Preface - Creativity and HCI: From Experience to Design in Education

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Data quality in biofilm high-throughput routine analysis: intralaboratory protocol adaptation and experiment reproducibility

Biofilm research is growing more diverse and dependent on high-throughput technologies and the large-scale production of results aggravates data substantiation. In particular, it is often the case that experimental protocols are adapted to meet the needs of a particular laboratory and no statistical validation of the modified method is provided. This paper discusses the impact of intra-laboratory adaptation and non-rigorous documentation of experimental protocols on biofilm data interchange and validation. The case study is a non-standard, but widely used, workflow for Pseudomonas aeruginosa biofilm development, considering three analysis assays: the crystal violet (CV) assay for biomass quantification, the XTT assay for respiratory activity assessment, and the colony forming units (CFU) assay for determination of cell viability. The ruggedness of the protocol was assessed by introducing small changes in the biofilm growth conditions, which simulate minor protocol adaptations and non-rigorous protocol documentation. Results show that even minor variations in the biofilm growth conditions may affect the results considerably, and that the biofilm analysis assays lack repeatability. Intra-laboratory validation of non-standard protocols is found critical to ensure data quality and enable the comparison of results within and among laboratories.We thank the Portuguese Foundation for Science and Technology (FCT) the strategic funding of UID/BIO/04469/2013 unit. The authors also acknowledge the support by FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects AntiPep PTDC/SAU-SAP/113196/2009 (FCOMP-01-0124-FEDER-016012), RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), the Projects "BioHealth-Biotechnology and Bioengineering approaches to improve health quality," NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER, and the Agrupamento INBIOMED from DXPCTSUG-FEDER unha maneira de facer Europa (2012/273). The research leading to these results has received funding from the European Union's Seventh Framework Programme FP7/REGPOT-2012-2013.1 under grant agreement No. 316265, BIOCAPS. This document reflects only the author's views, and the European Union is not liable for any use that may be made of the information contained herein. The authors also acknowledge the Ph.D. Grant of Paula Jorge, Ref. SFRH/BD/88192/2012

Antimicrobial combination therapies : a network perspective

The growing number of resistant strains and biofilm-related infections emerging in healthcare settings and in the general community is a major biomedical concern. Currently, antimicrobial studies are revisiting the potential of old products and looking for new products with alternative modes of action. Most notably, antimicrobial peptides (AMPs) are receiving a lot of attention because of the widespread availability, multiple mechanisms of action, non-specific molecular targets, and anti-biofilm capabilities. Considering that most of the results obtained in these studies lay in scientific literature, and manual curation is time and resource consuming, the development of bioinformatics approaches for the systematic screening of the literature is of obvious interest. In particular, the reconstruction of drug interaction networks reflecting in vitro and in vivo results is considered useful to identify the most promising candidates for the development of alternative antimicrobial therapies, such as antimicrobial combinations. Such networks can aid in profiling and interpreting the activity of AMPs and the added value of antimicrobial combinations, and thus, help exploit their potential. As a first contribution to this line of analysis, this work presents a novel network reconstruction for results obtained by AMP-drug combinations in fighting Pseudomonas aeruginosa infections. This network contains information about strains, combination methodologies, mode of growth, compound description (with drug and AMP database cross-linking) and quantification values (MICs, FICs, log reduction, etc.). So far, the network comprises 239 combinations, such that 83 % of the interactions pair an AMP with a non-AMP compound (antibiotics, enzymes, etc.), mainly traditional antibiotics. The majority (82 %) of the studies focused on the use of combinations on planktonic cells, and surprisingly enough, only 3 % of the studies tested the combination in biofilms. Furthermore, the network is dominated by a small number of highly connected nodes, namely the peptides colistin and polimyxin B. These are the products that are more often tested in antimicrobial combinations. The network is publicly available, and may be further explore using graph-based analysis tools. Hopefully, this will be a valuable resource to the design of new experiments, unveiling different mechanisms of action and helping in the prediction of new combinations

Antimicrobial peptide combinations against Pseudomonas aeruginosa and Staphylococcus aureus

Today, we are facing a major challenge regarding the development of new strategies and the discovery of new compounds with effective antimicrobial outcomes. The emergence of resistance is a preoccupant health threat and conventional antibiotics are being rendered ineffective. Researchers are now focusing in alternatives, such as the discovery of new antimicrobials with different modes of action, and the combination of agents potentiating their efficacy. AMPs are an example of new antimicrobials with promising applications, since they have different and sometimes unspecific mechanisms of action compared to traditional antibiotics, reducing the chance of acquired resistance. This work analyses AMP combinations against major pathogenic bacteria, Pseudomonas aeruginosa and Staphylococcus aureus, currently great contributors for resistance development and responsible for chronic infections, such as cystic fibrosis pneumonia. We present a screening of combinations of colistin with temporin A, citropin 1.1, tachyplesin I, lactoferricin B, magainin II and G10KHc, against these pathogens, including references and clinical isolate strains. Results show that most combinations have synergetic activities, which means that AMP combinations should be a viable way for the development of new antimicrobial treatments, thus reducing their toxicity and side effects, while maintaining efficacy. Some of the best combinations will be tested on biofilms of these bacteria, in order to test their prophylactic and therapeutic action against this different and more resilient mode of growth

Anti-biofilm peptide combinations against Pseudomonas aeruginosa and Staphylococcus aureus

Today, we are facing a major challenge regarding the development of new strategies and the discovery of new compounds with effective antimicrobial outcomes. The emergence of resistance is a preoccupant health threat and conventional antibiotics are being rendered ineffective [1]. Specifically, biofilm related infections are becoming a serious threat, being highly related to chronic infections but also nosocomial and biomaterial related infections, and they are considered the major cause of dissemination of antibiotic resistance in the nosocomial scenario. Researchers are now focusing in alternatives, such as the discovery of new antimicrobials with different modes of action, and the combination of agents potentiating their efficacy. AMPs are an example of new antimicrobials with promising applications, since they have different and sometimes unspecific mechanisms of action compared to traditional antibiotics, reducing the chance of acquired resistance, and are showing promising results in the biofilm area. A growing interest has been emerging for the use of antimicrobial combinations as a strategy to increase the antimicrobial spectrum, prevent the emergence of resistance, reduce toxicity and side effects and provide synergistic activity. Because of this, in this work we analyse AMP combinations against major pathogenic bacteria, Pseudomonas aeruginosa and Staphylococcus aureus, currently great contributors for resistance development and responsible for chronic infections, such as cystic fibrosis pneumonia. We present a screening of combinations of the AMP antibiotic colistin with the AMPs temporin A, citropin 1.1 and tachyplesin I against these pathogens, including references and clinical isolated strains. Planktonic and biofilm mode of growth were implemented and results show that most combinations have addictive and synergetic activities, including total inhibition of biofilm formation for some of the combinations tested. This means that AMP combinations should be a viable way for the development of new antimicrobial treatments, thus reducing their toxicity and side effects, while maintaining efficacy

Anti-biofilm peptide combinations in the eradication of pre-established biofilms of Pseudomonas aeruginosa and Staphylococcus aureus

The emergence of resistance is a preoccupant health threat and the development of new strategies and the use of novel compounds are in demand. Specifically, biofilm infections are a serious threat, causing chronic, nosocomial and biomaterial related infections, being related to dissemination of antibiotic resistance. Pseudomonas aeruginosa and Staphylococcus aureus are great developers of resistance and their ability to form biofilms makes them responsible for severe chronic infections. In this work, we associated the use of novel compounds antimicrobial peptides (AMPs) with a combination strategy. The AMP/antibiotic colistin was combined with three other AMPs (linear tachyplesin I; temporin A; citropin 1.1). Previous results showed the ability of these AMPs to combine synergistically against these bacteria. Here, we test these combinations against single pre-established biofilms and the results show promise. Currently, we are testing these combinations on mixed biofilms to assess their use in polymicrobial infections

Purification of plasmids using aqueous two-phase systems with amino affinity ligands

The increasing development and future application of molecular therapies such as gene therapy and DNA vaccination is expected to have a great impact in health care. However for their wide application large amounts of plasmid DNA (pDNA) are required with a stringent clearance of impurities. This prompted the development of new, efficient and cost-effective large-scale processes for the production and purification of pDNA. Most of the purification processes described are based on chromatography but dispite their high resolution, frequently they are difficult to scale-up, have low capacity and present low yields. In order to overcome these disadvantages other methodologies are also being developed. Aqueous two-phase systems (ATPS) are one of the most promising approaches for pDNA purification given their several advantages like easy scale-up, high capacity and the possibility of continuous operation. Despite their great potential ATPS have low selectivity, which limits the purification outcome. The addition of certain molecules with affinity for the target molecules (pDNA in this case) may increase their selectivity. In this work it was studied the possibility of using amino ligands for the affinity purification of pDNA from bacterial alkaline lysates. Two free amino acids, lysine and arginine, their respective Polyethylene glycol (PEG) conjugates, PEG-lysine and PEGarginine, and PEGamine were tested. The system used was composed of 16,2% (w/w) PEG 600 and 17,4% (w/w) dextran 100 (DEX) and it was evaluate the ability of each ligand to steer the pDNA to the phase where less impurities are accumulated (PEG rich phase). The results show that free amino acids did not have any effect on pDNA partitioning but the PEG conjugates were able to steer the pDNA to the PEG phase, at low concentrations. With the addition of 0,2% of PEG-lysine, or 0,5% of PEG-arginine or 4% of PEG-amine in relation to the total PEG, all the pDNA is recovered in the PEG phase. However it presents some RNA contamination, that could be removed by re-extracting with a new phase containing 30% of ammonium sulphate (NH4)2S04. The purified pDNA is obtained in the bottom phase of this new system with no measurable presence of RNA or proteins

Initial screening of poly(ethylene glycol) amino ligands for affinity purification of plasmid DNA in aqueous two-phase systems

Gene therapy and DNA vaccination are among the most expected biotechnological and medical advances for the coming years. However, the lack of cost-effective large-scale production and purification of pharmaceutical-grade plasmid DNA (pDNA) still hampers their wide application. Downstream processing, which is mainly chromatography-based, of pDNA remains the key manufacturing step. Despite its high resolution, the scaling-up of chromatography is usually difficult and presents low capacity, resulting in low yields. Alternative methods that are based on aqueous two-phase systems (ATPSs) have been studied. Although higher yields may be obtained, its selectivity is often low. In this work, modified polymers based on poly(ethylene glycol) (PEG) derivatisation with amino groups (PEGamine) or conjugation with positively charged amino acids (PEGlysine, PEGarginine, and PEGhistidine) were studied to increase the selectivity of PEGdextran systems towards the partition of a model plasmid. A two-step strategy was employed to obtain suitable pure formulations of pDNA. In the first step, a PEGdextran system with the addition of the affinity ligand was used with the recovery of the pDNA in the PEG-rich phase. Then, the pDNA was re-extracted to an ammonium-sulphate-rich phase in the second step. After removing the salt, this method yielded a purified preparation of pDNA without RNA and protein contamination.Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the NMR Portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho) and strategic funding for CQUM (UID/QUI/00686/2020).info:eu-repo/semantics/publishedVersio

Polydopamine-mediated immobilization of PALM and DNAse to create an anti-adhesive and antimicrobial bi-functional coating

The development of novel approaches to prevent biomaterial-associated infections are in great demand in modern healthcare. A mussel-inspired coating strategy was applied to introduce both anti-adhesive and antimicrobial functionalities on silicone material. Substrates were immersed in an alkaline solution of dopamine to form a thin layer of polydopamine and then transferred into a solution containing different proportions of the antimicrobial lipopetide PALM-KGK-NH2 and the enzyme DNAse I. Surface characterization confirmed the immobilization of both compounds onto silicone. The immobilization efficiency of peptide was about 65 % and peptide did no detach from the surface for up to 5 days. The mono-functional enzymatic coating prevented Staphylococcus aureus adhesion while the coating functionalized with the lipopetide killed most of the adhered cells. Combination of both compounds resulted in a bi-functional coating able to prevent bacterial adhesion and kill the adherent ones. Moreover, cells adhered to these modified surfaces exhibited the same antibiotic susceptibility pattern as cells adhered to unmodified surfaces, suggesting no resistance development. In conclusion, silicone functionalization with enzymes and antimicrobial peptides holds great potential in the development of biomaterials to prevent biomaterial-associated infections

Developing timely insights into Pseudomonas aeruginosa quorum sensing therapeutics through text mining

Publicado em "Biofilms7: microbial works of art: book of abstracts". ISBN 978-989-97478-7-6The pervasive growth of antibiotic-resistant is pressing the development of novel strategies to control infectious diseases. Quorum sensing (QS) is a key communication mechanism that allows bacteria to regulate gene expression, and thus many physiological activities e.g. virulence, motility, and biofilm formation. Hence, QS inhibition or quorum quenching is being pursued as a promising strategy to control clinical pathogens.Most available information about drug interactions with QS genes and molecules is scattered in the vast and ever-growing biomedical bibliome. So, text mining and network mining are attractive solutions to identify relevant interactions and generate new hypothesis for antimicrobial research.Here, we describe the implementation of such an automated workflow that extracts key information on P. aeruginosa QS-focused antimicrobial strategies from PubMed records. The workflow produces an integrated network, capturing the effect of antimicrobial agents over QS genes, QS signals and virulence factors. Interactions are contextualised by information on the conducted experimental methods and details on the antimicrobials and QS entities retrieved. The public Web-based interface (http://pcquorum.org) enables users to navigate through the interactions and look for indirect, non-trivial antimicrobial-QS associations.Currently, the P. aeruginosa antimicrobial-QS network contains 439 interactions encompassing 170 different drugs and 72 different QS entities; but it is in continuous, semi-automated growth. It offers a comprehensive picture of emerging anti-QS findings and thus may help in gaining novel understanding and prioritising new antimicrobial experiments