Flow behaviour in microchannels of an innovative blood analogue fluid based on giant unilamellar vesicles

This work focused on the development of an innovative blood analogue, containing giant unilamellar vesicles (GUVs), to mimic the flow behaviour of red blood cells (RBCs). The rheological characterization of different blood analogue solutions was performed in a stress controlled rheometer and the results have shown a good agreement when compared with a sample containing 5% of RBCs.This work was supported by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UID/FIS/04650/2013. The authors also acknowledge the financial support provided by FCT through the project PTDC/QEQ-FTT/4287/2014.info:eu-repo/semantics/publishedVersio

Understanding dengue virus capsid protein interaction with key biological targets

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Supplementary information accompanies this paper at http://www.nature.com/srepDengue virus (DENV) causes over 500,000 hospitalizations and 20,000 deaths worldwide every year. Dengue epidemics now reach temperate regions due to globalization of trade and travel and climate changes. Currently, there are no successful therapeutic or preventive approaches. We previously developed a peptide drug lead, pep14-23, that inhibits the biologically relevant interaction of DENV capsid (C) protein with lipid droplets (LDs). Surprisingly, pep14-23 also inhibits DENV C interaction with very low-density lipoproteins (VLDL). We thus investigated the similarity between the proposed DENV C molecular targets in LDs and VLDL, respectively, the proteins perilipin 3 (PLIN3) and apolipoprotein E (APOE). APOE N-terminal and PLIN3 C-terminal regions are remarkably similar, namely APOE α -helix 4 (APOEα 4) and PLIN3 α -helix 5 (PLIN3α 5) sequences, which are also highly superimposable structurally. Interestingly, APOE α -helical N-terminal sequence and structure superimposes with DENV C α -helices α 1 and α 2. Moreover, the DENV C hydrophobic cleft can accommodate the structurally analogous APOEα 4 and PLIN3α 5 helical regions. Mirroring DENV C-LDs interaction (previously shown experimentally to require PLIN3), we experimentally demonstrated that DENV C-VLDL interaction requires APOE. Thus, the results fit well with previous data and suggest future drug development strategies targeting the above mentioned α –helical structures.We acknowledge the support of Fundação para a Ciência e Tecnologia – Ministério da Educação e Ciência (FCT-MEC, Portugal) project PTDC/SAU-ENB/117013/2010, and Calouste Gulbenkian Foundation (Portugal). AFF and ICM also acknowledge FCT-MEC fellowship SFRH/BD/77609/2011 and Investigador FCT Program research contract IF/00772/2013, respectively

Microfluidic deformability study of an innovative blood analogue fluid based on giant unilamellar vesicles

Blood analogues have long been a topic of interest in biofluid mechanics due to the safety and ethical issues involved in the collection and handling of blood samples. Although the current blood analogue fluids can adequately mimic the rheological properties of blood from a macroscopic point of view, at the microscopic level blood analogues need further development and improvement. In this work, an innovative blood analogue containing giant unilamellar vesicles (GUVs) was developed to mimic the flow behavior of red blood cells (RBCs). A natural lipid mixture, soybean lecithin, was used for the GUVs preparation, and three different lipid concentrations were tested (1 × 10−3 M, 2 × 10−3 M and 4 × 10−3 M). GUV solutions were prepared by thin film hydration with a buffer, followed by extrusion. It was found that GUVs present diameters between 5 and 7 µm which are close to the size of human RBCs. Experimental flow studies of three different GUV solutions were performed in a hyperbolic-shaped microchannel in order to measure the GUVs deformability when subjected to a homogeneous extensional flow. The result of the deformation index (DI) of the GUVs was about 0.5, which is in good agreement with the human RBC’s DI. Hence, the GUVs developed in this study are a promising way to mimic the mechanical properties of the RBCs and to further develop particulate blood analogues with flow properties closer to those of real blood.COMPETE2020, NORTE2020, PORTUGAL2020, FEDER; FCT Project PTDC/QEQ-FTT/4287/2014 (POCI-01-0145-FEDER-016861); FCT Project PTDC/EMD-EMD/29394/2017 (NORTE-01-0145-FEDER-029394); FCT Project PTDC/EME-SIS/30171/2017 (NORTE-01-0145-FEDER-000032); FCT Project PTDC/QUI-QFI/28020/2017 (POCI-01-0145-FEDER-028020); SFRH/BD/99696/2014 PhD Grant;info:eu-repo/semantics/publishedVersio

Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins

<p>Abstract</p> <p>Background</p> <p>In recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in <it>meso</it>-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (<it>Enterococcus faecalis</it>) and of a Gram (-) bacterium (<it>Escherichia coli</it>). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms.</p> <p>Results</p> <p>Bacterial suspension (10⁷CFU mL^-1) treated with different photosensitizers concentrations (0.5, 1.0 and 5.0 μM) were exposed to white light (40 W m^-2) for a total light dose of 64.8 J cm^-2. The most effective photosensitizers against both bacterial strains were the Tri-Py⁺-Me-PF and Tri-Py⁺-Me-CO₂Me at 5.0 μM with a light fluence of 64.8 J cm^-2, leading to > 7.0 log (> 99,999%) of photoinactivation. The tetracationic porphyrin also proved to be a good photosensitizer against both bacterial strains. Both di-cationic and the monocationic porphyrins were the least effective ones.</p> <p>Conclusion</p> <p>The number of positive charges, the charge distribution in the porphyrins' structure and the <it>meso</it>-substituent groups seem to have different effects on the photoinactivation of both bacteria. As the Tri-Py⁺-Me-PF porphyrin provides the highest log reduction using lower light doses, this photosensitizer can efficiently photoinactivate a large spectrum of environmental bacteria. The complete inactivation of both bacterial strains with low light fluence (40 W m^-2) means that the photodynamic approach can be applied to wastewater treatment under natural light conditions which makes this technology cheap and feasible in terms of the light source.</p

In utero tracheal occlusion: surgical approach associated to steroid treatment increase VEGF (Vascular Endothelial Growth Factor) receptors in lungs of fetal rats

Background/Purpose: Congenital diaphragmatic hernia (CDH) presents with hypoplastic lungs and usually leads to pulmonary hypertension and high neonatal mortality. Fetal tracheal occlusion (TO) and prenatal corticotherapy are alternatives to accelerate fetal pulmonary growth and decrease hypoplasia in CDH. VEGF (Vascular Endothelial Growth Factor) production and surfactant production by type II pneumocytes are related with pulmonary maturityand are altered in CDH, but little has been described about VEGF receptors. Our objective was to quantify the receptors of VEGF (VEGFR) and type II pneumocytes, verifying the effects of TO and corticotherapy on normal lungs of fetal rats. Methods: Six groups of 12 Spreague-Dawley rat fetuses (gestation=22 days) were compared:TO, S (sham), C (control), TO+Dex, S+Dex and C+Dex. On the 18.5 gestational day, TO was performed with and without corticotherapy, using dexamethasone. On the 21.5 gestational daybody and lung weights were measured. Immunohistochemistry was carried out for VEGFR-1(Flt-1) and VEGFR-2 (Flk-1), as well as anti-SP-A, microscopic analysis and quantification of immune system marking. Results: Body weight decreased in Sham and lung weight increased in TO and TO+Dex (p&lt;0.05) groups. VEGFR-1 and VEGFR-2 increased in TO and in TO+Dex (p&lt;0.05). Type II pneumocytes (SP-A) decreased both in the TO and in TO+Dex groups in comparison with the Control groupin absolute value (p&lt;0.05), with no differences in relative value to total cell count. Conclusion: TO in association with the use of corticosteroids increased VEGFR-1 and VEGFR-2, while the quantity of type II pneumocytes (SP-A) decreased in relation to the area considered, but not in relation to the total of pulmonary cells.Introdução: A hérnia diafragmática congênita (HDC) causa hipoplasia e hipertensão pulmonar e em geral leva a alta morbidade e mortalidade neonatal. Traqueo-oclusão fetal (TO) e corticoterapia pré-natal são alternativas para acelerar o crescimento pulmonar fetal e diminuir a hipoplasia na HDC. A produção de VEGF (Vascular Endothelial Growth Factor) está relacionada com a maturidade pulmonar e sofre alterações na HDC ainda não elucidadas. Materiais e métodos: Seis grupos de 12 fetos de ratos Spreague-Dawley foram comparados: TO, Sham, Controle, TO+Dex, Sham+Dex e Controle+Dex. No dia 18,5º foi realizada TO com e sem corticoterapia utilizando dexametasona. No 21,5º dia gestacional os pesos corporal e pulmonar foram mensurados. Realizou-se imunohistoquímica para VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), seguida de morfometria. Resultados: O VEGFR-1 estava aumentado no TO (p&lt;0.05)e na TO+Dex (p&lt;0.05). O VEGFR-2 teve aumento significativo quando comparamos TO e TO+Dex com o controle (p&lt;0,05). Conclusão: A TO associada ao uso de corticoesteróide aumentou o número de VEGFR-1 e VEGFR-2 em pulmões de fetos de ratas

The Bom Santo Cave (Lisbon, Portugal): catchment, diet, and patterns of mobility of a Middle Neolithic population

The study of the Bom Santo Cave (central Portugal), a Neolithic cemetery, indicates a complex social, palaeoeconomic, and population scenario. With isotope, aDNA, and provenance, analyses of raw materials coupled with stylistic variability of material culture items and palaeogeographical data, light is shed on the territory and social organization of a population dated to 3800-3400 cal BC, i.e. the Middle Neolithic. Results indicate an itinerant farming, segmentary society, where exogamic practices were the norm. Its lifeway may be that of the earliest megalithic builders of the region, but further research is needed to correctly evaluate the degree of this community's participation in such a phenomenon

A matter of life and death: the Middle Neolithic population from Bom Santo Cave (Lisbon, Portugal)

The study of the Bom Santo Cave (central Portugal), a Neolithic cemetery, indicated a complex social, palaeoeconomic and population scenario. With isotope, aDNA and provenience analyses of raw materials coupled with stylistic variability of material culture items and palaeogeographical data light is shed on the territory and social organization of a population dated to 3800–3400 cal BC, i.e. the middle phase of the period. Results indicate an itinerant farming, segmentary society, where exogamic practices were the norm and patrilocality probably predominated. Its lifeway may be that of the earliest megalithic builders of the region, but further research is needed to correctly evaluate the degree of participation in such phenomenon

Antimicrobial Photodynamic Therapy: Study of Bacterial Recovery Viability and Potential Development of Resistance after Treatment

Antimicrobial photodynamic therapy (aPDT) has emerged in the clinical field as a potential alternative to antibiotics to treat microbial infections. No cases of microbial viability recovery or any resistance mechanisms against it are yet known. 5,10,15-tris(1-Methylpyridinium-4-yl)-20-(pentafluorophenyl)-porphyrin triiodide (Tri-Py+-Me-PF) was used as photosensitizer. Vibrio fischeri and recombinant Escherichia coli were the studied bacteria. To determine the bacterial recovery after treatment, Tri-Py+-Me-PF (5.0 μM) was added to bacterial suspensions and the samples were irradiated with white light (40 W m−2) for 270 minutes. Then, the samples were protected from light, aliquots collected at different intervals and the bioluminescence measured. To assess the development of resistance after treatment, bacterial suspensions were exposed to white light (25 minutes), in presence of 5.0 μM of Tri-Py+-Me-PF (99.99% of inactivation) and plated. After the first irradiation period, surviving colonies were collected from the plate and resuspended in PBS. Then, an identical protocol was used and repeated ten times for each bacterium. The results suggest that aPDT using Tri-Py+-Me-PF represents a promising approach to efficiently destroy bacteria since after a single treatment these microorganisms do not recover their viability and after ten generations of partially photosensitized cells neither of the bacteria develop resistance to the photodynamic process