Protein-biomembrane interactions as therapeutic targets

Biological membranes are dynamic structures essential for several cellular phenomena. The scope of the work of the Institute of Molecular Medicine (IMM) Biomembranes Unit is the study of biochemical and biophysical processes occurring at the membrane level on human cells and on their viral and bacterial pathogens. On the viral context, we are primarily interested on HIV and dengue virus, and particularly on the two steps of their life cycle involving their interaction with host cell membranes: the viral entry into target cells and the assembly of new viral particles. A special focus will be given to the study of the role of biological membranes on the mechanism of action of the HIV entry (membrane fusion) inhibitors enfuvirtide and T-1249. We are also involved in assessing the molecular basis of the activity of microbicides, such as rBPI21, that bind to specific components of bacterial membranes. Additionally, our line of work on the binding of fibrinogen to erythrocytes, and its relevance as a cardiovascular risk factor will be presented. An approach to the latter problem by single-molecule force spectroscopy, using an atomic force microscope (AFM), allowed the molecular recognition, characterization and partial identification of the human erythrocyte receptor for fibrinogen.These lines of work were supported by Fundação para a Ciência e a Tecnologia – Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal; projects PTDC/SAU-OSM/73449/2006 and PTDC/ QUI-BIQ/104787/2008), by the FP7-PEOPLE IRSES (International Research Staff Exchange Scheme) project MEMPEPACROSS (EU), and by Fundação Calouste Gulbenkian (Portugal). MMD and PMM also thank FCT-MCTES for the PhD fellowships SFRH/BD/41750/2007 and SFRH/BD/42205/2007, respectively

Polar lipids of commercial Ulva spp. of different origins: profiling and relevance for seaweed valorization

Macroalgae of the genus Ulva have long been used as human food. Local environmental conditions, among other factors, can have an impact on their nutrient and phytochemical composition, as well as on the value of the seaweed for food and non-food applications. This study is the first to initiate a comparison between commercial Ulva spp. from different European origins, France (FR, wild-harvested Ulva spp.), and Portugal (PT, farm-raised Ulva rigida), in terms of proximate composition, esterified fatty acids (FA), and polar lipids. The ash content was higher in PT samples, while FR samples had higher levels of proteins, lipids, and carbohydrates and other compounds. The profile of esterified FA, as well as FA-containing polar lipids at the class and species levels were also significantly different. The FR samples showed about three-fold higher amount of n-3 polyunsaturated FA, while PT samples showed two-fold higher content of monounsaturated FA. Quantification of glycolipids and phospholipids revealed, respectively, two-fold and three-fold higher levels in PT samples. Despite the differences found, the polar lipids identified in both batches included some lipid species with recognized bioactivity, valuing Ulva biomass with functional properties, increasing their added value, and promoting new applications, namely in nutraceutical and food markets.UIDB/50011/2020+UIDP/50011/2020, UID/QUI/00062/2019, UIDB/50006/2020, UIDB/50017/2020+UIDP/50017/2020, LISBOA-01-0145-FEDER-402-022125, POCI-01-0145-FEDER-030962, BPD/UI51/5041/2017, BPD/UI51/5042/2018; EC/H2020/727892/EUinfo:eu-repo/semantics/publishedVersio

Intrinsically bioactive cryogels based on platelet lysate nanocomposites for hemostasis applications.

The currently used hemostatic agents are highly effective in stopping hemorrhages but have a limited role in the modulation of the wound-healing environment. Herein, we propose an intrinsically bioactive hemostatic cryogel based on platelet lysate (PL) and aldehyde-functionalized cellulose nanocrystals (a-CNCs). PL has attracted great attention as an inexpensive milieu of therapeutically relevant proteins; however, its application as a hemostatic agent exhibits serious constraints (e.g., structural integrity and short shelf-life). The incorporation of a-CNCs reinforced the low-strength PL matrix by covalent cross-linking its amine groups that exhibit an elastic interconnected porous network after full cryogelation. Upon blood immersion, the PL-CNC cryogels absorbed higher volumes of blood at a faster rate than commercial hemostatic porcine gelatin sponges. Simultaneously, the cryogels released biomolecules that increased stem cell proliferation, metabolic activity, and migration as well as downregulated the expression of markers of the fibrinolytic process. In an in vivo liver defect model, PL-CNC cryogels showed similar hemostatic performance in comparison with gelatin sponges and normal material-induced tissue response upon subcutaneous implantation. Overall, owing to their structure and bioactive composition, the proposed PL-CNC cryogels provide an alternative off-the-shelf hemostatic and antibacterial biomaterial with the potential to deliver therapeutically relevant proteins in situ.The authors thank Hospital da Prelada (Porto, Portugal) for providing adipose tissue samples and Instituto Portugues do Sangue e Transplantacio-IPST (Portugal) (Porto, Portugal) for providing platelet concentrates. The authors would like to thank Alain Morais and Isabel Pires for their support in the in vivo procedure and histological evaluation, respectively. The authors would like to thank the anonymous reviewers for all useful and helpful comments on our manuscript. This work was supported by the European Research Council grant agreement no. 772817, FCT/MCTES (Fundacio para a Ciencia e a Tecnologia/Ministerio da Ciencia, Tecnologia, e Ensino Superior) and the Fundo Social Europeu atraves do Programa Operacional do Capital Humano (FSE/POCH) in the framework of Ph.D. grant PD/59/2013-PD/BD/113807/2015 (BBM) and CEECIND/01375/2017 (MGF), Norwegian Research Council for project no. 287953

Supercritical fluid extraction of Eucalyptus globulus bark: a promising approach for triterpenoid production

Eucalyptus bark contains significant amounts of triterpenoids with demonstrated bioactivity, namely triterpenic acids and their acetyl derivatives (ursolic, betulinic, oleanolic, betulonic, 3-acetylursolic, and 3-acetyloleanolic acids). In this work, the supercritical fluid extraction (SFE) of Eucalyptus globulus deciduous bark was carried out with pure and modified carbon dioxide to recover this fraction, and the results were compared with those obtained by Soxhlet extraction with dichloromethane. The effects of pressure (100-200 bar), co-solvent (ethanol) content (0, 5 and 8% wt), and multistep operation were studied in order to evaluate the applicability of SFE for their selective and efficient production. The individual extraction curves of the main families of compounds were measured, and the extracts analyzed by GC-MS. Results pointed out the influence of pressure and the important role played by the co-solvent. Ethanol can be used with advantage, since its effect is more important than increasing pressure by several tens of bar. At 160 bar and 40 degrees C, the introduction of 8% (wt) of ethanol greatly improves the yield of triterpenoids more than threefold

Caracterización de la actividad enzimática del jugo gástrico de pulpo, Octopus vulgaris y de choco, Sepia officinalis a distintos pH. Digestibilidad in vitro de distintas dietas, con jugo gástrico de pulpo

Intensive culture of marine species has been increasing in many countries, such as Japan, Norway, Spain, France, and Greece. Recent research to improve the quality and quantity of aquatic animals production by understanding the process of feeding (ingestion, digestion, and absorption), with emphasis on the digestive enzymatic capabilities is important in order to understand metabolic processes and reduce costs and time on the development of such diets. The impetus to increase knowledge about suitable inert diets to partially or totally replace live feed would reduce this expensive part of the operation, particularly in new potential species, such as the octopus, Octopus vulgaris, and the cuttlefi sh, Sepia offi cinalis, which are two of the most promising cephalopods for large-scale culture. This work is divided into two parts: (1) Enzymatic characterization, by determination of protease activity of the gastric juice at pH ranges from 2 to 12, since this is the major responsible of digestion processes, and (2) In vitro evaluation of several prepared diets, as well as a selection of several natural animal protein sources, by means of a pH-Stat system The results of Part 1 showed that the digestive enzyme activity in total proteases from the gastric juice was higher at pH 7 and 9, therefore being a more alkaline digestion for these species. Total proteases activity was higher at pH 8, while the tripsin activity was higher at pH 7, for both species. For Part 2, the natural diets (natural or liophylized squid, blue whiting and mussel) promoted the highest digestibility, with the highest value being attained with liophylized squid (49%). From the prepared diets, based on raw blue whiting paste, those diets agglutinated with gelatine delivered the highest digestibility. The use of soy protein, as well as heating gelatine as agglutinants, promoted poor digestibility

Prototype of an affordable pressure-controlled emergency mechanical ventilator for COVID-19

We present a viable prototype of a simple mechanical ventilator intended as a last resort to ventilate COVID-19 patients. The prototype implements the pressure-controlled continuous mandatory ventilation mode (PC-CMV) with settable breathing rates, inspiration/expiration time ratios and FiO2 modulation. Although safe, the design aims to minimize the use of technical components and those used are common in industry, so its construction may be possible in times of logistical shortage or disruption or in areas with reduced access to technical materials and at a moderate cost, affordable to lower income countries. Most of the device can be manufactured by modest technical means and construction plans are provided.Comment: This version differs from version 2 in that it includes toxicological and bio-safety tests and updated electronic

Frequência e perfil de suscetibilidade aos carbapenêmicos de bastonetes Gram-negativos não fermentadores de glicose isolados de amostras clínicas entre 2007 e 2012

Introdução: Um dos grandes problemas nos serviços de saúde é a ocorrência de infecções relacionadas com assistência à saúde (IRAS) por microrganismos resistentes a vários antimicrobianos. Objetivos: Descrever a frequência e o perfil de suscetibilidade de Pseudomonas aeruginosa e Acinetobacter baumannii aos carbapenêmicos no hospital da Fundação Santa Casa de Franca, São Paulo, Brasil. Métodos: Retrospectivamente, a suscetibilidade de P. aeruginosa e A. baumannii aos carbapenêmicos foi analisada em 304 isolados clínicos entre 2007 e 2012, a partir de um banco de dados do setor de microbiologia do laboratório clínico do hospital da Fundação Santa Casa de Franca, São Paulo, Brasil. Resultados: Das cepas isoladas e identificadas, 236 (5,3%) P. aeruginosa eram suscetíveis a imipenem (2007 - 69,6% a 2012 - 41,7%) e meropenem (2007 - 63,3% a 2012 - 25%). Além disso, todos os 68 (1,7%) isolados de A. baumannii eram suscetíveis aos dois antibióticos. Conclusão: Não foi identificada resistência de A. baumannii aos carbapenêmicos, no entanto houve diminuição da suscetibilidade aos carbapenêmicos no decorrer dos anos para P. aeruginosa.Introduction: One of the major problems in health services is the occurrence of healthcare-associated infections (HAIs) by microorganisms resistant to various antimicrobials. Objectives: To describe the frequency and susceptibility profile of Pseudomonas aeruginosa and Acinetobacter baumannii to carbapenems in the hospital from Fundação Santa Casa de Franca, São Paulo, Brazil. Methods: The susceptibility of P. aeruginosa and A. baumannii to carbapenems from 304 clinical isolates between 2007 and 2012 was retrospectively analyzed from a microbiology database at the clinical laboratory of the hospital of Fundação Santa Casa de Franca, São Paulo, Brazil. Results: From isolated and identified strains, 236 (5.3%) P. aeruginosa were susceptible to imipenem (2007 - 69.6% to 2012 - 41.7%) and meropenem (2007 - 63.3% to 2012 - 25%). In addition, all 68 (1.7%) A. baumannii isolates were susceptible to both antibiotics. Conclusion: A. baumannii resistance to carbapenems was not identified; however, there was a decrease in susceptibility to carbapenems over the years for P. aeruginosa

Selection of Saccharomyces cerevisiae strains for efficient very high gravity bio-ethanol fermentation processes

An optimized very high gravity (VHG) glucose medium supplemented with low cost nutrient sources was used to evaluate bio-ethanol production by 11 Saccharomyces cerevisiae strains. The industrial strains PE-2 and CA1185 exhibited the best overall fermentation performance, producing an ethanol titre of 19.2% (v/v) corresponding to a batch productivity of 2.5 g l-1 h-1, while the best laboratory strain (CEN.PK 113-7D) produced 17.5% (v/v) ethanol with a productivity of 1.7 g l-1 h-1. The results presented here emphasize the biodiversity found within S. cerevisiae species and that naturally adapted strains, such as PE-2 and CA1185, are likely to play a key role in facilitating the transition from laboratory technological breakthroughs to industrialscale bio-ethanol fermentations.Fundação para a Ciência e a Tecnologia (FCT) - PTDC/BIO/66151/2006, SFRH/ BD/64776/2009, SFRH/BPD/44328/ 200

Fermentation of deproteinized cheese whey powder solutions to ethanol by engineered Saccharomyces cerevisiae : effect of supplementation with corn steep liquor and repeated-batch operation with biomass recycling by flocculation

The lactose in cheese whey is an interesting substrate for the production of bulk commodities such as bio-ethanol, due to the large amounts of whey surplus generated globally. In this work, we studied the performance of a recombinant Saccharomyces cerevisiae strain expressing the lactose permease and intracellular ß-galactosidase from Kluyveromyces lactis in fermentations of deproteinized concentrated cheese whey powder solutions. Supplementation with 10 g/l of corn steep liquor significantly enhanced whey fermentation, resulting in the production of 7.4% (v/v) ethanol from 150 g/l initial lactose in shake-flask fermentations, with a corresponding productivity of 1.2 g/l/h. The flocculation capacity of the yeast strain enabled stable operation of a repeated-batch process in a 5.5-l air-lift bioreactor, with simple biomass recycling by sedimentation of the yeast flocs. During five consecutive batches, the average ethanol productivity was 0.65 g/l/h and ethanol accumulated up to 8% (v/v) with lactose-toethanol conversion yields over 80% of theoretical. Yeast viability (>97%) and plasmid retention (>84%) remained high throughout the operation, demonstrating the stability and robustness of the strain. In addition, the easy and inexpensive recycle of the yeast biomass for repeated utilization makes this process economically attractive for industrial implementation.Fundação para a Ciência e a Tecnologia (FCT)LACTOGAL-Produtos Alimentares S.A.Companhia Portuguesa de Amidos, S.A