Predicting Antifouling Activity and Acetylcholinesterase Inhibition of Marine-Derived Compounds Using a Computer-Aided Drug Design Approach

UIDB/50006/2020 Norma transit?ria DL 57/2016 UIDP/04378/2020 LA/P/0140/2020Biofouling is the undesirable growth of micro-and macro-organisms on artificial waterimmersed surfaces, which results in high costs for the prevention and maintenance of this process (billion €/year) for aquaculture, shipping and other industries that rely on coastal and off-shore infrastructure. To date, there are still no sustainable, economical and environmentally safe solutions to overcome this challenging phenomenon. A computer-aided drug design (CADD) approach comprising ligand-and structure-based methods was explored for predicting the antifouling activities of marine natural products (MNPs). In the CADD ligand-based method, 141 organic molecules extracted from the ChEMBL database and literature with antifouling screening data were used to build the quantitative structure–activity relationship (QSAR) classification model. An overall predictive accuracy score of up to 71% was achieved with the best QSAR model for external and internal validation using test and training sets. A virtual screening campaign of 14,492 MNPs from Encinar’s website and 14 MNPs that are currently in the clinical pipeline was also carried out using the best QSAR model developed. In the CADD structure-based approach, the 125 MNPs that were selected by the QSAR approach were used in molecular docking experiments against the acetylcholinesterase enzyme. Overall, 16 MNPs were proposed as the most promising marine drug-like leads as antifouling agents, e.g., macrocyclic lactam, macrocyclic alkaloids, indole and pyridine derivatives.publishersversionpublishe

Can drug safety be predicted and animal experiments reduced by using isolated mitochondrial fractions?

Mitochondrial toxicity has resulted in the withdrawal of several drugs from the market. One particular example is nefazodone, an anti-depressant withdrawn in the USA due to hepatoxicity caused by drug-induced mitochondrial dysfunction. Drug development and safety testing can involve the use of large numbers of laboratory animals, which, without a decisive pre-screening for mitochondrial toxicity, are often unable to pre-empt higher mortality rates in some patient groups. The use of isolated mitochondria as a screening tool for drug safety can decrease the number of laboratory animals used in pre-clinical studies, thus improving animal welfare and healthcare outcomes and costs. Novel techniques involving high-throughput methods can be used to investigate whether a molecule is a mitochondrial toxicant. Moreover, these screens are mechanistically-based, since the effects of the drug on oxidative phosphorylation, calcium homeostasis and mitochondrial genetics can be assessed. This review is intended to demonstrate that isolated mitochondrial fractions are suitable for predicting drug and general chemical safety in toxicological screenings, thus contributing to the refinement and reduction of animal use in laboratory research

Developing a model for cystic fibrosis sociomicrobiology based on antibiotic and environmental stress

Cystic fibrosis (CF) infections are invariably biofilm-mediated and polymicrobial, being safe to assume that a myriad of factors affects the sociomicrobiology within the CF infection site and modulate the CF community dynamics, by shaping their social activities, overall functions, virulence, ultimately affecting disease outcome. This work aimed to assess changes in the dynamics (particularly on the microbial composition) of dual-/three-species biofilms involving CF-classical (Pseudomonas aeruginosa) and unusual species (Inquilinus limosus and Dolosigranulum pigrum), according to variable oxygen conditions and antibiotic exposure. Low fluctuations in biofilm compositions were observed across distinct oxygen environments, with dual-species biofilms exhibiting similar relative proportions and P. aeruginosa and/or D. pigrum populations dominating three-species consortia. Once exposed to antibiotics, biofilms displayed high resistance profiles, and microbial compositions, distributions, and microbial interactions significantly challenged. The antibiotic/oxygen environment supported such fluctuations, which enhanced for three-species communities. In conclusion, antibiotic therapy hugely disturbed CF communities dynamics, inducing significant compositional changes on multispecies consortia. Clearly, multiple perturbations may disturb this dynamic, giving rise to various microbiological scenarios in vivo, and affecting disease phenotype. Therefore, an appreciation of the ecological/evolutionary nature within CF communities will be useful for the optimal use of current therapies and for newer breakthroughs on CF antibiotherapy.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). The authors also acknowledge the financial support provided by FCT through the projects: PTDC/SAU-ESA/646091/2006/ FCOMP01-0124-FEDER-007480FCT; strategic project PEst-OE/EQB/LA0023/2013; “BioHealth – Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2–O Novo Norte), QREN, FEDER; RECI/BBB-EBI/0179/2012 – Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB, FCOMP-01-0124-FEDER-027462, FEDER; and the DNA mimics project PIC/IC/82815/2007. The FCT BPD fellowship of Susana P. Lopes SFRH/BPD/95616/2013 and the support of the COST-Action TD1004:Theragnostics for imaging and therapy is also acknowledged.info:eu-repo/semantics/publishedVersio

Quantitative assessment of individual populations within polymicrobial biofilms

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Selecting appropriate tools providing reliable quantitative measures of individual populations in biofilms is critical as we now recognize their true polymicrobial and heterogeneous nature. Here, plate count, quantitative real-time polymerase chain reaction (q-PCR) and peptide nucleic acid probe-fluorescence in situ hybridization (PNA-FISH) were employed to quantitate cystic fibrosis multispecies biofilms. Growth of Pseudomonas aeruginosa, Inquilinus limosus and Dolosigranulum pigrum was assessed in dual- and triple-species consortia under oxygen and antibiotic stress. Quantification methods, that were previously optimized and validated in planktonic consortia, were not always in agreement when applied in multispecies biofilms. Discrepancies in culture and molecular outcomes were observed, particularly for triple-species consortia and antibiotic-stressed biofilms. Some differences were observed, such as the higher bacterial counts obtained by q-PCR and/or PNA-FISH (?4 log10 cells/cm2) compared to culture. But the discrepancies between PNA-FISH and q-PCR data (eg D. pigrum limited assessment by q-PCR) demonstrate the effect of biofilm heterogeneity in method's reliability. As the heterogeneity in biofilms is a reflection of a myriad of variables, tailoring an accurate picture of communities? changes is crucial. This work demonstrates that at least two, but preferentially three, quantification techniques are required to obtain reliable measures and take comprehensive analysis of polymicrobial biofilm-associated infections.The authors thank the financial support from the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI-01–0145-FEDER-006684) and BioTecNorte operation (NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 - Programa Operacional Regional do Norte. This work was also the result of the projects: (i) POCI01-0145-FEDER-006939 (Laboratory for Processing Engineering, Environment, Biotechnology and Energy – UID/EQU/00511/2013) funded by the ERDF, through COMPETE2020 and by national funds, through FCT; (ii) NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, supported by North Portugal Regional Operational Programme (NORTE2020), under the Portugal2020 Partnership Agreement, through the ERDF; (iii) Coded-FISH PTDC/DTP-PIC/4562/2014/16678; (iv) POCI-01-0145-FEDER-029841, through COMPETE2020 - Programa Operacional Competitividade e Internacionalização and by national funds, through FCT). Also, the fellowship of Susana P. Lopes SFRH/BPD/95616/2013 is acknowledged. The authors would also like to thanks to Dr Michael Surette (Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, ON, Canada) for kindly providing the I. limosus strain used in this study.info:eu-repo/semantics/publishedVersio

Unravelling Pseudomonas aeruginosa and Candida albicans communication in coinfections scenarios: insights through network analysis

The Supplementary Material for this article canbe found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2020.550505/ full#supplementary-materialModern medicine is currently facing huge setbacks concerning infection therapeutics as microorganisms are consistently knocking down every antimicrobial wall set before them. The situation becomes more worrying when taking into account that, in both environmental and disease scenarios, microorganisms present themselves as biofilm communities that are often polymicrobial. This comprises a competitive advantage, with interactions between different species altering host responses, antimicrobial effectiveness, microbial pathogenesis and virulence, usually augmenting the severity of the infection and contributing for the recalcitrance towards conventional therapy. Pseudomonas aeruginosa and Candida albicans are two opportunistic pathogens often co-isolated from infections, mainly from mucosal tissues like the lung. Despite the billions of years of co-existence, this pair of microorganisms is a great example on how little is known about cross-kingdom interactions, particularly within the context of coinfections. Given the described scenario, this study aimed to collect, curate, and analyze all published experimental information on the molecular basis of P. aeruginosa and C. albicans interactions in biofilms, in order to shed light into key mechanisms that may affect infection prognosis, increasing this area of knowledge. Publications were optimally retrieved from PubMed and Web of Science and classified as to their relevance. Data was then systematically and manually curated, analyzed, and further reconstructed as networks. A total of 641 interactions between the two pathogens were annotated, outputting knowledge on important molecular players affecting key virulence mechanisms, such as hyphal growth, and related genes and proteins, constituting potential therapeutic targets for infections related to these bacterial-fungal consortia. Contrasting interactions were also analyzed, and quorum-sensing inhibition approaches were highlighted. All annotated data was made publicly available at www.ceb.uminho.pt/ISCTD, a database already containing similar data for P. aeruginosa and Staphylococcus aureus communication. This will allow researchers to cut on time and effort when studying this particular subject, facilitating the understanding of the basis of the inter-species and inter-kingdom interactions and how it can be modulated to help design alternative and more effective tailored therapies. Finally, data deposition will serve as base for future dataset integration, whose analysis will hopefully give insights into communications in more complex and varied biofilm communities.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The authors also acknowledge COMPETE2020 and FCT for the project POCI-01-0145-FEDER-029841 and FCT for the PhD Grant of TG [grant number SFRH/BD/136544/2018].info:eu-repo/semantics/publishedVersio

An explanation for another familial case of Rett syndrome: maternal germline mosaicism

Rett syndrome (RTT; OMIM#312750) is a severe neurodevelopmental disorder that affects mainly girls. It has an estimated incidence of 1:10,000-15,000 females. Mutations in the X-linked gene methyl CpG-binding protein 2 (MECP2) have been found in most patients. The most accepted explanation for the sex bias is that the Rett mutation in sporadic cases has its origin in the paternal germline X chromosome and can thus only be transmitted to females. The majority of cases are sporadic (99.5%) but some familial cases have been described. These cases can either be explained by germline mosaicism or by asymptomatic carrier mothers with skewing of X-inactivation towards the wild-type MECP2 allele. We describe one of the few familial cases of RTT in which a maternal germline mosaicism is the most likely explanation. The mutation p.Arg270fs (c.808delC) was identified in both a girl with classical RTT and her brother who had the severe neurological phenotype usually described in males. The mutation was absent in DNA extracted from blood of both parents. These type of events must be taken into consideration in the genetic counselling of families after the diagnosis of a first case of RTT in a female or a MECP2 mutation in a male

Total body percentage and motor coordination among Portuguese schoolchildren

Children’s motor coordination may affect their activity pattern and thereby influence their bodyfatness. The aim of the present study was to analyse the relationship between motor coordination and total body fat percentage, in a Portuguese sample of schoolchildren aged 9–12 years. Method: The sample comprised 596 urban schoolchildren (girls 46?9%), aged 9–12 years (mean 9?61 ( SD 0?55) years) from North of Portugal. Total body fat percentage was cal- culated with Tetrapolar Bioelectrical Impedance Analysis, model Tanita TBF-300. Age- and sex-adjusted Z-scores computed total body fat percentage. Motor coordination levels were assessed with the Ko¨rperkoordination Test fu¨r Kinder (KTK) and children were classified according to age and sex KTK criteria (Schiling 1974). Date of birth, gender and school socio-economic status (eligible for benefit A, B or not eligible) were extracted from the school administrations records systems. School socio-economic status was used as a proxy measure of family socio-economic status. Results: In motor coordination, 22?4 % girls showed disturbance of coordination; 37?7 % insufficiencies of coordination; 39?5 % normal coordination and 0?4 % good coordination. Corresponding figures for boys were 7?3 %; 36?5 %; 54?3 % and 1?9 %, respectively. Linear regression analysis showed that Z-scores total body fat percentage (unstandardized B 5 20?258, SE 0?021, P , 0?001) were negatively associated with motor coordination, after adjustment for socio-economic status. Conclusions: Low motor coordination levels are nega- tively associated with total body fat percentage Z-scores. The early identification of children with poor motor coor- dination and/or high body fat percentage is crucial in order to implement and develop health-related behaviours. Funding: Research relating to this abstract was funded by FCT-MCTES Grant (BD/43808/2008). Reference: Schiling, F (1974) Korperkoordination Test fur Kinder, KTK. Beltz Test Gmbh, Weinheim

Chemical characterization of “Alcaparras” stoned table olives from Northeast Portugal

Commercial stoned table olives named "alcaparras" from Trás-os-Montes (Portugal) were chemically characterized. During three consecutive years (2004-2006) 30 samples (10 per year) were examined for their nutritional value (moisture, crude protein, total fat, ash, carbohydrates, and energy), with a detailed report of the fatty acids and tocopherols composition. Water was the major constituent (72.5 ± 5.5%), followed by fat (14.6 ± 5.1%). The average amount of protein and ash were 1.1% and 3.4%, respectively, reporting unusual ash values for table olives, related to the technological process. One hundred grams of fresh stoned table olives presented an average energetic value of 156 kcal, lower than most table olives. The lipids are rich in oleic acid (average of 77.7 ± 2.0%), followed by palmitic acid and linoleic acid. Samples showed an average of total tocopherols of 1.2 mg/100 g of fresh weight, being α-tocopherol the most abundant. Table olives are important sources of MUFA, as olive oil, recognized as a preventive factor in diseases in which free radicals are implicated, complemented by the amounts of vitamin E, with both antioxidant and vitamin action.The authors are grateful to INTERREG III A Program, Project PIREFI for financial support of this work.info:eu-repo/semantics/publishedVersio

Impact of microfiltration on the manufacture of goat’s milk cheese - microbiological studies

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Synthesis, characterization and preliminary antibacterial evaluation against Staphylococcus aureus of a new 2,4,5-tri(hetero)arylimidazole derivative based on azaindole heterocycle

Imidazole derivatives are known for their numerous biological applications, such as antibacterial, antifungal, antioxidant, antiviral, antiparasitic and anticancer, among others. Therefore, several imidazole derivatives have been synthesized and developed in recent years as potential drugs in the treatment of several diseases. In this communication, we report the synthesis of a new imidazole derivative, substituted at positions 2, 4 and 5 with heterocyclic groups, using a simple synthetic methodology and an easy purification procedure. The new compound was characterized by the usual spectroscopic techniques (NMR, UV-Vis absorption and emission). The evaluation of the novel imidazole derivative as a potential antibiotic drug was carried out against the Gram-positive bacterium Staphylococcus aureus, using disk test diffusion method. Results showed a dose-response effect against the bacteria under study, revealing that the rational design of this imidazole derivative is quite promising to improve antibacterial activity of imidazole derivatives.This research was funded by the Foundation for Science and Technology (FCT) through CQ/UM (UIDB/00686/2020) and “Contrato‐Programa” UIDB/04050/2020 funded by national funds. Thanks are also due to FCT for financial support to the Portuguese NMR Network (PTNMR, Bruker Avance III 400‐Univ. Minho)info:eu-repo/semantics/publishedVersio