Microbacterium memoriense sp. nov., a member of the Actinomycetota from marine beach sediment of the north coast of Portugal.

The oceans harbour a myriad of unknown micro-organisms that remain unstudied because of a failure to establish the right growth conditions under laboratory conditions. To overcome this limitation, an isolation effort inspired by the iChip was performed using marine sediments from Memória beach, Portugal. A novel strain, PMIC_1C1BT, was obtained and subjected to a polyphasic study. Cells of strain PMIC_1C1BT were Gram-positive, rod-shaped, divided by binary fission and formed colonies that were shiny light-yellow. Based on its full 16S rRNA gene sequence, strain PMIC_1C1BT was phylogenetically associated to the genus Microbacterium and its closest relatives were Microbacterium aurum KACC 15219T (98.55 %), Microbacterium diaminobutyricum RZ63T (98.48 %) and Microbacterium hatanonis JCM 14558T (98.13 %). Strain PMIC_1C1BT had a genome size of 2 761 607 bp with 67.71 mol% of G+C content and 2582 coding sequences, which is lower than the genus average. Strain PMIC_1C1BT grew from 15 to 30 °C, optimally at 25 °C, at pH 6.0 to 11.0, optimally between pH 6.0 and 8.0, and from 0 to 5 % (w/v) NaCl, optimally between 2.0 and 3.0 %. It grew with casamino acids, glutamine, methionine, N-acetylglucosamine, sodium nitrate, tryptophan, urea and valine as sole nitrogen sources, and arabinose and cellobiose as sole carbon sources. The major cellular fatty acids were anteiso-C15 : 0, iso-C16 : 0 and iso-C17 : 0. Genome mining revealed the presence of four biosynthetic gene clusters (BGCs) with low similarities to other known BCGs. Based on the polyphasic data, strain PMIC_1C1BT is proposed to represent a novel species, for which the name Microbacterium memoriense sp. nov. (=CECT 30366T=LMG 32350T) is proposed.

Streptomyces meridianus sp. nov. isolated from brackish water of the Tagus estuary in Alcochete, Portugal.

An isolation effort focused on sporogenous Actinomycetota from the Tagus estuary in Alcochete, Portugal, yielded a novel actinomycetal strain, designated MTZ3.1T, which was subjected to a polyphasic taxonomic study. MTZ3.1T is characterised by morphology typical of members of the genus Streptomyces, with light beige coloured substrate mycelium, which does not release pigments to the culture medium and with helicoidal aerial hyphae that differentiate into spores with a light-grey colour. The phylogeny of MTZ3.1T, based on the full 16S rRNA gene sequence, indicated that its closest relatives were Streptomyces alkaliterrae OF1T (98.48 %), Streptomyces chumphonensis KK1-2T (98.41 %), Streptomyces albofaciens JCM 4342T (98.34 %), Streoptomyces paromomycinus NBRC 15454T (98.34 %) and Streptomyces chrestomyceticus NRBC 13444T (98.34 %). Moreover, average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridisation (dDDH) are below the species cutoff values (ANI 67.70 and 68.35 %, AAI 77.06 and 76.71 % and dDDH 22.10 and 21.50 % for S. alkaliterrae OF1T and S. chumphonensis KK1-2T, respectively). Whole genome sequencing revealed that MTZ3.1T has a genome of 5 644 485 bp with a DNA G+C content of 71.29 mol% and 5044 coding sequences. Physiologically, MTZ3.1T is strictly aerobic, able to grow at 15-37 °C, optimally at 25 °C and between pH5 and 8 and showed high salinity tolerance, growing with 0-10 %(w/v) NaCl. Major cellular fatty acids are C15 : 0, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. Furthermore, it was able to utilise a variety of nitrogen and carbon sources. Antimicrobial screening indicated that MTZ3.1T has potent anti-Staphylococcus aureus activity. On the basis of the polyphasic data, MTZ3.1T is proposed to represent a novel species, Streptomyces meridianus sp. nov. (= CECT 30416T = DSM 114037T=LMG 32463T).

Streptomyces marispadix sp. nov., isolated from marine beach sediment.

A novel actinomycetal strain, designated M600PL45_2T, was isolated from marine sediments obtained from Ingleses beach, Porto, on the Northern Coast of Portugal and was subjected to a polyphasic taxonomic characterisation study. The here described Gram-reaction-positive strain is characterised by the production of a brown pigment in both solid and liquid medium and forms typical helical hyphae that differentiate into smooth spores. The results of a phylogenetic analysis based on the 16S rRNA gene sequence indicated that M600PL45_2T has a high similarity to two members of the genus Streptomyces, Streptomyces bathyalis ASO4wetT (98.51 %) and Streptomyces daqingensis NEAU ZJC8T (98.44 %). The genome of M600PL45_2T has a size of 6 695 159 bp, a DNA G+C content of 70.71 mol% and 5538 coding sequences. M600PL45_2T grows at 15-37 °C and with a maximal growth rate between 25 °C and 30 °C. Growth at pH 6.0 to 9.0 with the optimal range between 6.0 and 7.5 was observed. M600PL45_2T showed a high salinity tolerance, growing with 0-10 % (w/v) NaCl, with best growth with 1-3% (w/v) NaCl. Major cellular fatty acids are iso-C15:0 (25.03 %), anteiso-C15:0 (17.70) and iso-C16:0 (26.90 %). The novel isolate was able to grow in media containing a variety of nitrogen and carbon sources. An antimicrobial activity screening indicated that an extract of M600PL45_2T has inhibitory activity against Staphylococcus aureus. On the basis of the polyphasic data, M600PL45_2T (= CECT 30365T = DSM 114036T) is introduced as the type strain of a novel species, that we named Streptomyces marispadix sp. nov.

Challenges in Nanomaterial Characterization - From Definition to Analysis.

Nanomaterials have outstanding properties and have several applications, ranging from foods, cosmetics, pharmaceuticals to energy, construction, etc. As with all novel products, the benefits of nanomaterials use must be weighed against its health and environmental impact. They have different origins, natural, incidental, or engineered, they are widespread, and they need to be classified and characterized for various purposes, including nanotoxicology studies and risk assessment, workplaces and environment safety evaluation, consumer products evaluation, as well as manufacturing process control. To properly characterize nanomaterials, a consensual definition of nanomaterial is needed, and several analyses using the available characterization techniques must be performed. Various properties are relevant in the characterization process and many of them, namely size, are still a challenge that the research community is facing. The measurement of physical and chemical properties is very important in the case of nanomaterials. In view of this, in this chapter, available analytical techniques are reviewed based on nanomaterials classification, regulatory demands and toxicology assessment. Additionally, some of the current major challenges and gaps in nanomaterials characterization are identified and listed.

Comparison of neutral lipid fatty acid composition in organisms from different trophic levels.

The profiles of total fatty acids (TFAs) and the neutral lipid fatty acids (NLFAs) were compared for the bacterium Rhodopirellula rubra and the alga Raphidocelis subcapitata (conventional food source for Daphnia magna). D. magna NLFAs were assessed when this crustacean was fed with bacterium and alga, individually or in combination. After NLFA extraction, the profiles of the various organisms were characterized by gas chromatography. Results evidenced the relevance of the different composition of the fatty acid (FAs) fractions in the different organisms, R. rubra and R. subcapitata. In these species, the NFLA analyses revealed high amounts of long chain FAs (C19). The FA profile of D. magna was influenced by the different diets provided although the preferred diet was the alga. D. magna showed the capacity to adapt to the available food resources as it defines its FA profile according to its needs, namely for the long chain FAs (C19).

Assessment of Rhodopirellula rubra as a supplementary and nutritional food source to the microcrustacean Daphnia magna.

The daily use of the planctomycete Rhodopirellula rubra as an alternative or supplementary food source for Daphnia magna and its feasibility in the nutrition of transgenerational populations were studied. The life history parameters, fatty acids (saturated, mono- and polyunsaturated; SFAs, MUFAs and PUFAs), glycogen and protein contents of organisms during feeding assays and of the first generation were analysed. An increase in the yields of D. magna with the increase of the cell concentration of R. rubra was evident, but overall, bacteria supplied as the only food source was nutritionally insufficient as observed for all the parameters analysed. However, when R. rubra was added as supplement to the microalgae Raphidocelis subcapitata a significant improvement in the life history parameters was observed namely in the reproductive output and the somatic growth rate. The identified SFAs, MUFAs and PUFAs were the fatty acids more abundant in daphniids, and the feed regimens influenced daphniids fatty acid profiles. Additionally, the mixed diet resulted in a larger number and size of offspring in the different F1 broods as also observed with the results of F0 generation. The pink colouration present in D. magna body and eggs confirmed that bacteria were absorbed, the pigment(s) retained and passed on to the next generation. Our results showed that R. rubra can play an essential role in D. magna diet as a nutritional supplement showing potential biotechnological applications.

Adequacy of planctomycetes as supplementary food source for Daphnia magna.

The nutritional quality of daphnids diet can influence their growth, reproduction and survival. In aquatic ecosystems, bacteria can contribute significantly to Daphnia diet by supporting, for instances, their high needs for phosphorus. The laboratory feeding of the model organisms Daphnia spp. is algal based, but should be improved to allow their better performance. The aim of this study was to evaluate the potential of two planctomycetes, Gemmata obscuriglobus and Rhodopirellula rubra, from exponential and stationary growth phases as alternative or supplementary food source for Daphnia magna. The actinobacterium Arthrobacter sp. was used for comparison. The feeding with only bacteria showed the inefficacy of both planctomycetes and actinobacteria as the only food source. However, when used in supplement to Raphidocelis subcapitata, a decrease in the age of first reproduction, a significant increase in reproductive output, in somatic growth and in rate of population increase was found for the highest cell densities of bacteria tested. The typical pink coloration of these bacteria present in daphnids body and eggs confirmed bacterial absorption and metabolization of their pigment. Planctomycetes yielded better results than the actinobacteria Arthrobacter but G. obscuriglobus that possesses sterols did not induce a better performance comparatively to R. rubra. No relation could be established between the feeding treatments that allowed improvement of Daphnia performance and the different kind of Daphnia' Fatty Acid Methyl Esters. The use of sonication to separate planctomycetal cells before feeding the daphnids proved to be efficient. We confirmed that R. subcapitata supplemented by bacteria allows a better growth performance of D. magna.

Assessment of planctomycetes cell viability after pollutants exposure.

In this study, the growth of six different planctomycetes, a particular ubiquitous bacterial phylum, was assessed after exposure to pollutants. In addition and for comparative purposes, Pseudomonas putida, Escherichia coli and Vibrio anguillarum were tested. Each microorganism was exposed to several concentrations of 21 different pollutants. After exposure, bacteria were cultivated using the drop plate method. In general, the strains exhibited a great variation of sensitivity to pollutants in the order: V. anguillarum > planctomycetes > P. putida > E. coli. E. coli showed resistance to all pollutants tested, with the exception of phenol and sodium azide. Copper, Ridomil® (fungicide), hydrazine and phenol were the most toxic pollutants. Planctomycetes were resistant to extremely high concentrations of nitrate, nitrite and ammonium but they were the only bacteria sensitive to Previcur N® (fungicide). Sodium azide affected the growth on plates of E. coli, P. putida and V. anguillarum, but not of planctomycetes. However, this compound affected planctomycetes cell respiration but with less impact than in the aforementioned bacteria. Our results provide evidence for a diverse response of bacteria towards pollutants, which may influence the structuring of microbial communities in ecosystems under stress, and provide new insights on the ecophysiology of planctomycetes.

Editorial on Special Issue "Lipid Nanosystems for Local Drug Delivery".

Nanosystems provide an attractive approach to pharmacological therapy, with the possibility of enhancing the performance and overcoming the constraints of conventional therapies, thus adding substantial value to some of the already available formulations [...].

Nanostructured Lipid Carriers Enriched Hydrogels for Skin Topical Administration of Quercetin and Omega-3 Fatty Acid.

Chronic skin exposure to external hostile agents (e.g., UV radiation, microorganisms, and oxidizing chemicals) may increase oxidative stress, causing skin damage and aging. Because of their well-known skincare and protective benefits, quercetin (Q) and omega-3 fatty acids (ω3) have attracted the attention of the dermocosmetic and pharmaceutical sectors. However, both bioactives have inherent properties that limit their efficient skin delivery. Therefore, nanostructured lipid carriers (NLCs) and enriched PFC® hydrogels (HGs) have been developed as a dual-approach vehicle for Q and/or ω3 skin topical administration to improve bioactives' stability and skin permeation. Two NLC formulations were prepared with the same lipid composition but differing in surfactant composition (NLC1-soy lecithin and poloxamer 407; NLC2-Tween® 80 and dioctyl sodium sulfosuccinate (DOSS)), which have an impact on physicochemical properties and pharmaceutical and therapeutic performance. Despite both NLCs presenting high Q loading capacity, NLC2's physicochemical properties make them more suitable for topical skin administration and ensure longer colloidal stability. Additionally, NLC2 demonstrated a more sustained Q release, indicating higher bioactive storage while improving permeability. The occlusive effect of NLCs-enriched HGs also has a positive impact on skin permeability. Q-loaded NLC2, with or without ω3, -enriched HGs demonstrated efficacy as antioxidant and photoprotective formulations as well as effective reduction in S. aureus growth, indicating that they constitute a promising approach for topical skin administration to prevent skin aging and other damaging cutaneous processes.

Determination of zeta potential in Planctomycetes and its application in heavy metals toxicity assessment.

Zeta potential of Planctomycetes was evaluated under different environmental conditions and correlated to cell viability. Phylogenetically distinct strains of the Planctomycetes presented different negative zeta potential values. More negative values were associated with Rhodopirellula spp. and related to the great amount of fimbriae in these species. Milli-Q water was chosen as the best dispersion media to perform the measurements. Zeta potential increased with ionic strength and varied with pH. In the physiological range of pH 5.0-9, zeta potential remained low and Rhodopirellula sp. strain LF2 cells were viable. Out of this range, zeta potential increased significantly and viability decreased. The effect on zeta potential of arsenic, cadmium, chromium, copper, lead, nickel, and zinc was assessed in Rhodopirellula sp. strain LF2. Zeta potential increased with increasing toxicity of the heavy metals in a dose-response way. This result was confirmed by the results observed for Rhodopirellula baltica strain SH1 under copper toxicity. Lead was the most toxic metal and zinc was the least toxic as observed by zeta potential and viability. The results support a correlation between zeta potential and cell viability which seem to indicate the possibility to use it as a viability predictor for the effects of heavy metals toxicity.

Does a Diet Rich in the Bacterium Rhodopirellula rubra Improve Daphnia magna Performance?

BACKGROUND: In the wild various organisms contribute to daphnids diet. This study, intendeds to evaluate the potential of the concentration of Rhodopirellula rubra as a single or supplementary food source for Daphnia magna. METHODS: Feeding assays were performed according to standard guidelines for chronic assays (21 days), and life-history parameters and several biomarkers (protein content, oxidative stress, energetic reserves and pigments) were measured. Five food regimens were conducted with 20 individual replicates (A - R. subcapitata; 0.2 - suspension of R. rubra at 0.2 arbitrary units (AU); 0.4 - suspension of R. rubra at 0.4 AU; 0.2+A - suspension of R. rubra at 0.2+alga; 0.2+A-suspension of R. rubra at 0.4 AU + alga). Additionally, the effects of three diets (A, 0.2, and 0.2+A) on the longevity of D. magna were assessed. RESULTS: The five diets showed a different C, N, and carotenoids composition, with an increase in the mixed diets. The results confirmed that the mixed diets improved D. magna life-history parameters. A decrease in glycogen, and the increase of haemoglobin, protein, and gluthione-S-transferase (GST) were observed. Furthermore, D. magna fed with bacterial single diets, presented worsen life history parameters and a decrease in the protein content. An induction of oxidative stress response (increased catalase and GST), and a significant decrease in lipid peroxidation and an accumulation of glycogen and carotenoids were observed. Overall, an increase in the amount of R. rubra provided to D. magna, from 0.2 AU to 0.4 AU, negatively impacted daphnid performance. No significant effects on Daphnia longevity (a 110-day assay) were observed among the three diets tested. However, a significant survival percentage and fertility (cumulative offspring is more than twice) was observed when D. magna was fed with the mixed diet. CONCLUSIONS: Results demonstrated that different diets provided a nutritional diversified food to the daphnids that induced differences in D. magna performance. The mixed diets proved to be beneficial (with increase in offspring) on D. magna performance, independently of the bacterial concentration tested. When in single diet, bacterial concentration is not nutritionally sufficient to raise D. magna even when in increased concentration.

Formulation, Characterization, and Cytotoxicity Evaluation of Lactoferrin Functionalized Lipid Nanoparticles for Riluzole Delivery to the Brain.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with a very poor prognosis. Its treatment is hindered by a lack of new therapeutic alternatives and the existence of the blood-brain barrier (BBB), which restricts the access of drugs commonly used in ALS, such as riluzole, to the brain. To overcome these limitations and increase brain targeting, riluzole-loaded nanostructured lipid carriers (NLC) were prepared and functionalized with lactoferrin (Lf), facilitating transport across the BBB by interacting with Lf receptors expressed in the brain endothelium. NLC were characterized with respect to their physicochemical properties (size, zeta potential, polydispersity index) as well as their stability, encapsulation efficiency, morphology, in vitro release profile, and biocompatibility. Moreover, crystallinity and melting behavior were assessed by DSC and PXRD. Nanoparticles exhibited initial mean diameters between 180 and 220 nm and a polydispersity index below 0.3, indicating a narrow size distribution. NLC remained stable over at least 3 months. Riluzole encapsulation efficiency was very high, around 94-98%. FTIR and protein quantification studies confirmed the conjugation of Lf on the surface of the nanocarriers, with TEM images showing that the functionalized NLC presented a smooth surface and uniform spherical shape. An MTT assay revealed that the nanocarriers developed in this study did not cause a substantial reduction in the viability of NSC-34 and hCMEC/D3 cells at a riluzole concentration up to 10 µM, being therefore biocompatible. The results suggest that Lf-functionalized NLC are a suitable and promising delivery system to target riluzole to the brain.

Novel Exosome Biomarker Candidates for Alzheimer's Disease Unravelled Through Mass Spectrometry Analysis.

Exosomes are small extracellular vesicles (EVs) present in human biofluids that can transport specific disease-associated molecules. Consequently blood-derived exosomes have emerged as important peripheral biomarker sources for a wide range of diseases, among them Alzheimer's disease (AD). Although there is no effective cure for AD, an accurate diagnosis, relying on easily accessible peripheral biofluids, is still necessary to discriminate this disease from other dementias, test potential therapies and even monitor rate of disease progression. The ultimate goal is to produce a cost-effective and widely available alternative, which can also be employed as a first clinical screen. In this study, EVs with exosome-like characteristics were isolated from serum of Controls and AD cases through precipitation- and column-based methods, followed by mass spectrometry analysis. The resulting proteomes were characterized by Gene Ontology (GO) and multivariate analyses. Although GO terms were similar for exosomes' proteomes of Controls and ADs, using both methodologies, a clear segregation of disease cases was obtained when using the precipitation-based method. Nine significantly different abundant proteins were identified between Controls and AD cases, representing putative biomarker candidate targets. Among them are AACT and C4BPα, two Aß-binding proteins, whose exosome levels were further validated in individuals from independent cohorts using antibody-based approaches. The findings discussed represent an important contribution to the identification of novel exosomal biomarker candidates useful as potential blood-based tools for AD diagnosis.

Omega-3- and Resveratrol-Loaded Lipid Nanosystems for Potential Use as Topical Formulations in Autoimmune, Inflammatory, and Cancerous Skin Diseases.

Resveratrol (RSV) and omega 3 (ω3), because of their biological favorable properties, have become subjects of interest for researchers in dermocosmetic and pharmaceutical industries; however, these bioactives present technological limitations that hinder their effective delivery to the target skin layer. To overcome the stability and skin permeation limitations of free bioactives, this work proposes a combined strategy involving two different lipid nanosystems (liposomes and lipid nanoparticles) that include ω3 in their lipid matrix. Additionaly, RSV is only encapsulated in liposomes that provid an adequate amphiphilic environment. Each formulation is thoroughly characterized regarding their physical-chemical properties. Subsequently, the therapeutic performance of the lipid nanosystems is evaluated based on their protective roles against lipid peroxidation, as well as inhibition of cicloxygenase (COX) and nitric oxid (NO) production in the RWA264.7 cell line. Finally, the lipid nanosystems are incorporated in hydrogel to allow their topical administration, then rheology, occlusion, and RSV release-diffusion assays are performed. Lipid nanoparticles provide occlusive effects at the skin surface. Liposomes provide sustained RSV release and their flexibility conferred by edge activator components enhances RSV diffusion, which is required to reach NO production cells and COX cell membrane enzymes. Overall, the inclusion of both lipid nanosystems in the same semisolid base constitutes a promising strategy for autoimmune, inflammatory, and cancerous skin diseases.

Potential of FTIR Spectroscopy Applied to Exosomes for Alzheimer's Disease Discrimination: A Pilot Study.

Alzheimer's disease (AD) diagnosis is based on psychological and imaging tests but can also include monitoring cerebrospinal fluid (CSF) biomarkers. However, CSF based-neurochemical approaches are expensive and invasive, limiting their use to well-equipped settings. In contrast, blood-based biomarkers are minimally invasive, cost-effective, and a widely accessible alternative. Blood-derived exosomes have recently emerged as a reliable AD biomarker source, carrying disease-specific cargo. Fourier-transformed infrared (FTIR) spectroscopy meets the criteria for an ideal diagnostic methodology since it is rapid, easy to implement, and has high reproducibility. This metabolome-based technique is useful for diagnosing a broad range of diseases, although to our knowledge, no reports for FTIR spectroscopy applied to exosomes in AD exist. In this ground-breaking pilot study, FTIR spectra of serum and serum-derived exosomes from two independent cohorts were acquired and analyzed using multivariate analysis. The regional UA-cohort includes 9 individuals, clinically diagnosed with AD, mean age of 78.7 years old; and the UMG-cohort comprises 12 individuals, clinically diagnosed with AD (based on molecular and/or imaging data), mean age of 73.2 years old. Unsupervised principal component analysis of FTIR spectra of serum-derived exosomes revealed higher discriminatory value for AD cases when compared to serum as a whole. Consistently, the partial least-squares analysis revealed that serum-derived exosomes present higher correlations than serum. In addition, the second derivative peak area calculation also revealed significant differences among Controls and AD cases. The results obtained suggest that this methodology can discriminate cases from Controls and thus be potential useful to assist in AD clinical diagnosis.

More than just exosomes: distinct Leishmania infantum extracellular products potentiate the establishment of infection.

The use of secretion pathways for effector molecule delivery by microorganisms is a trademark of pathogenesis. Leishmania extracellular vesicles (EVs) were shown to have significant immunomodulatory potential. Still, they will act in conjunction with other released parasite-derived products that might modify the EVs effects. Notwithstanding, the immunomodulatory properties of these non-vesicular components and their influence in the infectious process remains unknown. To address this, we explored both in vitro and in vivo the immunomodulatory potential of promastigotes extracellular material (EXO), obtained as a whole or separated in two different fractions: EVs or vesicle depleted EXO (VDE). Using an air pouch model, we observed that EVs and VDE induced a dose-dependent cell recruitment profile different from the one obtained with parasites, attracting significantly fewer neutrophils and more dendritic cells (DCs). Additionally, when we co-inoculated parasites with extracellular products a drop in cell recruitment was observed. Moreover, in vitro, while VDE (but not EVs) downregulated the expression of DCs and macrophages activation markers, both products were able to diminish the responsiveness of these cells to LPS. Finally, the presence of Leishmania infantum extracellular products in the inoculum promoted a dose-dependent infection potentiation in vivo, highlighting their relevance for the infectious process. In conclusion, our data demonstrate that EVs are not the only relevant players among the parasite exogenous products. This, together with the dose-dependency observed, opens new avenues to the comprehension of Leishmania infectious process. The approach presented here should be exploited to revisit existing data and considered for future studies in other infection models.

Exosome isolation from distinct biofluids using precipitation and column-based approaches.

The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations' purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.

Microcalorimetric evidence of hydrophobic interactions between hydrophobically modified cationic polysaccharides and surfactants of the same charge.

We synthesized and characterized a series of new polymers-hydrophobically modified cationic polysaccharides-based on dextran having pendant N-(2-hydroxypropyl)-N,N-dimethyl-N-alkylammonium chloride groups randomly distributed along the polymer backbone. These polymers are good candidates for studying the hydrophobic effect on polymer/surfactant association. In previous papers we reported their interactions with oppositely charged surfactants. For further insight into the relative importance of the hydrophobic interaction in the association process now we studied the thermodynamics of the interaction of these hydrophobically modified polymers with surfactants of the same charge (DMRX/CnTAC) by isothermal titration calorimetry (ITC). In order to try to discriminate the solution behavior of these polymer/surfactant systems, we analyzed separately the interaction of unmodified dextran with ionic surfactants and the interactions between the corresponding cationic surfactants. The interaction enthalpies for DMRX/CnTAC systems were derived from a proposed thermodynamic model with equations that describe the polymer-surfactant interactions. The thermodynamic parameters for the DMRX/CnTAC aggregation process as well as surfactant micellization in the presence of the polymer were also calculated. From all the results we were able to ascertain the effect on the interactions of changing the alkyl chain length of the polyelectrolyte pendant groups or the surfactant. The importance of the polymer aggregation state on the mechanism of interaction was also addressed.