The underrepresentation of women in the international courts

The underrepresentation of women in the international courts reduces the democratic, normative, and sociological legitimacy of their decisions. Although States are bound to multiple legal instruments which impose that they take measures to ensure gender equality and balance, women face gender stereotypes and suffer discrimination which in turn jeopardizes their attempts to achieve the international judiciary. The national nominations and international procedures suffer from a lack of transparency and impartial evaluation standards leading to the perpetuation of exclusive networks that prioritize political maneuvering over the technical qualification of candidates. To ensure a representative and legitimate international bench, measures must be taken to ensure de facto equality for women, mandatory gender quotas and aspirational language in the Statutes of the Courts are tools that may reduce the issue

Evaluation of antibacterial activity of caffeic acid encapsulated by -cyclodextrins

Context: Caffeic acid is described as antibacterial, but this bioactive molecule has some issues regarding solubility and stability to environmental stress. Thus, encapsulation devices are required. Objective: The aim of this work was to study the effect of the caffeic acid encapsulation by cyclodextrins on its antibacterial activity. Materials and methods: The interactions between the caffeic acid and three cyclodextrins (-cyclodextrin (CD), 2-hydroxypropyl--cyclodextrin (HPCD) and methyl--cyclodextrin were study. Results and discussion: The formation of an aqueous soluble inclusion complex was confirmed for CD and HPCD with a 1:1 stoichiometry. The CD/caffeic acid complex showed higher stability than HPCD/caffeic acid. Caffeic acid antibacterial activity was similar at pH 3 and pH 5 against the three bacteria (K. pneumoniae, S. epidermidis and S. aureus). Conclusions: The antibacterial activity of the inclusion complexes was described here for the first time and it was shown that the caffeic acid activity was remarkably enhanced by the cyclodextrins encapsulation.The authors have declared no conflict of interests. The authors are grateful for the FCT Strategic Project PEst-OE/EQB/LA0023/ 2013 and the Project ‘‘BioHealth – Biotechnology and Bioengineering approaches to improve health quality,’’ Ref. NORTE-07–0124-FEDER-000027, co-funded by the ‘‘Programa Operacional Regional do Norte’’ (ON.2 – O Novo Norte), QREN, FEDER. The authors also acknowledge the project ‘‘Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB’’, Ref. FCOMP-01– 0124-FEDER-027462. This work is, also, funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through FCT Foundation for Science and Technology under the project PEstC/CTM/UI0264/2011. Additionally, the authors would like to thank the FCT for the grant for E. Pinho (SFRH/BD/62665/2009)

Antimicrobial composite wound dressing

Nowadays, a wound dressing is no longer a passive material, it must interact with the wound and improve the healing process. In fact, the actual requirements for a wound dressing are quite challenging. To achieve these demanding goal, wound dressing’ research have been focus on the development of composites that combine the best of two or more polymeric materials. Thus, our group developed a composite material of cotton functionalized with hydroxypropyl methylcellulose / cyclodextrins hydrogel to be used as antimicrobial wound dressing. Hydrogel polymer will improve the wearability and the drug delivery capacity of cotton textiles. And cotton will enhance the mechanical properties of hydrogel, facilitating the handle process. The composite synthesis was performed by one-step chemical crosslinking. The reaction parameters, such as crosslinker concentration and polymeric solution concentration, were optimized. The obtained composites were characterized base on their physicochemical and biological properties. To the best of our knowledge, loading and release of gallic acid (as antibacterial agent) into composites wound dressings, and its release for control wound infections, have not been evaluated until now. The developed composites have the combined properties of cotton and hydrogel. The gallic acid was successfully loaded into the polymeric network, and it release was sustained for 48 h. The loaded composites can destroy bacterial cells preserving the gallic acid antibacterial activity. Thus, the developed composites showed suitable properties for the incorporation of gallic acid and utilization as antibacterial wound dressing.TSSiPRO—NORTE-01-0145-FEDER-000015—supported by the regional operational program NORTE 2020, under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund;info:eu-repo/semantics/publishedVersio

Development of an aptamer-based biosensor for the detection of food toxins

According to the last EFSA/ECDC report, bacterial toxins are the second leading causative agent (17.0%) of foodborne outbreaks in Europe [1]. Aptasensors, as biosensors that use aptamers are called, are seen as a simple, rapid and cost-effective assay format with high suitability for point-of-care testing, allowing a sensitive and mostly qualitative detection of analytes [2]. In this work, DNA aptamers previously selected by our group (Apt1, Apt2, Apt3, Apt4 and Apt5) for staphylococcal enterotoxin A (SEA), one of the most reported bacterial toxins, were applied as recognition molecules in a lateral flow assay. For this, lateral flow strips consisting of a sample pad with glass fibre, test zone with nitrocellulose membrane and absorbent pad with cellulose membrane were assembled. Gold nanoparticles (AuNPs) covalently attached with Apt5 were synthesized. Biotinylated aptamers (140 pmol of Apt1, Apt2, Apt3 and Apt4) were immobilized in the test zone using streptavidin as an anchor. A DNA probe (140 pmol) complementary to Apt5 was also immobilized as a test control. Then, SEA solutions (0.3 ng/L) as well as negative samples were prepared and incubated with Apt5- AuNPs (OD3) in binding buffer for 10 min. Different assay combinations (Apt5-AuNPs + Apt1/Apt2/Apt3/Apt4) were tested. The samples (60 L) were applied to the sample pad, allowing the solution to flow on the strip until the test lines were visualized by the accumulation of AuNPs. SEA samples were positively detected, with the combination of Apt5-AuNPs with Apt3, providing the best result, followed by Apt4, Apt2 and Apt1. Negative controls were validated by the control line. Further tests to determine the detection limit and improve the noise ratio are being carried out. These results show that aptasensors can be a simple and rapid alternative for the detection of SEA. Furthermore, this format assay can be easily adapted to any food toxin.This work was financially supported by: LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through FCT/MCTES (PIDDAC); project POCI01-0145- FEDER-028659, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI). The authors also thank FCT for the PhD Fellowship SFRH/BD/138883/2018.info:eu-repo/semantics/publishedVersio

Antimicrobial activity assessment of textiles : standard methods comparison

Antimicrobial fabrics are increasingly important in a great variety of applications and thus several standard methods to evaluate their efficiency have been developed. However, there is no consensus on the most adequate method to be used. Therefore, aim of this work was to compare the practical applicability of the best known standards: AATCC 147, ISO 20645:2004, AATCC:100 and JIS L 1902. Four samples, with different amounts of antimicrobial agents, were used. It was tested 3 qualitative methods (AATCC 147, ISO 20645 and JIS L 1902–Halo method) and 2 quantitative (AATCC 100 and JIS L 1902–Absorption method). For each method, both Gram-positive (Staphylococcus aureus) and Gram-negative (Klebsiella pneumoniae) bacteria were used. Textiles samples assayed did not present diffusible activity, thus only the qualitative results from the AATCC 147 and the Halo method could be analyzed and no differences were observed between them. Therefore, the AATCC 147 or the JIS L 1902–Halo method can be used for a simple and expedite screening of a large amount of samples with or without diffusible antimicrobial activity. In contrast, the ISO 20645 can only be used when diffusible antimicrobial agents are present. Concerning the two quantitative methods, the results showed that the JIS L 1902 method is more sensitive to the amount of antimicrobial agent than the AATCC 100 test. An additional assay also showed that the JIS L 1902 is sensitive enough to distinguish serial dilutions of the antimicrobial agent

Gallic acid and cyclodextrins : inclusion complexes and antimicrobial activity

Gallic acid (GA), or 3,4,5-trihydroxybenzoic acid, is the commonest and simplest phenolic acid, with just one aromatic ring. This phenolic molecule has been described as antioxidant, anti-inflammatory and anti-tumour, also, antimicrobial and anti-fungal activity has been attributed to it. Besides all the GA biological activities, this compound is cheap, due to the easy plant extraction, and non-toxic. Thus, this phenolic acid has been widely used in food, drugs and cosmetic industry. GA, as other phenolic compounds, is susceptible to environmental factors which may lead to the losing their structural integrity and bioactivity. This can be overcome by the encapsulation with cyclodextrins (CD). They are cyclic oligosaccharides arising from the degradation of starch; inexpensive and friendly to humans. CD are able to form an inclusion complex with a wide range of bioactive molecules, including hydrophobic ones, protect and modulate their release. In the present work, the formation of an inclusion complex (IC) between βCD, HPβCD (2-Hydroxypropyl-β-cyclodextrin) or MβCD (methyl-β-cyclodextrin) and GA was analysed by UV spectrophotometry. The antimicrobial activity of the complexes was also assessed, by qualitative and quantitative methods. The influence of the buffer and pH on the formation of the IC and on the GA antimicrobial activity was also tested. The IC formation was analysed in 2 buffer solutions (K2HPO4/ KH2PO4 and H3PO4/NaOH). The GA and IC absorbance spectrum showed different appearances depending on the buffer used. When K2HPO4/ KH2PO4 was used, the IC spectrum presented some alterations on the λmax comparatively to the GA spectrum. Since, the GA and ICs spectra obtained for the H3PO4/NaOH were similar, this buffer was selected. The IC formation between GA and βCD, HPβCD or MβCD was analysed at pH 5, 7 and 8. Based on the results it was clear that the pH used affected the IC formation in the conditions tested. The βCD formed IC 1:1 with GA for all pH values, being the pH5 the most favourable. The same was observed for MβCD, but these CD had the lowest values for the association constant, meaning that the IC formation between MβCD and GA is not very efficient. Regarding the HPβCD, in neutral pH the IC formed was 1:2 and for the others pH was 1:1, pH5 was the most favourable for the IC formation and pH7 the least. The antimicrobial activity was assessed for the best combinations of GA/CD (βCD pH5 and pH7; HPβCD pH5 and pH8). For all the IC tested, the antimicrobial activity of GA was improved or alike to the GA without encapsulation. To the authors knowledge, the IC formation between HPβCD or MβCD and GA has not been reported until know, as well as the antimicrobial activity of the IC βCD/GA and HPβCD/GA

Development of a new antimicrobial material for wound dressing

Tese de doutoramento em Engenharia BiomédicaNowadays, wound dressings are complex materials that, behind the mechanical protection, are also capable of interacting with the injury tissue. These complex products improve the healing process by maintaining suitable conditions, allowing skin to establish integrity with appropriate cosmetic results. The incidence of chronic wounds related with diseases, population ageing, and tissue infection caused by multi-resistant strains of bacteria, has been increasing. Thus, the wound dressing research is now focused on the development of new materials with higher specificity in order to improve the quality of patients’ life. One of the directions is the use of natural materials for wound dressing’s production for the development of a friendlier product. Hydrogels have been successfully used as wound dressings. They gather favourable properties for wound healing, such as good biocompatibility and ability to maintain the proper environmental humidity, which facilitate the ready removal of the material with minimal pain. However, hydrogels lack the capacity to load and control the release of bioactive molecules. Thus, the incorporation of cyclodextrins on the hydrogels allowed the improvement of the polymeric network ability to protect and modulate the release of bioactive molecules. Additionally, cyclodextrins are capable of complex with a wide range of molecules, due to their microheterogeneous environment (hydrophilic outside and hydrophobic cavity). Moreover, plants have been used as source for new therapeutic agents able to enhance the healing process. Polyphenolics, one of the groups of bioactive molecules, has been described as anti-oxidant, anticarcinogenic, anti-inflammatory and antimicrobial. However, their application by the pharmaceutical industry is limited by polyphenolics lower solubility and stability to environmental stress. Thus, cyclodextrins has been proposed as viable carrier for the maintenance of bioactive molecules’ structural integrity and bioactivity. Therefore, the present thesis reports the work developed to obtain a wound dressing, using natural compounds, capable of preventing infection of the injury tissues. The first step was to select phenolic compounds from natural extracts obtained from Portuguese wild plants. Gallic and caffeic acid revelled to be the most efficient antibacterial agents against bacteria commonly isolated from skin and soft tissue infections (Klebsiella pneumoniae, Staphylococcus epidermidis and Staphylococcus aureus). Besides, gallic acid has shown very low cytotoxicity, for concentrations lower than 0.01 mg.mL-1. In order to enhance gallic and caffeic acids solubility and stability, their encapsulation by cyclodextrins was evaluated. The inclusion complexes obtained were stable and capable of destroying the bacteria used. Although, gallic and caffeic acid had similar molecular structure, the cyclodextrins encapsulation behaviour was different. In the case of gallic acid, the hydroxypropyl-β-cyclodextrin formed a more stable inclusion complex at pH 3 (when compared with β-cyclodextrin and methyl-β-cyclodextrin) as well as presented a better antibacterial activity. Moreover, gallic acid carboxylic group was positioned towards the smaller cyclodextrins ’cavity and hydroxyl groups were placed near the wider opening. The gallic acid neutral form, positioned inside the cyclodextrins, preserved gallic acid antibacterial activity. Otherwise, the caffeic acid carboxylic group was project to the water phase and the aromatic part inside the cyclodextrins cavity. Additionally, the native cyclodextrin was better for the encapsulation of this phenolic at pH 5 and it antibacterial activity was enhanced by the inclusion complex formation. Hence, the incorporation of gallic and caffeic acid in a polymeric network was, also, studied. A hydrogel based on cyclodextrins and hydroxypropyl methylcellulose, using 1,4-butanediol diglycidyl ether as cross-linking agent, was synthetized. Only the β-cyclodextrin and hydroxypropyl-β-cyclodextrin permitted the formation of hydrogels with good mechanical properties. Both networks behaved with superabsorbent materials and were capable of loading and release gallic and caffeic acid by a control mechanism. The differences between the 2 phenolic acids found in the study of the inclusion complexes were similar to the load hydrogels. The gel with hydroxypropyl-β-cyclodextrin had better properties regarding the gallic acid and the gel with β-cyclodextrin was more suitable in the caffeic acid case. Thus, cyclodextrins were the major mechanism involved on the load and release of the gallic and caffeic acid. Furthermore, the phenolic acids antibacterial activity and effect on the fibroblasts proliferation was maintained after their incorporation on the hydrogels. In conclusion, the work developed proved that natural antibacterial agents can be used to control the growth of the most common bacteria isolated from infected wounds, and their incorporation in cyclodextrins-based hydrogels preserved the selected phenolics biological activities. Thus, the hydrogels synthesised could be useful as natural antibacterial agent’s delivery systems device for the treatment of wound infections.Atualmente, os pensos utilizados no tratamento de feridas são materiais complexos que oferecem proteção mecânica e promovem cicatrização, mantendo as condições mais favoráveis para o restabelecimento da integridade da pele. A incidência de feridas crónicas como consequência de doenças, do envelhecimento da população e de infeções provocadas por bactérias multirresistentes, tem aumentado nos últimos anos. Esta evolução obrigou a indústria a direcionar os seus esforços para o desenvolvimento de materiais com maior especificidade, contribuindo para melhorar a qualidade de vida dos pacientes. Os hidrogéis reúnem propriedades que favorecem a cicatrização de feridas, nomeadamente biocompatibilidade e capacidade para manter a humidade, o que facilita a remoção dos dispositivos com reduzido trauma ou dor para o paciente. Por isso mesmo, têm sido aplicados com sucesso no tratamento de feridas crónicas. Contudo, a sua capacidade para adsorver e libertar fármacos de um modo controlado é reduzida. Assim, a incorporação de ciclodextrinas em matrizes poliméricas tem sido investigada de modo a melhorar a capacidade dos hidrogéis para proteger e modular a libertação de biomoléculas. As ciclodextrinas apresentam, ainda, a vantagem de conseguirem encapsular uma grande variedade de moléculas, pois possuem uma superfície externa hidrofílica e uma cavidade hidrofóbica. A procura de novos agentes antimicrobianos tem crescido nos últimos anos como consequência do decréscimo de eficiência dos antibióticos contra bactérias multirresistentes. As plantas possuem uma gama alargada de moléculas, por exemplo polifenólicos, capazes de promoverem a cicatrização e prevenirem infeções. Os polifenólicos têm sido descritos como antioxidante, anticarcinogénico, anti-inflamatório e antimicrobiano. No entanto, a sua utilização pela indústria farmacêutica é limitada, devido à sua baixa solubilidade e estabilidade. Assim sendo, a encapsulação de compostos polifenólicos com ciclodextrinas tem sido proposta como opção viável, pois a sua integridade estrutural e a bioatividade são preservadas após inclusão. A presente tese resume o trabalho desenvolvido na conceção de um penso para o tratamento de feridas com compostos naturais, capaz de melhorar a cicatrização e de prevenir infeções. Inicialmente, procedeu-se à seleção de compostos fenólicos obtidos a partir de extratos de plantas do Nordeste português. Os ácidos gálico e cafeico foram os compostos polifenólicos que apresentaram maior eficiência no controle da proliferação de bactérias, normalmente isoladas de feridas infetadas (Klebsiella pneumoniae, Staphylococcus epidermidis and Staphylococcus aureus). Além do mais, o ácido gálico apresentou reduzida citotoxicidade para concentrações abaixo de 0.01 mg.mL-1. No sentido de melhorar as propriedades de estabilidade e solubilidade dos ácidos gálico e cafeico procedeu-se ao estudo da sua encapsulação com ciclodextrinas. Os complexos de inclusão obtidos demostraram ser estáveis e capazes de manter a atividade antibacteriana dos fenólicos. Apesar dos ácidos gálico e cafeico apresentarem uma estrutura molecular semelhante, o comportamento de encapsulação com ciclodextrinas divergiu. No caso do ácido gálico, a hidroxipropil-β-ciclodextrina permitiu a formação de complexos mais estáveis a pH 3 e com melhor atividade antibacteriana. E, supõe-se que o ácido gálico encapsulado apresente o grupo carboxílico orientado para a abertura mais pequena da ciclodextrina e os grupos hidroxilos voltados para o interior da cavidade. Pelo contrário, o grupo carboxílico do ácido cafeico estará projetado para fora da ciclodextrina e a parte aromática para o interior da cavidade. Além disso, a β-ciclodextrina foi a mais eficiente na complexação do ácido cafeico a pH 5 e observou-se um reforço das propriedades antibacterianas deste fenólico depois da inclusão. Após a confirmação da complexação dos ácido fenólicos pelas ciclodextrinas com preservação das propriedades antibacterianas, estudou-se a incorporação dos ácidos gálico e cafeico num hidrogel. Para tal, produziu-se uma matriz polimérica utilizando ciclodextrinas e hidroxipropil metil celulose como monómeros e 1,4-butanediol diglicidil éter como agente de reticulação. A formação de hidrogéis com propriedades mecânicas aceitáveis só foi confirmada com a utilização da β-ciclodextrina e da hidroxipropil-β-ciclodextrina. Em ambos os casos obteve-se materiais superabsorventes, em que as ciclodextrinas desempenharam um papel crucial na capacidade de modelar a adsorção e a libertação dos ácidos fenólicos. Além disso, a atividade antibacteriana e o efeito sobre a proliferação de fibroblastos manteve-se após incorporação dos ácidos fenólicos nos hidrogéis. Em conclusão, o trabalho desenvolvido demostrou ser possível utilizar ácidos fenólicos de origem natural para controlar o crescimento das bactérias normalmente isoladas de feridas infetadas. Comprovou-se, ainda, a possibilidade de incorporação destes compostos antimicrobianos em hidrogéis com ciclodextrinas mantendo as suas propriedades biológicas. Assim sendo, os hidrogéis antibacterianos sintetizados poderão ser úteis na prevenção e tratamento de infeções em feridas.Project “BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. The authors also acknowledge the project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through FCT - Foundation for Science and Technology under the project PEst-C/CTM/UI0264/2011 Ref. FCOMP-01-0124-FEDER-027462 and the FCT grant SFRH/ BD/ 62665/2009

Assessment of antimicrobial activity of textiles for wound dressing: methodology optimization

Normally, the skin is capable of restore the tissue integrity, after wound injury. However, the deposition of bacteria on the wound site results on infection causing pain and healing delay. To control bacteria proliferation, antimicrobial textiles have been developed, and the assessment of their activity is a required step. Although, several standard methods were published to assess textiles antimicrobial activity, they are time and material consuming and have some shortcomings with regard to the real conditions of use. Therefore, the aim of this work was to optimize the method described on JIS L 1902:2008-Testing for antibacterial activity and efficacy on textile products, the most commonly used standard. Two textile samples were used: A-cotton without treatment (control) and B-cotton with 10% of the recommend concentration of Ruco-bac AGP. The microorganism used was Staphylococcus aureus-ATCC 6538. The first improvement was sample size. On the qualitative method, square samples with 1x1cm2 were used instead 2.5x2.5cm2 (suggested by the standard). For sample A no antimicrobial activity was observed and for sample B the halo size was similar for both sizes used. For the quantitative method, the samples used had 0.4g (standard suggestion) and 0.1g. Sample A had the same bacterial growth before and after contact with the fabric and sample B had no bacterial growth. With this improvement, the amount of sample and solutions need for the test was reduced four times. To reduce the use of disposable material, instead of 50mL falcons, 6 well plates were used. In this case, no bacteria were recovered from the sample A after incubation period on 6 well plates. These means, that the centrifugation is a crucial step to detach all bacteria from the fabric. The effect of the bacterial inoculum volume was also assessed. Three inoculum volumes (250, 100 and 50µL) were added to 0,1g samples. No significant differences were observed for both samples. A healthy skin has 105bacteria/cm2 and up to this value it is considered that the skin is infected. Therefore, 3 inoculum concentrations were tested-3x105, 3x106, 3x107cell/mL. The results showed that the inoculum concentration had no significant changes for both samples after the incubation period. In conclusion, it is possible to use samples 4 times smaller than the standard suggestion, use higher inoculum volume to simulate wound exudate and higher concentration, to accurately predict the sample behaviour on an infected skin