Estimation of Structural Sensitivity of Intrinsically Disordered Regions in Response to Hyperosmotic Stress in Living Cells Using FRET.

Intrinsically disordered regions (IDRs) are protein domains that participate in crucial cellular processes. During stress conditions, the physicochemical properties of the cellular environment change, directly impacting the conformational ensemble of IDRs. IDRs are inherently sensitive to environmental perturbations. Studying how the physicochemical properties of the cell regulate the conformational ensemble of IDRs is essential for understanding the environmental control of their function. Here, we describe a step-by-step method for measuring the structural sensitivity of IDRs in living Saccharomyces cerevisiae cells in response to hyperosmotic stress conditions. We present the use of ensemble fluorescence resonance energy transfer (FRET) to estimate how the global dimensions of IDRs change during a progressive increase of hyperosmotic stress imposed on cells with any osmolyte. In addition, we provide a script for processing fluorescence measurements and comparing structural sensitivity for different IDRs. By following this method, researchers can obtain valuable insights into the conformational changes that IDRs undergo in the complex intracellular milieu upon changing environments.

Study of antimycobacterial, cytotoxic, and mutagenic potential of polymeric nanoparticles of copper (II) complex.

This study aimed to encapsulate and characterise a potential anti-tuberculosis copper complex (CuCl2(INH)2.H2O:I1) into polymeric nanoparticles (PNs) of polymethacrylate copolymers (Eudragit®, Eu) developed by nanoprecipitation method. NE30D, S100 and, E100 polymers were tested. The physicochemical characterisations were performed by DLS, TEM, FTIR, encapsulation efficiency and, in vitro release studies. Encapsulation of I1 in PN-NE30D, PN-E100, and PN-S100 was 26.3%, 94.5%, 22.6%, respectively. The particle size and zeta potentials were 82.3 nm and -24.5 mV for PNs-NE30D, 304.4 nm and +18.7 mV for PNs-E100, and 517.9 nm and -6.9 mV for PNs-S100, respectively. All PDIs were under 0.5. The formulations showed an I1 controlled release at alkaline pH with 29.7% from PNs-NE30D, 7.9% from PNs-E100 and, 28.1% from PNs-S100 at 1 h incubation. PNs were stable for at least 3 months. Particularly, PNs-NE30D demonstrated moderate inhibition of M. tuberculosis and low cytotoxic activity. None of the PNs induced mutagenicity.

Variability of Omega-3/6 Fatty Acid Obtained Through Extraction-Transesterification Processes from Phaeodactylum tricornutum.

The effect of direct transesterification methods on the omega-3/6 composition of extracts from Phaeodactylum tricornutum was studied. The aim of this work was to identify an extraction method which allowed to obtain the most suitable profile of fatty acids in terms of its potential benefits to health, particularly if further used in the food industry. Seven methods using acids, alkalis, and heterogeneous-catalysts, (namely methods from 1 to 7, abbreviated as M1-M7) were performed to determine α-linolenic (ALA), linoleic (LA), docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids. The composition of fatty acids was in all cases characterized by the major abundance of palmitic (23.95-34.08%), palmitoleic (30.94-35.56%), oleic acids (3.00-7.41%), and EPA (0.5-6.45%). Unsaturated fatty acids extraction yield was higher with a two-step transesterification process (M6, 63.65%). The total fatty acid methyl ester content (FAME) obtained with acid-transesterification (M1) reached about 21% wt, and 60% w/w total lipids. ALA higher relative content (ALA/LA ratio) was obtained when a lipid pre-extraction step was performed prior to acid-catalysis (M4). The transesterification method based on alkali-catalyst (M3, KOH catalyst) led to obtain higher DHA relative contents (DHA/EPA ratio up to 0.11), although its FAME content was 3.75-fold lower than that obtained with acid-transesterification (M1). Overall, this study shows that direct transesterification with alkali-catalyst (M3) improves the determination of PUFA content from the diatom through a more efficient transesterification-based extraction process, and thus allow to assess the value of the biomass more accurately for application in the food industry.

Binary Medical Nanofluids by Combination of Polymeric Eudragit Nanoparticles for Vehiculization of Tobramycin and Resveratrol: Antimicrobial, Hemotoxicity and Protein Corona Studies.

The development of smart nanoparticles (NPs) became a trend to enhance the delivery of drugs. In the present work, Tobramycin (TB), an aminoglycoside antibiotic that displays several undesirable side effects, has been encapsulated into cationic Eudragit®E100 (E100) NPs for the treatment of infections caused by Pseudomonas aeruginosa. Combination with neutral Eudragit®NE30D (NE30D) NPs containing resveratrol (RSV), a strong natural antioxidant, increased the antimicrobial activity of TB (75% higher than free TB). NPs were stabilized with 1.0% (w/v) poloxamer 188 (P188) or poloxamer 407 (P407) as surfactants. E100 NPs showed 83.3 ± 8.5%, and 70.1 ± 2.7 encapsulation efficiency (EE) of TB with P188 and P407 coatings, respectively. The presence of NPs was confirmed by DLS and TEM studies. TB was controlled released from NPs for 6 h. Hemotoxicity tests of NPs in the range of MIC values on human blood gave negative results. Analysis of Surface Plasmon Resonance verified that NE30D/P407/RSV does not interact with plasma proteins BSA, IgG or fibrinogen, besides E100/P188/TB interact with BSA, findings that are compatible with a negligible in vivo clearance of the nanovehicles. The obtained results show a potential binary fluid composed of two NPs to highly improve the effectiveness of conventional antibiotics.