Influence of saliva and mucin on the adhesion of Candida oral clinical isolates

Objectives: This research work intends to clarify the role of artificial saliva, in particularly the role of mucin, a salivary protein, on the surface properties and adhesion ability of Candida spp. oral clinical isolates to abiotic surfaces. Methods: Four oral clinical isolates of Candida spp. were used: two Candida albicans strains (AC; AM) and two Candida parapsilosis strains (AD; AM2). The strains were isolated from patients using oral prosthesis. The microorganisms were cultured in the absence or presence of mucin and artificial saliva, and their adhesion to an abiotic surface (coated with mucin and artificial saliva) was evaluated. Results: The presence of mucin per se onto the abiotic surface decreased the adhesion of all strains, although the combination of mucin with artificial saliva had reduced this effect. No direct correlation between adhesion and the surface free energies of adhesion of the microorganisms was found. Significance: Candida spp. were human commensal microorganisms that became pathogenic when the host immune defenses were compromised. Medical devices were colonized by Candida spp. particularly, oral prostheses, which might lead to the degradation of the prostheses and systemic infections. The salivary secretions that constantly cover the oral cavity influenced Candida spp. adhesion process. Therefore, it was important to understand the interactions between Candida spp., salivary proteins and the characteristic of oral prosthesis when developing materials for oral prostheses.The authors thank 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 would also like to thank the Fundação para a Ciência e Tecnologia for the Strategic Project Pest-OE/EQB/LA0023/2013 and Fundação para a Ciência e Tecnologia (FCT) for Ana Oliveira PhD Grant (SFRH/BD/68588/2010) and Catarina L. Seabra PhD Grant (SFRH/BD/89001/2012). The authors would also like to acknowledge Professora Rosário Oliveira, which is no longer with us, for her exceptional contribution and dedication to this work

Development of dioctadecyldimethylammonium bromide/monoolein liposomes for gene delivery

The artificial introduction of nucleic acids (NA) into mammalian cells (transfection) has become, in recent years, a well-established procedure in basic and applied research, which allowed the study of gene function and regulation. The advances in this area have made possible the use of these methods for gene-based medicines, which constitute alternative therapeutic approaches. One of the most prominent methods is lipofection that uses cationic liposome/NA complexes (a.k.a. lipoplexes) for the complexation, transport and release of therapeutic sequences into target cells. Although yielding lower transfection efficiencies compared with viral gene delivery, lipofection vectors are much safer for medical applications because no significant mutational or toxicological risk exist. Dioctadecyldimethylammonium Bromide (DODAB)/Monoolein (MO) liposomes have recently been described as a new promising alternative to common transfection reagents, due to the pioneering application of MO as helper lipid in lipoplex formulations. In this chapter, we will review the effect of MO on the physicochemical properties of DODAB/MO liposomes and pDNA/DODAB/MO lipoplexes. How lipoplex properties may affect the interaction with different extracellular components and their cell uptake and trafficking will be discussed. The importance of lipoplex biocompatibility towards efficient gene therapy will also be approached presenting pDNA/DODAB/MO system as a lipoplex model, supporting the use of MO as new helper lipid in lipofection.FCTCOMPETEThis work was supported by FCT research project PTDC/QUI/69795/2006, which is cofunded by the program COMPETE from QREN with co-participation from the European Community fund FEDER; CFUM [PEst-C/FIS/UI0607/2011]; CBMA [Pest C/BIA/UI4050/2011]; J.P.N. Silva holds a PhD Grant (SFRH/BD/46968/2008); A. C.N. Oliveira holds a PhD grant (SFRH/BD/68588/2010)

Monoolein-based nanocarriers for enhanced folate receptor-mediated RNA delivery to cancer cells

We report the development and characterization of a novel nanometric system for specific delivery of therapeutic siRNA for cancer treatment. This vector is based on a binary mixture of the cationic surfactant dioctadecyldimethylammonium chloride (DODAC) and the helper lipid monoolein (MO). These liposomes were previously validated by our research group as promising non-viral vectors for nucleic acid delivery. In this work, the DODAC:MO vesicles were for the first time functionalized with polyethylene glycol and PEG-folate conjugates to achieve both maximal stability in biological fluids and increase selectivity toward folate receptor α expressing cells. The produced DODAC:MO:PEG liposomes were highly effective in RNA complexation (close to 100%), and the resulting lipoplexes also demonstrated high stability in conditions simulating their administration by intravenous injection (physiological pH, high NaCl, heparin and fetal bovine serum concentrations). In addition, cell uptake of the PEG-folate-coated lipoplexes was significantly greater in folate receptor α positive breast cancer cells (39% for 25 µg/mL of lipid and 31% for 40 µg/mL) when compared with folate receptor α negative cells (31% for 25 µg/mL of lipid and 23% for 40 µg/mL) and to systems without PEG-folate (≈13% to 16% for all tested conditions), supporting their selectivity towards the receptor. Overall, the results support these systems as appealing vectors for selective delivery of siRNA to cancer cells by folate receptor α-mediated internalization, aiming at future therapeutic applications of interest

A novel approach for the production of DODAB:MO lipoplexes : the influence of temperature on the physicochemical characteristics and cell transfection efficiency

Cationic liposomes/ DNA complexes (lipoplexes) have been widely used as nano-carriers for animal cell transfection, with the neutral lipid (helper) playing a determinant role for the efficiency of this process due to the formation of non-lammellar intermediates that are akin to membrane fusion process [1]. We have developed a novel formulation containing the cationic agent dioctadecyldimethylammonium bromide (DODAB) and 1-monooleoyl-rac-glycerol (MO) as helper lipid [2]. In previous studies, we have demonstrated a strong dependence of the DNA complexation rate with several structural parameters such as the monoolein content or the DNA/cationic lipid charge ratio (+/-) [3]. The preparation method itself influences the structural properties of the lipoplexes formed as well as their lipofection capacity. This study addressed the effect of preparation temperature (25ºC or 50ºC) on DODAB:MO lipoplexes (molar ratios 4:1 and 2:1) physicochemical properties, as well as on their cell transfection efficiency (TE).Fundação para a Ciência e a Tecnologia (FCT) - PTDC/QUI/69795/2006, SFRH/BD/46968/200

Monoolein-based lipoplexes (DODAB/MO/DNA) as non-viral vector for transfection- from physicochemical characterization to biological application

Cationic liposomes/DNA (lipoplexes) have been widely used as non-viral vectors for transfection, the role of the neutral lipid in liposome formulation being determinant for the efficiency of this process [1,2]. In this work, we studied the potential of monoolein (MO) as helper lipid for cellular transfection. Lipoplexes composed of pDNA and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at different molar ratios (4:1, 2:1 and 1:1) were investigated, as well as different cationic lipid/DNA ratios. The physicochemical properties of the lipoplexes (size and charge), the formation of the lipoplexes, the effect of MO on pDNA condensation and the effect of heparin on percentage of pDNA release from the lipoplexes were also studied by Ethidium Bromide (EtBr) exclusion assays, Dynamic Light Scattering (DLS), Zeta Potential (æ) and electrophoresis. The cytotoxicity, transfection efficiency, as well as the intracellular localization of labeled DNA were evaluated on 293T cells. It was found that the presence of MO not only increases the efficiency of pDNA compactation, but also affects the physicochemical properties of lipoplexes, which could possibly interfere with lipoplex-cell interactions. The DODAB:MO (2:1) and (4:1) formulations were capable of efficiently mediate in vitro cell transfection. These results were consistent with fluorescence microscopy studies, which illustrated that lipoplexes were able to gain entry into the cytosol and deliver pDNA to the nucleus. Understanding the structure–activity relationship of MO based lipoplexes will give direction toward the development of safe and efficient gene delivery systems.Portuguese Foundation for Science and Technology (FCT) for financial support to Center of Physics and Center of Molecular & Environmental Biology and funding through projects PTDC/QUI/69795/2006 and SFRH/BD/46968/2009

Monoolein as helper lipid for non-viral transfection in mammals

Lipoplexes composed of pDNA and DODAB/MO at different molar ratios (4:1, 2:1 and 1:1) and different cationic lipid/DNA charge ratios were investigated. The physicochemical properties of the lipoplexes (size and charge), the pDNA complexation, and the effect of heparin on pDNA release, were studied by Dynamic Light Scattering, Zeta Potential, and Ethidium Bromide exclusion assays. The cytotoxicity, transfection efficiency and the intracellular localization of DNA were evaluated on 293T cells.The Portuguese Foundation for Science and Technology (FCT) for the financial support to the Center of Physics and Center of Molecular & Environmental Biology and funding through projects PTDC/QUI/69795/2006 and SFRH/BD/46968/2009 are acknowledged

Effects of preparation method on the physicochemical characteristics and cell transfection efficiency of non‐viral nanocarriers

The success of gene therapy is decisively dependent on the development of effective carrier systems, able to compact and thus protect the genetic material as to avoid both in vitro and in vivo barriers for transgene delivery [1]. Given the problems of safety encountered with viral vectors, non-viral carrier systems are a safer alternative for the therapeutic delivery of genes [2]. Cationic liposomes/DNA (lipoplexes) have been widely used as non-viral vectors for cell transfection, with the neutral lipid (helper) of the liposome formulation playing a determinant role for the efficiency of this process. The lipoplex preparation method itself influences the structural properties of the produced lipoplex, affecting its final lipofection capability [3, 4]. In this work, we have studied the potential for cell transfection of a new liposomal formulation containing monoolein (MO) as helper lipid, using various preparation methods. Lipoplexes composed of pSV-β-galactosidase and dioctadecyldimethylammonium bromide (DODAB)/1-monooleoyl-rac-glycerol (MO) at (4:1) molar ratio and at cationic lipid/DNA ratio of 4.0 were tested. Ethidium Bromide (EtBr) exclusion assays, Dynamic Light Scattering (DLS), and Zeta Potential (ζ) were used to study plasmidic DNA complexation and physicochemical properties of the formed lipoplexes (their size and electrical charge). Transfection efficiency was also evaluated on 293T cells by determining the activity of β-galactosidase, the reporter gene. The results indicate that the lipoplexes’ physicochemical properties are strongly dependent on the preparation method (one-step or multi-step complexation, at 25ºC or 50ºC), with resulting mean sizes varying from 350 to 1700 nm and superficial charge densities ranging from +9 mV to +30 mV. No clear correlation was found, however, between lipoplex physicochemical properties and observed cell transfection efficiencies, suggesting that other parameters, such as structure (degree of DNA condensation/compaction) of the complex and the mode of internalization by the cell may also weigh in on cell transfection. Nevertheless, it was possible to determine that lipoplex preparation methods can greatly affect DNA uptake by cells. Optimal nanotherapy requires the therapeutic molecule to be protected from degradation and reach its target cell and intracellular location. This work demonstrates that the MO-based system is a viable nanocarrier for plasmidic DNA and that the conditions to prepare the carriers for gene therapy application can be modulated to achieve maximal delivery efficiency

Counter ions and constituents combination affect DODAX: MO nanocarriers toxicity in vitro and in vivo

Liposomes have received extensive attention as nanocarriers for bioactive compounds due to their good biocompatibility, possibility of targeting and incorporation of hydrophilic and hydrophobic compounds. Although generally considered as safe, detailed knowledge of the effects induced in cells and tissues with which they interact is still underexplored. The aim of this study is to gain insight into the toxicity profile of dioctadecyldimethylammonium (DODAX) : monoolein(MO) liposomes (X is bromide or chloride), previously validated for gene therapy, by evaluating the effect of the counter ions Br− or Cl−, and of the cationic : neutral lipid molar fraction, both in vitro and in vivo. Effects on cellular metabolism and proliferation, plasma membrane integrity, oxidative stress, mitochondrial membrane potential dysfunction and ability to trigger apoptosis and necrosis were evaluated in a dose-/time-dependent manner in normal human skin fibroblasts. Also, newly fertilized zebrafish zygotes were exposed to liposomes, permitting a fast-track evaluation of the morphophysiological modifications. In vitro data showed that only very high doses of DODAX : MO induce apoptosis and necrosis, inhibit cell proliferation, and affect the metabolism and plasma membrane integrity of fibroblasts in a dose-/time-dependent manner. Furthermore, liposomes affected mitochondrial function, increasing ROS accumulation and disturbing mitochondrial membrane potential. DODAC-based liposomes were consistently more toxic when compared to DODAB-based formulations; furthermore, the inclusion of MO was found to reduce toxicity, in contrast to liposomes with cationic DODAX only, especially in DODAB : MO (1 : 2) nanocarriers. These results were corroborated, in a holistic approach, by cytotoxicity profiling in five additional human cell lines, and also with the zebrafish embryotoxicity testing, which constitutes a sensitive and informative tool and accurately extends cell-based assays.This work was supported by the strategic programme UID/BIA/ 04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI); by FEDER through POFCCOMPETE; and by national funds from FCT through PEstC/FIS/UI0607/2013 (CFUM) and PTDC/QUI/69795/2006. Ana Oliveira held the scholarship, SFRH/BD/68588/2010. Marisa P. Sárria holds a Marie Curie COFUND fellowship funding from the European Union’s7 th Framework Programme for research, technological development and demonstration under grant agreement 600375. The authors are grateful to the technical support of Marinnova – Marine and Environmental Innovation, Technology and Services, a R&I company anchored to the Centre of Marine and Environmental Research (CIIMAR – University of Porto, Portugal), focused at providing innovative services and products in the field of marine and environmental sciences

Role of counter-ion and helper lipid content in the design and properties of nanocarrier systems: a biophysical study in 2D and 3D lipid assemblies

There is a direct correlation between the physicochemical properties of nanocarrier systems and their biological performance, including stability under physiological conditions, cellular internalization and transfection efficiency. Therefore, understanding the biophysical aspects that affect self-assembled nanocarriers is determinant for a rational design of efficient formulations. In this study, a comprehensive evaluation of the effects of each component on the molecular organization of aggregates formed by the cationic lipids dioctadecyldimethylammonium bromide and chloride (DODAB and DODAC) and the neutral lipid monoolein (MO) was made. Specifically, the effects of the helper lipid content (MO) and the role of the counter-ion of the cationic lipids were evaluated in 2D and 3D assemblies by Langmuir surface pressure–molecular area (π–A) isotherms, Brewster Angle Microscopy (BAM), infrared reflection absorption spectroscopy (IRRAS), confocal Raman microscopy, and Small Angle X-ray Scattering (SAXS). The results show that MO has a different distribution on the DODAC and DODAB bilayers, and a fluidizing effect dependent on the MO content. For low MO molar ratios, the fluidizing effect was more pronounced in DODAC : MO mixtures, indicating a more homogeneous distribution of MO in DODAC than in DODAB bilayers. For high MO molar ratios, packing of membranes was similar for both cationic lipids, and the effect of the counter-ion is attenuated. The distribution of MO in the two cationic systems is closely related with the efficiency of the counter-ions in the screening of the charged group.We acknowledge DAAD/FCT that provided the financial support required to gather the Portuguese and the German coworkers. This work was further supported by FEDER through POFC-COMPETE and by national funds from FCT, through the projects PEst-OE/BIA/UI4050/2014 (CBMA) and PEst-C/FIS/UI0607/2013 (CFUM). Marlene Lúcio acknowledges FCT for the financial support provided by the exploratory project IF/00498/2012. C.R.-A. is grateful to the European Union through the Operational Programme for Cross-border Cooperation: Spain-Portugal under Grant POCTEP 2007-2013 and to European Regional Development Fund for research funding (Innovation in Nanomedicine Project). The authors would also like to acknowledge Irina Berndt and Claudia Botelho

MAMMALS IN PORTUGAL : A data set of terrestrial, volant, and marine mammal occurrences in P ortugal

Mammals are threatened worldwide, with 26% of all species being includedin the IUCN threatened categories. This overall pattern is primarily associatedwith habitat loss or degradation, and human persecution for terrestrial mam-mals, and pollution, open net fishing, climate change, and prey depletion formarine mammals. Mammals play a key role in maintaining ecosystems func-tionality and resilience, and therefore information on their distribution is cru-cial to delineate and support conservation actions. MAMMALS INPORTUGAL is a publicly available data set compiling unpublishedgeoreferenced occurrence records of 92 terrestrial, volant, and marine mam-mals in mainland Portugal and archipelagos of the Azores and Madeira thatincludes 105,026 data entries between 1873 and 2021 (72% of the data occur-ring in 2000 and 2021). The methods used to collect the data were: live obser-vations/captures (43%), sign surveys (35%), camera trapping (16%),bioacoustics surveys (4%) and radiotracking, and inquiries that represent lessthan 1% of the records. The data set includes 13 types of records: (1) burrowsjsoil moundsjtunnel, (2) capture, (3) colony, (4) dead animaljhairjskullsjjaws, (5) genetic confirmation, (6) inquiries, (7) observation of live animal (8),observation in shelters, (9) photo trappingjvideo, (10) predators dietjpelletsjpine cones/nuts, (11) scatjtrackjditch, (12) telemetry and (13) vocalizationjecholocation. The spatial uncertainty of most records ranges between 0 and100 m (76%). Rodentia (n=31,573) has the highest number of records followedby Chiroptera (n=18,857), Carnivora (n=18,594), Lagomorpha (n=17,496),Cetartiodactyla (n=11,568) and Eulipotyphla (n=7008). The data setincludes records of species classified by the IUCN as threatened(e.g.,Oryctolagus cuniculus[n=12,159],Monachus monachus[n=1,512],andLynx pardinus[n=197]). We believe that this data set may stimulate thepublication of other European countries data sets that would certainly contrib-ute to ecology and conservation-related research, and therefore assisting onthe development of more accurate and tailored conservation managementstrategies for each species. There are no copyright restrictions; please cite thisdata paper when the data are used in publications.info:eu-repo/semantics/publishedVersio