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

NMR and molecular modelling studies on elastase inhibitor-peptides for wound management

Proteases play an important and critical role in the physiological process of wound repair. However, excessive and unregulated release of proteolytic enzymes (e.g., elastase) mediates abnormal degradation of healthy tissues, which leads to inflammatory disorders such as chronic wounds. Thus, it is of therapeutic interest to develop novel synthetic inhibitor-peptides of elastase, which can restore the balance between the free enzyme and the endogenous inhibitors in chronic wounds. In previous works, we have reported two different drug delivery systems to release novel elastase inhibitors to the wound site. In both systems synthetic peptides (KRCCPDTCGIKCL-Pep4 and KRMMPDTMGIKML-Pep4M) based on the primary structure of the endogenous elastase inhibitor, secretory leucocyte protease inhibitor, were used as active material. Phosphorylation of the reported peptides prompts significant structural differences, which reflects in distinct inhibitory capacity towards elastase. These structural modifications were prompted by electrostatic interactions and hydrogen bonds established from the peptide phosphoresidue. The current study was also extended to another synthetic peptide (WCTASVPPQCY-PepBBI) that is based on the reactive loop of another elastase inhibitor, the Bowmen-Birk inhibitor. PepBBI, phosphorylated and non-phosphorylated, displays similar behaviour to Pep4 and Pep4M. The structural modifications reported herein were evaluated by two-dimensional nuclear magnetic resonance and molecular modelling approaches.The authors gratefully acknowledge the financial support of the Portuguese Foundation for Science and Technology (scholarship SFRH/BD/36522/2007 and PEst-OE/EQB/LA0004/2011), FEDER (European Fund for Regional Development)-COMPETE-QREN-EU and the European Project Lidwine - Multifunctional medical textiles for wound (e.g. Decubitus). We acknowledge CERMAX at ITQB-UNL and Rede Nacional de RMN for access to the facilities. Rede Nacional de RMN is supported with funds from FCT, Projecto de Re-equipamento Cientifico contract REDE/1517/RMN/2005, Portugal. Micaelo, N.M. acknowledges the contract research program "Compromisso corn a Ciencia" reference: C2008-UMINHO-CQ-03 and access to the Minho University GRIUM cluster

A setup for integral measurements of multiple scattering angular distributions by 10- to 100-keV electrons

A dedicated setup has been developed to study the angular distributions of electrons traversing thin films employing the low-energy beam available from the gun of the São Paulo Microtron. In this first stage, only integral measurements are possible using the fraction of the electron beam current collected in a Faraday cup and in a ring surrounding the entrance of the former. The overall normalisation is provided by measuring the beam current collected by the scattering chamber as well, thus covering the full solid angle. In this work, important construction specifications are presented. The experience gained by operating this system is also discussed regarding its critical aspects like: avoiding cross talk between the cup and the ring both at the physical and electronic levels, measurements of small charges deposited on large objects (the chamber in particular), and conditioning of the various surfaces involved. Finally, some selected results are compared to the well-known theory by Goudsmit and Saunderson with good agreement

Thermal, mechanical and chemical analysis of poly(vinyl alcohol) multifilament and braided yarns

Poly(vinyl alcohol) (PVA) in multifilament and braided yarns (BY) forms presents great potential for the design of numerous applications. However, such solutions fail to accomplish their requirements if the chemical and thermomechanical behaviour is not sufficiently known. Hence, a comprehensive characterisation of PVA multifilament and three BY architectures (6, 8, and 10 yarns) was performed involving the application of several techniques to evaluate the morphological, chem- ical, thermal, and mechanical features of those structures. Scanning electron microscopy (SEM) was used to reveal structural and morphological information. Differential thermal analysis (DTA) pointed out the glass transition temperature of PVA at 76 °C and the corresponding crystalline melt- ing point at 210 °C. PVA BY exhibited higher tensile strength under monotonic quasi-static loading in comparison to their multifilament forms. Creep tests demonstrated that 6BY structures present the most deformable behaviour, while 8BY structures are the least deformable. Relaxation tests showed that 8BY architecture presents a more expressive variation of tensile stress, while 10BY of- fered the least. Dynamic mechanical analysis (DMA) revealed storage and loss moduli curves with similar transition peaks for the tested structures, except for the 10BY. Storage modulus is always four to six times higher than the loss modulus.This work was funded by European Regional Development funds (FEDER) through the Competitiveness and Internationalization Operational Program (POCI)—COMPETE andby Na-tional Funds through Portuguese Fundação para a Ciência e Tecnologia (FCT) under the project UID/EMS/50022/2020, UID/EEA/04436/2019 andUID/ CTM/00264/2019. Andrea Zille acknowledges financial support of the FCT through the project PTDC/CTM-TEX/28295/2017,and Nuno Dourado acknowledges financial support of the FCT through the project PTDC/EME-SIS/28225/2017. M.F.S.M. de Moura acknowledges the ‘Laboratório Associado de Energia, Transportes e Aeronáutica’ (LAETA) for the financial support

Fauna de abelhas nativa em plantações de tomate : uma comparação de métodos de amostragem ativa e de armadilha

O tomate é amplamente cultivado em todo o mundo e requer polinização por abelhas nativas ou manejadas para realizar o pleno potencial de produção dos frutos. Para investigar a riqueza e abundância de espécies de abelhas nativas em plantações de tomate do Centro-Oeste do Brasil, dois métodos de amostragem (armadilhas pan-trap e amostragem ativa) foram utilizados em nove propriedades de junho a setembro de 2011. Um total de 465 indivíduos de 44 espécies foi coletado. A composição das espécies de abelhas amostradas diferiu dependendo do método utilizado. Vinte e duas espécies foram capturadas exclusivamente em armadilhas, 13 outras por meio de amostragem ativa e nove por ambos os métodos. A maioria das espécies de abelhas capturadas neste estudo pode ser considerada polinizadores eficazes do tomate, porque elas podem executar a polinização por vibração. Vibrando seus músculos torácicos, essas abelhas podem liberar o pólen das anteras para seus próprios corpos e para os estigmas da mesma flor, uma vez que eles estão dentro do cone de anteras da variedade do tomate estudado. Ambos os métodos amostraram espécies exclusivas de abelhas vibradoras. No entanto, as armadilhas capturaram abelhas vibradoras e não vibradoras indiscriminadamente e o método ativo amostrou principalmente a abelha vibradora. As coletas utilizando armadilhas e amostragens ativas foram complementares. O uso de apenas um método de amostragem não fornece um entendimento completo da riqueza de espécies de polinizadores de tomate no campo.The tomato is widely cultivated throughout the world and requires pollination by wild or managed bees to realize its full-potential fruit production. Two different sampling methods (pan trapping and active sampling) were employed in nine different properties from June to September of 2011 to investigate the richness and abundance of native bee species present in tomato crops of Center-West Brazil. A total of 465 individuals of 44 species were collected, with the composition of sampled bee species differing between the methods used. Twenty-two species were exclusively captured in pan traps, 13 others through active sampling and nine by both methods. Most of the sampled bee species can be considered effective pollinators of the tomato because they can perform buzz-pollination. By vibration, these bees can liberate pollen from anthers into the air or onto their own bodies and the stigmas of the same flower because the stigmas of the studied tomato variety are within the anther cone. Both methods exclusively sampled some species of buzz-pollinating bees, however, pan-trapping captured buzzing and non-buzzing visitors indiscriminately while active sampling captured more buzzing bees. Pan-trapping and active sampling appear to complement each other, and so the use of only one or the other would not provide a full understanding of the species richness of tomato pollinators in the field

Active neutralizing mats for corrosive chemical storage

Laboratories and industries that handle chemicals are ubiquitously prone to leakages. These may occur in storage rooms, cabinets or even in temporary locations, such as workbenches and shelves. A relevant number of these chemicals are corrosive, thus commercial products already exist to prevent material damage and injuries. One strategy consists of the use of absorbing mats, where few display neutralizing properties, and even less a controlled neutralization. Nevertheless, to the authors’ knowledge, the commercially available neutralizing mats are solely dedicated to neutralizing acid or alkali solutions, never both. Therefore, this work describes the development and proof of a completely novel concept, where a dual component active mat (DCAM) is able to perform a controlled simultaneous neutralization of acid and alkali leakages by using microencapsulated active components. Moreover, its active components comprise food-grade ingredients, embedded in nonwoven polypropylene. The acid neutralizing mats contain sodium carbonate (Na2CO3 ) encapsulated in sodium alginate microcapsules (MC-ASC). Alkali neutralizing mats possess commercial encapsulated citric acid in hydrogenated palm oil (MIRCAP CT 85-H). A DCAM encompasses both MC-ASC and MIRCAP CT 85-H and was able to neutralize solutions up to 10% (v/v) of hydrochloric acid (HCl) and sodium hydroxide (NaOH). The efficacy of the neutralization was assessed by direct titration and using pH strip measurement tests to simulate the leakages. Due to the complexity of neutralization efficacy evaluation based solely on pH value, a thorough conductivity study was performed. DCAM reduced the conductivity of HCl and NaOH (1% and 2% (v/v)) in over 70%. The composites were characterized by scanning electron microscopy (SEM), differential calorimetry (DSC) and thermogravimetric analysis (TGA). The size of MC-ASC microcapsules ranged from 2 µm to 8 µm. Finally, all mat components displayed thermal stability above 150 ◦C

Fracture characterisation of bone-cement bonded joints under mode I loading

Over the years, many techniques have been developed for the stabilisation of bone fractures. The study of the adhesion of bone-to-bone cement is an important step towards the development of new immobilization systems. Although bone cement has been used for more than fifty years, very few studies have been performed regarding the evaluation of fracture properties. In this work, numerical and experimental investigations were conducted to evaluate the strain energy release rate under mode I loading in a bone-cement bonded joint, using the Double Cantilever Beam (DCB) test. Cohesive zone laws were also measured combining the finite element method with non-linear elastic fracture mechanics. This has been made in a cortical bone bonded joint with poly- methylmethacrylate (PMMA). Consistent results have been obtained regarding fracture toughness in a widely used bone-to-bone cement joint in many biomedical applications.The first author acknowledges the Portuguese (FCT) for the conceded financial support through the reference grant PTDC/EME-SIS/28225/2017. M.F.S.M. de Moura acknowledges the ‘Laboratório Associado de Energia, Transportes e Aeronáutica’ (LAETA) for the financial support by the project UID/EMS/50022/2020. The corresponding author acknowledges FCT for the conceded financial support through the reference projects PTDC/EME-SIS/28225/2017 and UID/EEA/04436/2019.Funding: Portuguese Foundation for Science and Technology for MsC grant of the first author, and research project PTDC/EME-SIS/28225/2017

Knitted textile KTPs for instrumented underwater building systems

[Excerpt] The main scope of this study was to assess the potential of knitted textiles for underwater instrumented underwater building systems, using textile knitted preforms (multi-shape; multimaterial; etc…), using natural fibres (Hemp, Linen and Cotton)