Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%

Biodegradation of textile azo dyes by Phanerochaete chrysosporium

Instituto de Biotecnologia e Química Fina / Universidade do Minho (IBQF/UM) - Praxis XXI, Praxis XXI 2/2.1/QUI/44/94, Praxis XXI/BD/9120/96

Climate- and fire-smart landscape scenarios call for redesigning protection regimes to achieve multiple management goals

Integrated management of biodiversity and ecosystem services (ES) in heterogeneous landscapes requires considering the potential trade-offs between conflicting objectives. The UNESCO's Biosphere Reserve zoning scheme is a suitable context to address these trade-offs by considering multiple management zones that aim to minimise conflicts between management objectives. Moreover, in Mediterranean ecosystems, management and planning also needs to consider drivers of landscape dynamics such as wildfires and traditional farming and forestry practices that have historically shaped landscapes and the biodiversity they host. In this study, we applied a conservation planning approach to prioritise the allocation of management zones under future landscape and climate scenarios. We tested different landscape management scenarios reflecting the outcomes of climate-smart and fire-smart policies. We projected the expected landscape dynamics and associated changes on the distribution of 207 vertebrate species, 4 ES and fire hazard under each scenario. We used Marxan with Zones to allocate three management zones, replicating the Biosphere Reserves zoning scheme (“Core area”, “Buffer zone” and “Transition area”) to address the various management objectives within the Biosphere Reserve. Our results show that to promote ES supply and biodiversity conservation, while also minimising fire hazard, the reserve will need to: i) Redefine its zoning, especially regarding Core Areas, which need a considerable expansion to help mitigate changes in biodiversity and accommodate ES supply under expected changes in climate and species distribution. ii) Revisit current management policies that will result in encroached landscapes prone to high intensity, uncontrollable wildfires with the potential to heavily damage ecosystems and compromise the supply of ES. Our results support that both climate- and fire-smart policies in the Meseta Ibérica can help develop multifunctional landscapes that help mitigate and adapt to climate change and ensure the best possible maintenance of biodiversity and ES supply under uncertain future climate conditions.This study was supported by national funds - Portuguese Foundation for Science and Technology, under the “FirESmart” project (PCIF/MOG/0083/2017), and the project UIDB/04033/2020. CCS is supported by the “Financiamento Programático” UIDP/04050/2020 funded by national funds through the FCT I.P. VH was funded by the Junta de Andalucía through an Emergia contract (EMERGIA20_00135). AR is supported by ‘Juan de la Cierva’ fellowship program funded by the Spanish Ministry of Science and Innovation (IJC2019-041033-I). ÂS received support from the Portuguese Foundation for Science and Technology (FCT) through Ph.D. Grant SFRH/BD/132838/2017, funded by the Ministry of Science, Technology and Higher Education, and by the European Social Fund - Operational Program Human Capital within the 2014–2020 EU Strategic Framework.info:eu-repo/semantics/publishedVersio

Biodegradation of bioaccessible textile azo dyes by Phanerochaete chrysosporium

Azo dyes are important chemical pollutants of industrial origin. Textile azo dyes with bioaccessible groups for lignin degrading fungi, such as 2-methoxyphenol (guaiacol) and 2,6-dimethoxyphenol (syringol), were synthesised using different aminobenzoic and aminosulphonic acids as diazo components. The inocula of the best biodegradation assays were obtained from a pre-growth medium (PAM), containing one of the synthesised dyes. The results of the dye biodegradation assays were evaluated every 7 days, by the decrease of the absorbance at the maximum wavelength of the dye, by the decrease of the sucrose concentration in the culture medium and by the increase of the biomass during the 28 days of assay. It was observed that the extent of dye biodegradation depended on the sucrose concentration, on the degraded dye structure and, on the dye present in the PAM medium.Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XXI/BD/15878/98, PRAXIS XXI/BD/9120/96.Instituto de Biotecnologia e Química Fina (IBQF)

Estudos de biodegradabilidade de corantes azo de aplicação têxtil por Phanerochaete chrysosporium

Sintetizaram-se corantes azo de aplicação têxtil, usando como componentes diazo ácidos aminobenzóicos e aminossulfónicos e como componentes de acoplamento 2- metoxifenol e 2,6-dimetoxifenol. A utilização destas componentes de acoplamento teve como objectivo aumentar a bioacessibilidade dos corantes ao fungo lenhinolítico da podridão branca Phanerochaete chrysosporium, já que estes grupos estão presentes na estrutura da lenhina e têm sido referidos como pontos de acesso para o sistema enzimático lenhinolítico do fungo. As experiências de biodegradação realizaram-se em meio líquido, com sacarose e em condições limitantes de azoto, com agitação e temperatura controladas. A biodegradação dos corantes foi acompanhada por espectrofotometria UV-Visível, quantificando a diminuição da intensidade da banda de absorção máxima (λmax) do corante. Em estudos preliminares estabeleceram-se condições experimentais optimizadas relativamente ao meio de préadaptação e à concentração de sacarose utilizada. Estabeleceram-se algumas correlações entre a estrutura química dos corantes e a sua biodegradação.Praxis XX

Fatigue detection through physiological assessment during real-life occupational situations: Preliminary results

Background: Fatigue is a significant health and safety-related problem among workers. In general, it decreases performance and physical strength, causing incidents and accidents in operational situations. During military activities, soldiers often encounter severe conditions, which combined lead to fatigue manifestations affecting their health and performance. Continuous monitoring of their overall health status would prevent its adverse effects. Objective: This work aimed to present the preliminary results of a retrospective assessment of military training physiological recordings using an alert-based fatigue detection algorithm to validate its accurate functioning. Methods: Three case studies from soldiers participating in military training tests were recruited for evaluation. The referred algorithm was developed to manage fatigue through the combined assessment of physiological variables and determine different fatigue levels warnings to advisetimely interventions and prevent potential health impact. Each examined case included the continuous recording of heart rate, breathing rate and core temperature. The algorithm translated physiological sensory data into minute alarms according to fatigue levels determined through the conjunction of normative and related research criteria. Results and Discussion: Outcomes revealed that the algorithm could evidence the different stages of training and the resulting physical demands on soldiers using their physiological response throughout the exercises. Retrieved fatigue alarms showed the high physiological cost of military practices and helped to overview the impact of each training period. Finally, results also demonstrated the importance of individual and contextualised assessment for accurately characterise the subject's fatigue status. Conclusions: It is concluded that the developed decision model can improve the management of real-time fatigue, allowing early detection of potential indicators of further physical impairments. Furthermore, it can lead to the enhancement of work-rest cycles, not only for tactical personnel but also for any safety-sensitive occupation. For future work, its validity will be tested through more participants, and other variableswill be added to improve its accuracy.</jats:p

Endogenous cathelicidin production limits inflammation and protective immunity to Mycobacterium avium in mice

The production of antimicrobial peptides, such as the cathelicidins, plays a prominent role in the innate immune response against microbial pathogens. Cathelicidins are widely distributed amongst living organisms, and the antimicrobial peptides generated by proteolysis of the precursor forms are typically cationic and [alpha]-helical, a structure that facilitates their interaction and insertion into anionic bacterial cell walls and membranes, causing damage and promoting microbial death. Here, we found that mouse cathelicidin (Camp) expression was induced in bone marrow-derived macrophages by infection with Mycobacterium avium in a TLR2- and TNF-dependent manner. However, the endogenous production of the cathelin-related antimicrobial peptide (CRAMP) was not required for the bacteriostasis of M. avium either in primary cultures of macrophages or in vivo, as shown by the use of CRAMP-null mice. In contrast, the lack of Camp led to a transient improvement of M. avium growth control in the spleens of infected mice while at the same time causing an exacerbation of the inflammatory response to infection. Our data highlight the anti-inflammatory effects of CRAMP and suggests that virulent mycobacteria may possess strategies to escape its antimicrobial activity.Funded through project PTDC/BIA-BCM/112138/2009FCOMP-01-0124-FEDER014185

Chemical characterization and bioactive properties of Prunus avium L.: The widely studied fruits and the unexplored stems

The aim of this study was to characterize sweet cherry regarding nutritional composition of the fruits, and individual phytochemicals and bioactive properties of fruits and stems. The chromatographic profiles in sugars, organic acids, fatty acids, tocopherols and phenolic compounds were established. All the preparations (extracts, infusions and decoctions) obtained using stems revealed higher antioxidant potential than the fruits extract, which is certainly related with its higher phenolic compounds (phenolic acids and flavonoids) concentration. The fruits extract was the only one showing antitumor potential, revealing selectivity against HCT-15 (colon carcinoma) (GI50~74 μg/mL). This could be related with anthocyanins that were only found in fruits and not in stems. None of the preparations have shown hepatotoxicity against normal primary cells. Overall, this study reports innovative results regarding chemical and bioactive properties of sweet cherry stems, and confirmed the nutritional and antioxidant characteristics of their fruits.The authors are grateful to Foundation for Science and Technology (FCT, Portugal) for financial support to the research centre CIMO (strategic project PEst OE/AGR/UI0690/2011) and L. Barros researcher contract under “Programa Compromisso com Ciência – 2008”

Sustainable strategy based on induced precipitation for the purification of phycobiliproteins

Phycobiliproteins are fluorescent proteins mainly produced by red macroalgae and cyanobacteria. These proteins, essential to the survival of these organisms, find application in many fields of interest, from medical, pharmaceutical, and cosmetic to food and textile industries. The biggest obstacle to their use is the lack of simple environmental and economical sustainable methodologies to obtain these proteins with high purity. In this work, a new purification process is proposed based on the induced precipitation of the target proteins followed by ultrafiltration. Purities of 89.5% of both phycobiliproteins and 87.3% of R-phycoerythrin were achieved using ammonium sulfate and poly(acrylic acid) sodium salts as precipitation agents (followed by an ultrafiltration step), while maintaining high recovery yields and protein structure stability. Environmental analysis performed to evaluate the proposed process shows that the carbon footprint for the proposed process is much lower than that reported for alternative methodology, and the economic analysis reveals the cost-effective character associated to its high performance. This work is a step toward more sustainable and effective methodologies/processes with high industrial potential.publishe

Sustainable lysis of Bacillus subtilis biomass to recover the biopharmaceutical L-asparaginase

The first-line biopharmaceutical used to treat Acute lymphoblastic leukemia (ALL), Oncaspar, is based on the enzyme L-asparaginase (ASNase), and has annual sales of ca. USD $100 million. In addition to other sources, genetically modified Bacillus subtilis is regarded as one of the most promising hosts for the ASNase production. The Aliivibrio fischeri ASNase type II, which has anti-tumour activity due its higher specific affinity for L-asparagine, expressed in B. subtillis is located in the periplasm. Therefore, cell lysis is required for the ASNase recovery. Nevertheless, typical cell lysis approaches, e.g. chemical methods with surfactants lead to some biocompatibility concerns and the need of extra purification steps. To overcome this drawback, in this work, ultrasound sonication (USS) conditions were studied to develop a greener and more biocompatible method for ASNase recovery from B. subtilis cell lysis. The USS cell lysis was optimized regarding the amplitude of USS pulse, number of lysis cycles and mass of cells/volume of solvent ratio. The identification and quantification of ASNase and major impurities present in the cell extract after lysis were investigated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high-performance liquid chromatography (SE-HPLC). ASNase activity was determined by monitoring the hydrolysis of the substrate, L-asparagine. The results obtained show that the ideal conditions for B. subtilis cell lysis are an amplitude of USS pulse of 60%, 40 cycles of lysis and 10 mL of phosphatebuffered saline (PBS) per 1 g of cells. Overall, an optimized sustainable B. subtilis cell lysis method was developed, avoiding the use of surfactants and with low energy consumption.publishe