAQV, financed by national funds from FCT/MCTES (UID/QUI/50006/2019). The financial support from the European Union (FEDER funds through COMPETE) and National Funds (Fundação para a Ciência e Tecnologia-FCT) through project PTDC/ASP-PES/29547/2017, by FCT/MEC with national funds and co-funded by FEDER, is also acknowledged. J.A. Loureiro post-doc grant was supported by NORTE-01-0145-FEDER-000005 – LEPABE-2 ECO-INNOVATION, from North Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Optical Studies in Red/NIR Persistent Luminescent Cr‐Doped Zinc Gallogermanate (ZGGO:Cr)

Zn1+xGa2‐2xGexO4 (ZGGO:Cr)‐persistent phosphor, with a molar fraction, x, of x = 0.1, doped with a 0.5% molar of chromium, was synthesised via solid‐state reaction at 1350 °C for 36 h. X‐ray diffraction measurements and Raman spectroscopy evidence a single crystalline phase corresponding to the cubic spinel structure. Room temperature (RT) photoluminescence (PL) and afterglow decay profiles were investigated using above and below bandgap excitation. In both cases, persistent PL was observed for almost 8 h, mainly originating from a Cr3+ defect, the so‐called N2 optical centre. RT PL excitation and diffuse reflectance allow identification of the best pathways of Cr3+ red/NIR emission, as well as estimation of the ZGGO bandgap energy at 4.82 eV. An in‐depth investigation of the observed luminescence at 15 K and temperature‐dependent PL under site‐selective excitation reveals the spectral complexity of the presence of several optically active Cr3+ centres in the ZGGO host that emit in almost the same spectral region. Furthermore, the temperature dependence of the R‐lines’ intensity indicates the existence of thermal populating processes between the different optical centres. Such observations well account for a wide distribution of defect trap levels available for carrier capture/release, as measured by the persistent luminescence decay, from which the carriers are released preferentially to the N2 Cr3+‐related optical centre.publishersversionpublishe

Spent yeast waste streams as a sustainable source of bioactive peptides for skin applications

Spent yeast waste streams are a byproduct obtained from fermentation process and have been shown to be a rich secondary source of bioactive compounds such as phenolic compounds and peptides. The latter are of particular interest for skin care and cosmetics as they have been shown to be safe and hypoallergenic while simultaneously being able to exert various effects upon the epidermis modulating immune response and targeting skin metabolites, such as collagen production. As the potential of spent yeast’s peptides has been mainly explored for food-related applications, this work sought to understand if peptide fractions previously extracted from fermentation engineered spent yeast (Saccharomyces cerevisiae) waste streams possess biological potential for skin-related applications. To that end, cytotoxic effects on HaCat and HDFa cells and whether they were capable of exerting a positive effect upon the production of skin metabolites relevant for skin health, such as collagen, hyaluronic acid, fibronectin and elastin, were evaluated. The results showed that the peptide fractions assayed were not cytotoxic up to the highest concentration tested (500 µg/mL) for both cell lines tested. Furthermore, all peptide fractions showed a capacity to modulate the various target metabolites production with an overall positive effect being observed for the four fractions over the six selected targets (pro-collagen IαI, hyaluronic acid, fibronectin, cytokeratin-14, elastin, and aquaporin-9). Concerning the evaluated fractions, the overall best performance (Gpep > 1 kDa) was of an average promotion of 41.25% over the six metabolites and two cell lines assessed at a concentration of 100 µg/mL. These results showed that the peptide fractions assayed in this work have potential for future applications in skin-related products at relatively low concentrations, thus providing an alternative solution for one of the fermentation industry’s waste streams and creating a novel and highly valuable bioactive ingredient with encompassing activity to be applied in future skin care formulations.info:eu-repo/semantics/publishedVersio

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples

The online version contains supplementary material available at: https://doi.org/10.1007/s00253-023-12627-9.The renewable, abundant, and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples' microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited $\beta$-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples.Open access funding provided by FCT|FCCN (b-on). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, the projects LIGNOZYMES—Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01–0145-FEDER-029773), and B3iS—Biodiversity and Bioprospecting of Biosurfactants in Saline Environments (PTDC/BII-BIO/5554/2020); and by RNCA Advanced Computing Project MetaLignoZymes, metagenomic analysis of lignocellulosic residues towards the discovery of novel enzymes (CPCA/A0/408464/2021).info:eu-repo/semantics/publishedVersio

Clinical application of next-generation sequencing of plasma cell-free DNA for genotyping untreated advanced non-small cell lung cancer

Simple Summary Plasma ctDNA is a material source for molecular analysis particularly useful when tissue is not available or sufficient. NGS-based plasma genotyping should be integrated into the clinical workup of newly diagnosed advanced NSCLC. Background: Analysis of circulating tumor DNA (ctDNA) has remarkable potential as a non-invasive lung cancer molecular diagnostic method. This prospective study addressed the clinical value of a targeted-gene amplicon-based plasma next-generation sequencing (NGS) assay to detect actionable mutations in ctDNA in patients with newly diagnosed advanced lung adenocarcinoma. Methods: ctDNA test performance and concordance with tissue NGS were determined, and the correlation between ctDNA findings, clinical features, and clinical outcomes was evaluated in 115 patients with paired plasma and tissue samples. Results: Targeted-gene NGS-based ctDNA and NGS-based tissue analysis detected 54 and 63 genomic alterations, respectively; 11 patients presented co-mutations, totalizing 66 hotspot mutations detected, 51 on both tissue and plasma, 12 exclusively on tissue, and 3 exclusively on plasma. NGS-based ctDNA revealed a diagnostic performance with 81.0% sensitivity, 95.3% specificity, 94.4% PPV, 83.6% NPV, test accuracy of 88.2%, and Cohen's Kappa 0.764. PFS and OS assessed by both assays did not significantly differ. Detection of ctDNA alterations was statistically associated with metastatic disease (p = 0.013), extra-thoracic metastasis (p = 0.004) and the number of organs involved (p = 0.010). Conclusions: This study highlights the potential use of ctDNA for mutation detection in newly diagnosed NSCLC patients due to its high accuracy and correlation with clinical outcomes

Covalent organic frameworks as catalyst support: A case study of thermal, hydrothermal, and mechanical pressure stability of β-ketoenamine-linked TpBD-Me2

Covalent organic frameworks (COFs) are crystalline, ordered networks, that, due to their high surface areas and the opportunity for periodic placement of catalytically active sites, are interesting materials for catalysis. Despite the great interest in the use of COFs for this application, there is currently a lack of fundamental understanding on how catalytically relevant conditions affect the integrity of the materials. To gain insight into the stability of COFs as catalyst supports, we herein subjected a β-ketoenamine-linked COF to thermal treatment at high temperatures, to autogenous pressure in water at different temperatures, and to mechanical pressure during pelletizing, after which the materials were thoroughly characterized to gain insight into the structural changes occurring during these catalytically relevant treatments. The COF was largely stable under all hydrothermal conditions studied, highlighting the applicability of β-ketoenamine-linked COFs under aqueous and vapor conditions. On the other hand, thermal and pressure treatments led to a rapid decline in the surface area already at the lowest temperatures and pressures studied. Theoretical calculations indicated this loss to stem from interlayer rearrangement or buckling of the COF layers induced by the applied conditions. This study demonstrates the suitability of β-ketoenamine-linked COFs for use under hydrothermal conditions, and sheds light on the degradation pathways under thermal and pressure treatments, opening the path to the design of COFs with increased stability under such conditions.Fundação para a Ciência e a Tecnologia | Ref. UTA-EXPL/NPN/0055/2019Fundação para a Ciência e a Tecnologia | Ref. PTDC/QUI-OUT/2095/2021Fundação para a Ciência e a Tecnologia | Ref. PTDC/EQU-EQU/1707/2020Agencia Estatal de Investigación | Ref. RYC2020-030414-IUniversidade de Vigo/CISU