Ana Cristina Norberto Gonçalves Oliveira

Polysaccharide production and biofilm formation by Pseudomonas fluorescen: effects of pH and surface material

Abstract Although the synthesis of extracellular polysaccharides was first recognized in certain bacterial cultures a long time ago, its role in bacterial adhesion is still subject to some debate. Several fermentation batch cultures were performed under different conditions of pH (pH 7, maintained with NaOH and HCl; pH 7 in phosphate buffer, and without pH control) in order to study the relation between the production of extracellular polysaccharides and biofilm formation on polymeric slides suspended in the culture medium. The polymers used were polystyrene, polypropylene, polyethylene and poly(vinyl chloride). The maximum amount of exopolysaccharides in the culture medium occurs at pH 7, although slightly thicker biofilms seem to be formed when there is no pH control. The biofilms were analysed by scanning electron microscopy and by wavelength dispersion spectroscopy. Biofilm morphology seems to be much more dependent on the type of substratium than on the pH of the medium; for different pH values, a polymeric network can be more clearly observed on biofilms formed on all surfaces except poly(vinyl chloride).

Dioctadecyldimethylammonium:monoolein nanocarriers for efficient in vitro gene silencing.

This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.

Molecular study of congenital erythrocytosis in 70 unrelated patients revealed a potential causal mutation in less than half of the cases (Where is/are the missing gene(s)?)

Congenital erythrocytosis can be classified as primary, when the defect is intrinsic to the RBC progenitors and independent of the serum erythropoietin (Epo) concentration, or secondary, when the erythrocytosis is the result of an upregulation of Epo production. Primary erythrocytosis is associated with mutations in the EPOR gene, secondary CE can de due to mutations that stabilize the hemoglobin in the oxygenated form or to mutations in the genes that control the transcriptional activation of the EPO gene – VHL, EGLN1, EPAS1. Chuvash polycythemia, caused by mutations in VHL gene, shares features of both primary and secondary erythrocytosis, with increased Epo production but also hypersensitivity of progenitors to Epo.

Tunable pDNA/DODAB:MO lipoplexes : the effect of incubation temperature on pDNA/DODAB:MO lipoplexes structure and transfection efficiency

Dioctadecyldimethylammonium bromide (DODAB):1-monooleoyl-rac-glycerol (MO) cationic liposomes were reported as a promising alternative to common transfection agents, showing superior effectiveness on the transfection of the 293T mammalian cell line with pSV-β-gal plasmid DNA. The study of DODAB:MO aggregates in the absence of DNA has indicated that their morphology depends on the balance between DODABs tendency to form bilayer structures and MOs propensity to form inverted non-lamellar structures. Other parameters, such as the temperature have proved to be crucial in the definition of the morphology of the developed nanocarrier. Therefore, in this work, a step forward to the current gene carrier system will be given by studying the effect of the tunable parameters (incubation temperature and MO content) on the structure of pDNA:DODAB:MO lipoplexes. More importantly, the implications that these tunable parameters could have in terms of lipoplex transfection efficiency will be investigated. Dynamic light scattering (DLS), zeta (ζ) potential, cryo-transmission electron microscopy (cryo-TEM) and ethidium bromide (EtBr) exclusion were used to assess the formation, structure and destabilization of pDNA:DODAB:MO lipoplexes at DODAB molar fractions of (1:1) and above equimolarity (2:1, 4:1) prepared at incubation temperatures from 25 to 50°C. Experimental results indicate that pDNA:DODAB:MOs structure is sensitive to the lipoplex incubation temperature, resulting in particles of distinct size, superficial charge and structure. These variations are also visible on the complexation dynamics of pDNA, and subsequent release upon incubation with the model proteoglycan heparin (HEP), at 25 and 50°C. Increase in temperature leads to re-organization of DODAB and MO molecules within the liposomal formulation, causing a positive charge re-localization in the lipoplex surface, which not only alters its structure but also its transfection efficiency. Altogether, these results confirm that in the DODAB:MO carriers, an increase in the incubation temperature has a similar effect on aggregate morphology as the observed with an increase in MO content. This conclusion is extended to the pDNA:DODAB:MO lipoplexes morphology and subsequent transfection efficiency defining new strategies in lipoplexes preparation that could be used to modulate the properties of other lipid formulations for nonviral gene delivery applications.

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

Abstract 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.

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.

Familial thrombotic risk based on the genetic background of Protein C Deficiency in a Portuguese Study.

Inherited protein C (PC) deficiency is a well‐known risk factor for venous thrombosis (VT). Plasma PC levels are reliable in moderate to severe deficiencies; however, in mildly deficient individuals, the levels may overlap with those considered normal. Genetic studies of PROC, which encodes PC, could help identify carriers; genome‐wide association studies (GWAS) have shown that approximately 50% of phenotypic variation in PC deficiency is caused by the cumulative effects of mutations in several other loci, namely in the PROCR.

Combined study of ADAMTS13 and complement genes in the diagnosis of thrombotic microangiopathies using next-generation sequencing

Essentials The differential diagnosis of acute thrombotic microangiopathy (TMA) is challenging. To the ADAMTS13 activity < or >10% was added a next‐generation sequencing (NGS) gene panel. The ADAMTS13 mutation p.Cys754Arg was frequent in hereditary thrombotic thrombocytopenic purpura. We identified novel complement gene mutations and this procedure improved our diagnostic strategy.

VoxLaps: A Free Symbol-Based AAC Application for Brazilian Portuguese

This paper aims to highlight the need for access to assistive technologies focused on augmentative and alternative communication (AAC), especially those available for the Brazilian Portuguese language, and the problems involved, as well as provide answers to these difficulties through the VoxLaps software, a free graphical symbol-based AAC application for the Android platform, developed under the supervision and support from a multidisciplinary rehabilitation team. This group of students and professionals evaluated the software functionality and usability, as well as other ACC tools, in order to compare their performances through the observation-based method and usability tests.