Efficient extraction of the RuBisCO enzyme from spinach leaves using aqueous solutions of biocompatible ionic liquids

Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) is the most abundant protein on the planet, being present in plants, algae and various species of bacteria, with application in the pharmaceutical, chemical, cosmetic and food industries. However, current extraction methods of RuBisCO do not allow high yields of extraction. Therefore, the development of an efficient and selective RuBisCOs’ extraction method is required. In this work, aqueous solutions of biocompatible ionic liquids (ILs), i.e., ILs derived from choline and analogues of glycine-betaine, were applied in the RuBisCO’s extraction from spinach leaves. Three commercial imidazolium-based ILs were also investigated for comparison purposes. To optimize RuBisCO’s extraction conditions, response surface methodology was applied. Under optimum extraction conditions, extraction yields of 10.92 and 10.57 mg of RuBisCO/g of biomass were obtained with the ILs cholinium acetate ([Ch][Ac]) and cholinium chloride ([Ch]Cl), respectively. Circular dichroism (CD) spectroscopy results show that the secondary structure of RuBisCO is better preserved in the IL solutions when compared to the commonly used extraction solvent. The obtained results indicate that cholinium-based ILs are a promising and viable alternative for the extraction of RuBisCO from vegetable biomass.publishe

Integrated platform resorting to ionic liquids comprising the extraction, purification and preservation of DNA

The large-scale production of therapeutically targeted-deoxyribonucleic acid (DNA) has passed through several challenges, postponing the tangible implementation of an effective, economic and sustainable manufacturing system. Such challenges comprise the need to develop an integrative downstream process able to extract, purify and long-term preserve DNA, whilst reducing the risk of degradation by endonucleases that would compromise their effectiveness as therapeutic products. In this work, three-phase partitioning (TPP) systems formed by the application of aqueous biphasic systems (ABS) composed of several biocompatible cholinium-based ionic liquids (ILs), are proposed for the separation of double stranded DNA (dsDNA) from the endonuclease deoxyribonuclease I (DNase I). By taking advantage of the tailor-made properties of ILs, dsDNA can be completely extracted to the IL-rich phase, whereas DNase I is precipitated at the ABS interphase. The ABS/TPP formed by IL cholinium glycolate ([N 111(2OH) ][Gly]) fulfills the aim of this work, i.e. at ensuring the technical viability of IL-based ABS/ TPP for the “one-pot” extraction, purification and long-term preservation of dsDNA. The results reveal the potential of this system to be applied in the bioprocessing of DNA, particularly relevant when envisioning DNA- based therapeutic products.publishe

Insights on the laccase extraction and activity in ionic-liquid-based aqueous biphasic systems

Due to their catalytic properties, selectivity, and efficiency, enzymes are excellent biocatalysts. In particular, laccases are versatile multi-copper oxidases with great interest for a wide plethora of biotechnological and environmental applications. Even though several laccase-catalysed processes have been reported at an industrial level, the high costs of their downstream processing required to provide biocatalysts with high purity levels, stability and activity remains one of the main drawbacks when economically evaluating the overall processes. Aqueous biphasic systems based on ionic liquids (ILs) can be foreseen as a promising alternative approach for the extraction and activity maintenance/improvement of enzymes, essentially due to the designer solvents ability of ionic liquids. However, to take advantage of this feature and to use the full potential of IL-based aqueous biphasic systems, it is necessary to understand the effect of ILs as phase-forming constituents and how they affect the enzymes extraction and activity. In order to overcome the lack of information on this topic in the literature, in this work, IL-based aqueous biphasic systems were investigated to extract and enhance the laccase activity, in order to gather evidences that could be used to improve the enzymes downstream processing. To this end, a wide screening of imidazolium-, pyridinium-, pyrrolidinium-, piperidinium-, tetraalkylphosphonium-, and tetraalkylammonium-based ILs as phase-forming components of ABS was carried out. Furthermore, these ILs were used to create ABS combined with salts, polymers and used as adjuvants in polymer-based ABS. Most ABS comprising ILs revealed to be highly efficient extraction platforms, allowing the complete extraction of laccase for all the conditions tested, and with an enzyme activity enhancement by more than 50%. Overall, the obtained results demonstrate that laccase preferentially partitions to the most hydrophilic phase in ABS comprising ILs, both used as adjuvants or as phase-forming components, corresponding to the phase in which the IL is enriched. Furthermore, the IL chemical structure of the IL plays a significant role in the enzyme activity, where ILs with a higher number of hydroxyl groups seem to be relevant to improve the laccase activity.publishe

A proposed adaptation of the European Foundation for Quality Management Excellence Model to physical activity programmes for the elderly - development of a quality self-assessment tool using a modified Delphi process

<p>Abstract</p> <p>Background</p> <p>There has been a growing concern in designing physical activity (PA) programmes for elderly people, since evidence suggests that such health promotion interventions may reduce the deleterious effects of the ageing process. Complete programme evaluations are a necessary prerequisite to continuous quality improvements. Being able to refine, adapt and create tools that are suited to the realities and contexts of PA programmes for the elderly in order to support its continuous improvement is, therefore, crucial. Thus, the aim of this study was to develop a self-assessment tool for PA programmes for the elderly.</p> <p>Methods</p> <p>A 3-round Delphi process was conducted via the Internet with 43 national experts in PA for the elderly, management and delivery of PA programmes for the elderly, sports management, quality management and gerontology, asking experts to identify the propositions that they considered relevant for inclusion in the self-assessment tool. Experts reviewed a list of proposed statements, based on the criteria and sub-criteria from the European Foundation for Quality Management Excellence Model (EFQM) and PA guidelines for older adults and rated each proposition from 1 to 8 (disagree to agree) and modified and/or added propositions. Propositions receiving either bottom or top scores of greater than 70% were considered to have achieved consensus to drop or retain, respectively.</p> <p>Results</p> <p>In round 1, of the 196 originally-proposed statements (best practice principles), the experts modified 41, added 1 and achieved consensus on 93. In round 2, a total of 104 propositions were presented, of which experts modified 39 and achieved consensus on 53. In the last round, of 51 proposed statements, the experts achieved consensus on 19. After 3 rounds of rating, experts had not achieved consensus on 32 propositions. The resulting tool consisted of 165 statements that assess nine management areas involved in the development of PA programmes for the elderly.</p> <p>Conclusion</p> <p>Based on experts' opinions, a self-assessment tool was found in order to access quality of PA programmes for the elderly. Information obtained with evaluations would be useful to organizations seeking to improve their services, customer satisfaction and, consequently, adherence to PA programmes, targeting the ageing population.</p

Antimicrobial photodynamic therapy alone or in combination with antibiotic local administration against biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis

Antimicrobial photodynamic therapy (aPDT) kills several planktonic pathogens. However, the susceptibility of biofilm-derived anaerobic bacteria to aPDT is poorly characterized. Here, we evaluated the effect of Photodithazine (PDZ)-mediated aPDT on Fusobacterium nucleatum and Porphyromonas gingivalis biofilms. In addition, aPDT was tested with metronidazole (MTZ) to explore the potential antimicrobial effect of the treatment. The minimum inhibitory concentration (MIC) of MTZ was defined for each bacterial species. Single-species biofilms of each species were grown on polystyrene plates under anaerobic conditions for five days. aPDT was performed by applying PDZ at concentrations of 50, 75 and 100 mg/L, followed by exposure to 50 J/cm2 LED light (660 nm) with or without MTZ. aPDT exhibited a significant reduction in bacterial viability at a PDZ concentration of 100 mg/L, with 1.12 log10 and 2.66 log10 reductions for F. nucleatum and P. gingivalis in biofilms, respectively. However, the antimicrobial effect against F. nucleatum was achieved only when aPDT was combined with MTZ at 100× MIC. Regarding P. gingivalis, the combination of PDZ-mediated aPDT at 100 mg/L with MTZ 100× MIC resulted in a 5 log10 reduction in the bacterial population. The potential antimicrobial effects of aPDT in combination with MTZ for both single pathogenic biofilms were confirmed by live/dead staining. These results suggest that localized antibiotic administration may be an adjuvant to aPDT to control F. nucleatum and P. gingivalis biofilms

Detection of Chlamydial {DNA} from Mediterranean Loggerhead Sea Turtles in Southern Italy

Chlamydiae are obligate intracellular bacteria that include pathogens of human and veterinary importance. Several reptiles were reported to host chlamydial agents, but pathogenicity in these animals still needs clarification. Given that only one report of chlamydiosis was described in sea turtles, and that chlamydiae might also be detected in hosts without clinical signs, the current study examined asymptomatic Mediterranean loggerhead sea turtles for the presence of chlamydial DNA. Twenty loggerhead sea turtles, rehabilitated at the Marine Turtle Research Centre (Portici, Italy), were examined collecting ocular-conjunctival, oropharyngeal and nasal swabs. Samples were processed through quantitative and conventional PCR analyses to identify Chlamydiales and Chlamydiaceae, with particular attention to C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis. Although it was not possible to determine the species of chlamydiae involved, the detection of chlamydial DNA from the collected samples suggests that these microorganisms might act as opportunistic pathogens, and underlines the role of sea turtles as potential carriers. This study highlights the presence of chlamydial agents in sea turtles, and encourages further research to fully characterize these microorganisms, in order to improve the management of the health and conservation of these endangered species, and prevent potential zoonotic implications

Characterization of the canine CD20 as a therapeutic target for comparative passive immunotherapy

Research Areas: Science & TechnologyAnti-CD20 therapies have revolutionized the treatment of B-cell malignancies. Despite these advances, relapsed and refractory disease remains a major treatment challenge. The optimization of CD20-targeted immunotherapies is considered a promising strategy to improve current therapies. However, research has been limited by the scarcity of preclinical models that recapitulate the complex interaction between the immune system and cancers. The addition of the canine lymphoma (cNHL) model in the development of anti-CD20 therapies may provide a clinically relevant approach for the translation of improved immunotherapies. Still, an anti-CD20 therapy for cNHL has not been established stressing the need of a comprehensive target characterization. Herein, we performed an in-depth characterization on canine CD20 mRNA transcript and protein expression in a cNHL biobank and demonstrated a canine CD20 overexpression in B-cell lymphoma samples. Moreover, CD20 gene sequencing analysis identifed six amino acid diferences in patient samples (C77Y, L147F, I159M, L198V, A201T and G273E). Finally, we reported the use of a novel strategy for the generation of anti-CD20 mAbs, with human and canine cross-reactivity, by exploring our rabbit derived singledomain antibody platform. Overall, these results support the rationale of using CD20 as a target for veterinary settings and the development of novel therapeutics and immunodiagnostics.info:eu-repo/semantics/publishedVersio

Seagrass connectivity on the west coast of Africa supports the hypothesis of grazer-mediated seed dispersal

Population connectivity influences the distribution of genetic diversity and divergence along a species range, as the likelihood of extinction or differentiation increases in isolated populations. However, there is still poor understanding of the processes mediating interpopulation dispersal in marine species that are sessile and lack planktonic life stages. One such case is the seagrass species Halodule wrightii, which produces basal seeds, although detached plants can drift. Along the tropical western coast of Africa, this species occurs in distant discontinuous habitats, raising the question of how interpopulation dispersal is mediated. The species is a key source of ecosystem functions including feeding large migratory grazers. This study aims to infer whether genetic differentiation of the seagrass H. wrightii along the western coast of Africa supports a hypothesis of predominant transportation of rafting seagrass by ocean currents, versus the hypothesis of biotic vectors of dispersal. Additional hypotheses were addressed concerning range-wide clonality and genetic diversity, assessed with microsatellite markers on populations of the west coast of Africa from Mauritania to Angola. Population genetic diversity and structure were compared with predictions from biophysical models of dispersal by oceanographic currents. The genetic data revealed low divergence among most populations, in strong contrast with predictions of very low probability of connectivity mediated by currents along the western African coastline. Moderate to high genotypic diversity showed important seed recruitment, but genetic and genotypic diversities were lower at range edges. Populations north and south of the equator were differentiated, and remarkably, so were neighboring equatorial populations despite their proximity. These results reveal independent sources of colonization of meadows in these islands, which are major habitat for migratory grazing green turtles, also supporting the hypothesis of biotically mediated seed transport. The importance of seagrass for conservation of endangered macrofauna has been widely reported; here we report evidence supporting the reciprocal role, that macrofauna protection can also plays a role in long-term survival and reproductive success of seagrass.Fundação para a Ciência e Tecnologia - FCTinfo:eu-repo/semantics/publishedVersio

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.Peer Reviewe