Biodegradation of Plastics and Formation of PHA Bioplastics—A Circular Bioeconomy Approach

This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, IP, in the scope of the project UIDP/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB, and UID/CTM/50025/2020-2023 of the Research Unit on Materials Science—CENIMAT–I3N. FCT/MCTES through project grants PTDC/QUIQUI/119116/2010, PTDC/GEO-FIQ/5162/2014, , and PTDC/EEI-EEE/0415/2021.Plastics are present in the majority of daily-use products worldwide. Due to society’s production and consumption patterns, plastics are accumulating in the environment, causing global pollution issues and intergenerational impacts. Our work aims to contribute to the development of solutions and sustainable methods to mitigate this pressing problem, focusing on the ability of marine-derived actinomycetes to accelerate plastics biodegradation and produce polyhydroxyalkanoates (PHAs), which are biodegradable bioplastics. The thin plastic films’ biodegradation was monitored by weight loss, changes in the surface chemical structure (Infra-Red spectroscopy FTIR-ATR), and by mechanical properties (tensile strength tests). Thirty-six marine-derived actinomycete strains were screened for their plastic biodegradability potential. Among these, Streptomyces gougerotti, Micromonospora matsumotoense, and Nocardiopsis prasina revealed ability to degrade plastic films—low-density polyethylene (LDPE), polystyrene (PS) and polylactic acid (PLA) in varying conditions, namely upon the addition of yeast extract to the culture media and the use of UV pre-treated thin plastic films. Enhanced biodegradation by these bacteria was observed in both cases. S. gougerotti degraded 0.56% of LDPE films treated with UV radiation and 0.67% of PS films when inoculated with yeast extract. Additionally, N. prasina degraded 1.27% of PLA films when these were treated with UV radiation, and yeast extract was added to the culture medium. The main and most frequent differences observed in FTIR-ATR spectra during biodegradation occurred at 1740 cm−1, indicating the formation of carbonyl groups and an increase in the intensity of the bands, which indicates oxidation. Young Modulus decreased by 30% on average. In addition, S. gougerotti and M. matsumotoense, besides biodegrading conventional plastics (LDPE and PS), were also able to use these as a carbon source to produce degradable PHA bioplastics in a circular economy conceptpublishersversionpublishe

Hydrophobic DES Based on Menthol and Natural Organic Acids for Use in Antifouling Marine Coatings

Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.Marine biofouling negatively impacts industries with off-shore infrastructures, such as naval, oil, and aquaculture. To date, there are no ideal sustainable, economic, and environmentally benign solutions to deal with this phenomenon. The advances achieved in green solvents, as well as its application in different industries, such as pharmaceutical and biotechnology, have promoted the emergence of deep eutectic systems (DES). These eutectic systems have applications in various fields and can be revolutionary in the marine-based industrial sector. In this study, the main objective was to investigate the potential use of hydrophobic DES (HDES) based on menthol and natural organic acids for their use as marine antifouling coatings. Our strategy encompassed the physicochemical characterization of different formulations, which allowed us to identify the most appropriate molar ratio and intermolecular interactions for HDES formations. The miscibility of the resulting HDES with the marine coating has been evaluated and proven to be successful. The Men/OL (1:1) system proved to be the most promising in terms of cost-production and thus was the one used in subsequent antifouling tests. The cytotoxicity of this HDES was evaluated using an in vitro cell model (HaCat cells) showing no significant toxicity. Furthermore, the application of this system incorporated into coatings that are used in marine structures was also studied using marine species (Mytilus edulis mussels and Patella vulgata limpets) to evaluate both their antifouling and ecotoxicity effects. HDES Men/OL (1:1) incorporated in marine coatings was promising in reducing marine macrofouling and also proved to be effective at the level of microfouling without viability impairment of the tested marine species. It was revealed to be more efficient than using copper oxide, metallic copper, or ivermectin as antifouling agents. Biochemical assays performed on marine species showed that this HDES does not induce oxidative stress in the tested species. These results are a strong indication of the potential of this HDES to be sustainable and efficiently used in marine fouling control technologies.publishersversionpublishe

Analysis of a Cell Wall Mutant Highlights Rho-Dependent Genome Amplification Events in Staphylococcus aureus

This work was financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB. This work was also supported by FCT through grants PTDC/BIA-MIC/31645/2017 (awarded to R.G.S.) and PTDC/CVT-CVT/29510/2017 (awarded to M.M.); Projects LISBOA-01-0145-FEDER007660 (Microbiologia Molecular, Estrutural e Celular) and UID/Multi/04378/2019 funded by FEDER funds through COMPETE2020—Programa Operacional de Competitividade e Internacionalização (POCI); and by ONEIDA project (LISBOA-01-0145-FEDER-016417) co-funded by FEEI—“Fundos Europeus Estruturais e de Investimento” from “Programa Operacional Regional Lisboa2020” and by national funds through FCT.In a study of antibiotic resistance in Staphylococcus aureus, specific cell wall mutants were previously generated for the peptidoglycan biosynthesis gene murF, by the insertion of an integrative plasmid. A collection of 30 independent mutants was obtained, and all harbored a variable number of copies of the inserted plasmid, arranged in tandem in the chromosome. Of the 30 mutants, only 3, F9, F20 and F26, with a lower number of plasmid copies, showed an altered peptidoglycan structure, lower resistance to β-lactams and a different loss-of-function mutation in rho gene, that encodes a transcription termination factor. The rho mutations were found to correlate with the level of oxacillin resistance, since genetic complementation with rho gene reestablished the resistance and cell wall parental profile in F9, F20 and F26 strains. Furthermore, complementation with rho resulted in the amplification of the number of plasmid tandem repeats, suggesting that Rho enabled events of recombination that favored a rearrangement in the chromosome in the region of the impaired murF gene. Although the full mechanism of reversion of the cell wall damage was not fully elucidated, we showed that Rho is involved in the recombination process that mediates the tandem amplification of exogeneous DNA fragments inserted into the chromosomepublishersversionpublishe

Distinct phenotypic and genomic signatures underlie contrasting pathogenic potential of Staphylococcus epidermidis clonal lineages

Copyright © 2019 Espadinha, Sobral, Mendes, Méric, Sheppard, Carriço, de Lencastre and Miragaia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Background: Staphylococcus epidermidis is a common skin commensal that has emerged as a pathogen in hospitals, mainly related to medical devices-associated infections. Noteworthy, infection rates by S. epidermidis have the tendency to rise steeply in next decades together with medical devices use and immunocompromized population growth. Staphylococcus epidermidis population structure includes two major clonal lineages (A/C and B) that present contrasting pathogenic potentials. To address this distinction and explore the basis of increased pathogenicity of A/C lineage, we performed a detailed comparative analysis using phylogenetic and integrated pangenome-wide-association study (panGWAS) approaches and compared the lineages's phenotypes in in vitro conditions mimicking carriage and infection. Results: Each S. epidermidis lineage had distinct phenotypic signatures in skin and infection conditions and differed in genomic content. Combination of phenotypic and genotypic data revealed that both lineages were well adapted to skin environmental cues. However, they appear to occupy different skin niches, perform distinct biological functions in the skin and use different mechanisms to complete the same function: lineage B strains showed evidence of specialization to survival in microaerobic and lipid rich environment, characteristic of hair follicle and sebaceous glands; lineage A/C strains showed evidence for adaption to diverse osmotic and pH conditions, potentially allowing them to occupy a broader and more superficial skin niche. In infection conditions, A/C strains had an advantage, having the potential to bind blood-associated host matrix proteins, form biofilms at blood pH, resist antibiotics and macrophage acidity and to produce proteases. These features were observed to be rare in the lineage B strains. PanGWAS analysis produced a catalog of putative S. epidermidis virulence factors and identified an epidemiological molecular marker for the more pathogenic lineage. Conclusion: The prevalence of A/C lineage in infection is probably related to a higher metabolic and genomic versatility that allows rapid adaptation during transition from a commensal to a pathogenic lifestyle. The putative virulence and phenotypic factors associated to A/C lineage constitute a reliable framework for future studies on S. epidermidis pathogenesis and the finding of an epidemiological marker for the more pathogenic lineage is an asset for the management of S. epidermidis infections.DE and CM were supported by Ph.D. grants PD/BD/52206/2013 and SFRH/BD/129483/2017, respectively, from the Fundação para a Ciência e Tecnologia (FCT). This work was partially supported by project PTDC/FIS-NAN/0117/2014, project PTDC/CVT-CVT/29510/2017, project PTDC/BIA-MIC/31645/2017, and project EXPOSE - SAICT-POL/23222/2016 from FCT; Projects LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) and UID/Multi/04378/2019) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI); by ONEIDA project (LISBOA-01-0145-FEDER- 016417) co-funded by FEEI - “Fundos Europeus Estruturais e de Investimento” from “Programa Operacional Regional Lisboa2020” and by national funds through FCT; Operacional Competitividade e Internacionalização, Programa Operacional Regional de Lisboa (FEDER) and Fundação para a Ciência e a Tecnologia.info:eu-repo/semantics/publishedVersio

Rotational tumbling of escherichia coli aggregates under shear

Growing living cultures of Escherichia coli bacteria are investigated using real-time in situ rheology and rheoimaging measurements. In the early stages of growth (lag phase) and when subjected to a constant stationary shear, the viscosity slowly increases with the cell's population. As the bacteria reach the exponential phase of growth, the viscosity increases rapidly, with sudden and temporary abrupt decreases and recoveries. At a certain stage, corresponding grossly to the late phase of growth, when the population stabilizes, the viscosity also keeps its maximum constant value, with drops and recoveries, for a long period of time. This complex rheological behavior, which is observed to be shear strain dependent, is a consequence of two coupled effects: the cell density continuous increase and its changing interacting properties. Particular attention is given to the late phase of growth of E. coli populations under shear. Rheoimaging measurements reveal, near the static plate, a rotational motion of E. coli aggregates, collectively tumbling and flowing in the shear direction. This behavior is interpreted in the light of a simple theoretical approach based on simple rigid body mechanics.info:eu-repo/semantics/publishedVersio

Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan

Fundacao para a Ciencia e a Tecnologia (FCT) through grants PTDC/FIS-NAN/ 0117/2014 and PTDC/BIA-MIC/31645/2017. project UID/Multi/04378/2019 (Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO), funded by FCT/MCTES; project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular), funded by FEDER through COMPETE2020-Programa Operacional Competitividade e Internacionalizacao (POCI); national funds through FCT; by project ONEIDA (LISBOA-01-0145-FEDER-016417), cofunded by FEEI (Fundos Europeus Estruturais e de Investimento) from the Programa Operacional Regional Lisboa 2020; and by national funds from FCT. Funding was also provided by European Society of Clinical Microbiology and Infectious Diseases research grant 2015, awarded to R.G.S. B.V.G., T.A.F., and I.R.G. were supported by fellowships SFRH/BD/131623/2017 respectively. J.S.D. acknowledges the National NMR Network (PTNMR) and Infrastructure Project ROTEIRO/0031/2013-PINFRA/22161/2016 (cofinanced by FEDER through COMPETE 2020, POCI, PORL, and FCT through PIDDAC).Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus. It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to β-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth.publishersversionpublishe

Unveiling the mechanism of action of 7α-acetoxy-6β-hydroxyroyleanone on an mrsa/visa strain: Membrane and cell wall interactions

UIDB/00100/2020 PTDC/MED-QUI/29036/2017 CEECIND/03414/2018 UIDB/04378/2020 PTDC/BIA-MIC/31645/2017 UID/DTP/04138/2019 UID/DTP/04567/2019 CBIOS/PRUID/BI1/2017 UIDB/04567/2020 UID/AMB/50017 UIDP/50017/2020 UIDB/50017/2020The number of cases of failure in the treatment of infections associated with resistant bacteria is on the rise, due to the decreasing efficacy of current antibiotics. Notably, 7α-Acetoxy-6β-hydroxyroyleanone (AHR), a diterpene isolated from different Plectranthus species, showed antibacterial activity, namely against Methicillin-resistant Staphylococcus aureus (MRSA) strains. The high antibacterial activity and low cytotoxicity render this natural compound an interesting alternative against resistant bacteria. The aim of this study is to understand the mechanism of action of AHR on MRSA, using the MRSA/Vancomycin-intermediate S. aureus (VISA) strain CIP 106760, and to study the AHR effect on lipid bilayers and on the cell wall. Although AHR interacted with lipid bilayers, it did not have a significant effect on membrane passive permeability. Alternatively, bacteria treated with this royleanone displayed cell wall disruption, without revealing cell lysis. In conclusion, the results gathered so far point to a yet undescribed mode of action that needs further investigation.publishersversionpublishe

Comparative Chemical Profiling and Antimicrobial/Anticancer Evaluation of Extracts from Farmed versus Wild Agelas oroides and Sarcotragus foetidus Sponges

This study was implemented in the framework of the research project SPINAQUA (Grant No 239) funded by the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT) under the “1st call for H.F.R.I. Research Projects for the support of Post-doctoral Researchers”. This publication is based upon work from COST Action CA18238 (Ocean4Biotech), supported by COST (European Cooperation in Science and Technology) program, which provided Short Term Scientific Mission (STSM) grant support to D.V.-M. to perform the experimental work at NOVA-FCT. C.M.P.R. is financially supported by La Caixa Foundation (Grant No LCF/PR/HR21/52410028). Publisher Copyright: © 2023 by the authors.Marine sponges are highly efficient in removing organic pollutants and their cultivation, adjacent to fish farms, is increasingly considered as a strategy for improving seawater quality. Moreover, these invertebrates produce a plethora of bioactive metabolites, which could translate into an extra profit for the aquaculture sector. Here, we investigated the chemical profile and bioactivity of two Mediterranean species (i.e., Agelas oroides and Sarcotragus foetidus) and we assessed whether cultivated sponges differed substantially from their wild counterparts. Metabolomic analysis of crude sponge extracts revealed species-specific chemical patterns, with A. oroides and S. foetidus dominated by alkaloids and lipids, respectively. More importantly, farmed and wild explants of each species demonstrated similar chemical fingerprints, with the majority of the metabolites showing modest differences on a sponge mass-normalized basis. Furthermore, farmed sponge extracts presented similar or slightly lower antibacterial activity against methicillin-resistant Staphylococcus aureus, compared to the extracts resulting from wild sponges. Anticancer assays against human colorectal carcinoma cells (HCT-116) revealed marginally active extracts from both wild and farmed S. foetidus populations. Our study highlights that, besides mitigating organic pollution in fish aquaculture, sponge farming can serve as a valuable resource of biomolecules, with promising potential in pharmaceutical and biomedical applications.publishersversionpublishe

Proteomics to Identify New Blood Biomarkers for Diagnosing Patients With Acute Stroke

Funding Information: UCIBIO-UID/Multi/04378/2019 also supported by FCT (CEEC Funding Information: This work was financed by Fundação para a Ciência e a Tecnologia (FCT) with PTDC/MEC-NEU/28750/2017 grant, LA/P/0140/2020 funding,iNOVA4Health—UIDB/04462/2020, and UCIBIO-UID/Multi/04378/2019 units. A.S.C. is supported by FCT. R.M. is also supported by FCT (CEEC position, 2019–2025 investigator). Funding Information: This work was financed by Fundação para a Ciência e a Tecnologia (FCT) with PTDC/MEC-NEU/28750/2017 grant, LA/P/0140/2020 funding, Publisher Copyright: © 2023 The Authors.BACKGROUND: Blood biomarkers are a potential tool for early stroke diagnosis. We aimed to perform a pilot and exploratory study on untargeted blood biomarkers in patients with suspected stroke by using mass spectrometry analysis. METHODS AND RESULTS: This was a prospective observational study of consecutive patients with suspected stroke admitted within 6 hours of last being seen well. Blood samples were collected at admission. Patients were divided into 3 groups: ischemic stroke (IS), intracerebral hemorrhage (ICH), and stroke mimics. Quantitative analysis from mass spectrometry data was performed using a supervised approach. Biomarker-based prediction models were developed to differentiate IS from ICH and ICH+stroke mimics. Models were built aiming to minimize misidentification of patients with ICH as having IS. We included 90 patients, one-third within each subgroup. The median age was 71 years (interquartile range, 57–81 years), and 49 participants (54.4%) were women. In quantitative analysis, C3 (complement component 3), ICAM-2 (intercellular adhesion molecule 2), PLGLA (plasminogen like A), STXBP5 (syntaxin-binding protein 5), and IGHV3-64 (immunoglobulin heavy variable 3-64) were the 5 most significantly dysregulated proteins for both comparisons. Biomarker-based models showed 88% sensitivity and 89% negative predictive value for differentiating IS from ICH, and 75% sensitivity and 95% negative predictive value for differentiating IS from ICH+stroke mimics. ICAM-2, STXBP5, PLGLA, C3, and IGHV3-64 displayed the highest importance score in our models, being the most informative for identifying patients with stroke. CONCLUSIONS: In this proof-of-concept and exploratory study, our biomarker-based prediction models, including ICAM-2, STXBP5, PLGLA, C3, and IGHV3-64, showed 75% to 88% sensitivity for identifying patients with IS, while aiming to minimize misclassification of ICH. Although our methodology provided an internal validation, these results still need validation in other cohorts and with different measurement techniques.publishersversionpublishe