Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library.

Xylanases are key biocatalysts in the degradation of the ß-1,4-glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-ß-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. KEY POINTS: • A GH10 endo-1,4-ß-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB's in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis.

Current and novel approaches in yeast cell death research.

The study of cell death mechanisms in fungi, particularly yeasts, has gained substantial interest in recent decades driven by the potential for biotechnological advancements and therapeutic interventions. Examples include the development of robust yeast strains for industrial fermentations and high-value compound production, novel food preservation strategies against spoilage yeasts, and the identification of targets for treating fungal infections in the clinic. In this review, we discuss a wide range of methods to characterize cellular alterations associated with yeast cell death, noting the advantages and limitations. We describe assays to monitor reversible events versus those that mark a commitment to cell death (point-of-no-return), as these distinctions are important to decipher the underlying regulatory mechanisms. Several well-known challenges remain, including the varied susceptibilities to death within a cell population and the delineation of detailed cell death mechanisms. The identification and characterization of morphologically distinct subsets of dying yeast cells within dynamic yeast populations provides opportunities to reveal novel vulnerabilities and survival mechanisms. Elucidating the intricacies of yeast regulated cell death (yRCD) will contribute to the advancement of scientific knowledge and foster breakthrough discoveries with broad-ranging implications.

The fungicide cymoxanil impairs respiration in Saccharomyces cerevisiae via cytochrome c oxidase inhibition.

Cymoxanil (CYM) is a widely used synthetic acetamide fungicide, but its biochemical mode of action remains elusive. Since CYM inhibits cell growth, biomass production, and respiration in Saccharomyces cerevisiae, we used this model to characterize the effect of CYM on mitochondria. We found it inhibits oxygen consumption in both whole cells and isolated mitochondria, specifically inhibiting cytochrome c oxidase (CcO) activity during oxidative phosphorylation. Based on molecular docking, we propose that CYM blocks the interaction of cytochrome c with CcO, hampering electron transfer and inhibiting CcO catalytic activity. Although other targets cannot be excluded, our data offer valuable insights into the mode of action of CYM that will be instrumental in driving informed management of the use of this fungicide.

Enhancement of Acetate-Induced Apoptosis of Colorectal Cancer Cells by Cathepsin D Inhibition Depends on Oligomycin A-Sensitive Respiration.

Colorectal cancer (CRC) is a leading cause of death worldwide. Conventional therapies are available with varying effectiveness. Acetate, a short-chain fatty acid produced by human intestinal bacteria, triggers mitochondria-mediated apoptosis preferentially in CRC but not in normal colonocytes, which has spurred an interest in its use for CRC prevention/therapy. We previously uncovered that acetate-induced mitochondrial-mediated apoptosis in CRC cells is significantly enhanced by the inhibition of the lysosomal protease cathepsin D (CatD), which indicates both mitochondria and the lysosome are involved in the regulation of acetate-induced apoptosis. Herein, we sought to determine whether mitochondrial function affects CatD apoptotic function. We found that enhancement of acetate-induced apoptosis by CatD inhibition depends on oligomycin A-sensitive respiration. Mechanistically, the potentiating effect is associated with an increase in cellular and mitochondrial superoxide anion accumulation and mitochondrial mass. Our results provide novel clues into the regulation of CatD function and the effect of tumor heterogeneity in the outcome of combined treatment using acetate and CatD inhibitors.

Exosomes modified with anti-MEK1 siRNA lead to an effective silencing of triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is a highly heterogenous disease not sensitive to endocrine or HER2 therapy and standardized treatment regimens are still missing. Therefore, development of novel TNBC treatment approaches is of utmost relevance. Herein, the potential of MAPK/ERK downregulation by RNAi-based therapeutics in a panel of mesenchymal stem-like TNBC cell lines was uncovered. Our data revealed that suppression of one of the central nodes of this signaling pathway, MEK1, affects proliferation, migration, and invasion of TNBC cells, that may be explained by the reversion of the epithelial-mesenchymal transition phenotype, which is facilitated by the MMP-2/MMP-9 downregulation. Moreover, an exosome-based system was successfully generated for the siRNA loading (iExoMEK1). Our data suggested absence of modification of the physical properties and general integrity of the iExoMEK1 comparatively to the unmodified counterparts. Such exosome-mediated downregulation of MEK1 led to a tumor regression accompanied by a decrease of angiogenesis using the chick chorioallantoic-membrane model. Our results highlight the potential of the targeting of MAPK/ERK cascade as a promising therapeutic approach against TNBC.

Bovine Lactoferrin-Loaded Plasmonic Magnetoliposomes for Antifungal Therapeutic Applications.

Bovine lactoferrin (bLf) is a milk-derived protein that exhibits potent broad-spectrum antifungal activity against multiple fungi. bLf is susceptible to degradation, while some of its properties depend on the tertiary structure. So, the encapsulation of bLf in stimuli-responsive therapeutic formulations provides an added value to enhance its biological activities. Plasmonic magnetoliposomes (PMLs) arise as promising nanocarriers for dual hyperthermia (magneto-photothermia) and local chemotherapy, since the combination of magnetic and gold nanoparticles (NPs) in a single nanosystem (multifunctional liposomes) enables the targeting and controlled release of loaded drugs. In this work, plasmonic magnetoliposomes (PMLs) containing manganese ferrite nanoparticles (28 nm size) and gold nanoparticles (5-7.5 nm size), functionalized with 11-mercaptoundecanoic acid or octadecanethiol, were prepared and loaded with bLf. The NPs' optical, magnetic and structural properties were measured via UV/vis/NIR absorption spectroscopy, SQUID and TEM, respectively. The Specific Absorption Rate (SAR) was calculated to assess the capabilities for magnetic and photothermal hyperthermia. Finally, the antifungal potential of bLf-loaded PMLs and their mechanism of internalization were assessed in Saccharomyces cerevisiae by counting the colony forming units and using fluorescence microscopy. The results demonstrate that PMLs are mainly internalized through an energy- and temperature-dependent endocytic process, though the contribution of a diffusion component cannot be discarded. Most notably, only bLf-loaded plasmonic magnetoliposomes display cytotoxicity with an efficiency similar to free bLf, attesting their promising potential for bLf delivery in the context of antifungal therapeutic interventions.

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples.

The renewable, abundant , and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples' microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited ß-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples. KEY POINTS: • Sequence- and function-based metagenomics were used to find CAZymes in compost samples. • Thermophilic composts proved to be rich in bacterial GH3, GH5, and GH9 enzymes. • Compost-derived fosmid libraries are enriched in clones with ß-glucosidase activity.

Synthesis and Cytotoxicity Assessment of Citrate-Coated Calcium and Manganese Ferrite Nanoparticles for Magnetic Hyperthermia.

Calcium-doped manganese ferrite nanoparticles (NPs) are gaining special interest in the biomedical field due to their lower cytotoxicity compared with other ferrites, and the fact that they have improved magnetic properties. Magnetic hyperthermia (MH) is an alternative cancer treatment, in which magnetic nanoparticles promote local heating that can lead to the apoptosis of cancer cells. In this work, manganese/calcium ferrite NPs coated with citrate (CaxMn1-xFe2O4 (x = 0, 0.2, 1), were synthesized by the sol-gel method, followed by calcination, and then characterized regarding their crystalline structure (by X-ray diffraction, XRD), size and shape (by Transmission Electron Microscopy, TEM), hydrodynamic size and zeta potential (by Dynamic Light Scattering, DLS), and heating efficiency (measuring the Specific Absorption Rate, SAR, and Intrinsic Loss Power, ILP) under an alternating magnetic field. The obtained NPs showed a particle size within the range of 10 nm to 20 nm (by TEM) with a spherical or cubic shape. Ca0.2Mn0.8Fe2O4 NPs exhibited the highest SAR value of 36.3 W/g at the lowest field frequency tested, and achieved a temperature variation of ~7 °C in 120 s, meaning that these NPs are suitable magnetic hyperthermia agents. In vitro cellular internalization and cytotoxicity experiments, performed using the human cell line HEK 293T, confirmed cytocompatibility over 0-250 µg/mL range and successful internalization after 24 h. Based on these studies, our data suggest that these manganese-calcium ferrite NPs have potential for MH application and further use in in vivo systems.

Lactoferrin perturbs intracellular trafficking, disrupts cholesterol-rich lipid rafts and inhibits glycolysis of highly metastatic cancer cells harbouring plasmalemmal V-ATPase.

The milk-derived bovine lactoferrin (bLf) is an iron-binding glycoprotein with remarkable selective anticancer activity towards highly metastatic cancer cells displaying the proton pump V-ATPase at the plasma membrane. As studies aiming to dissect the bLf mechanisms of action are critical to improve its efficacy and boost its targeted clinical use, herein we sought to further uncover the molecular basis of bLf anticancer activity. We showed that bLf co-localizes with V-ATPase and cholesterol-rich lipid rafts at the plasma membrane of highly metastatic cancer cells. Our data also revealed that bLf perturbs cellular trafficking, induces intracellular accumulation of cholesterol and lipid rafts disruption, downregulates PI3K, and AKT or p-AKT and inhibits glycolysis of cancer cells harbouring V-ATPase at the plasma membrane lipid rafts. Altogether, our results can lay the foundation for future bLf-based targeted anticancer strategies as they unravel a novel cascade of molecular events that explains and further reinforces bLf selectivity for cancer cells displaying plasmalemmal V-ATPase.

Acetic acid triggers cytochrome c release in yeast heterologously expressing human Bax.

Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochrome c (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cyt c release, closely resembling the natural Bax function in the cellular context. This regulated cell death process was reverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3ß. This novel system mimics human Bax α regulation by GSK3ß and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation.

Saccharomyces cerevisiae Cells Lacking the Zinc Vacuolar Transporter Zrt3 Display Improved Ethanol Productivity in Lignocellulosic Hydrolysates.

Yeast-based bioethanol production from lignocellulosic hydrolysates (LH) is an attractive and sustainable alternative for biofuel production. However, the presence of acetic acid (AA) in LH is still a major problem. Indeed, above certain concentrations, AA inhibits yeast fermentation and triggers a regulated cell death (RCD) process mediated by the mitochondria and vacuole. Understanding the mechanisms involved in AA-induced RCD (AA-RCD) may thus help select robust fermentative yeast strains, providing novel insights to improve lignocellulosic ethanol (LE) production. Herein, we hypothesized that zinc vacuolar transporters are involved in vacuole-mediated AA-RCD, since zinc enhances ethanol production and zinc-dependent catalase and superoxide dismutase protect from AA-RCD. In this work, zinc limitation sensitized wild-type cells to AA-RCD, while zinc supplementation resulted in a small protective effect. Cells lacking the vacuolar zinc transporter Zrt3 were highly resistant to AA-RCD, exhibiting reduced vacuolar dysfunction. Moreover, zrt3Δ cells displayed higher ethanol productivity than their wild-type counterparts, both when cultivated in rich medium with AA (0.29 g L-1 h-1 versus 0.11 g L-1 h-1) and in an LH (0.73 g L-1 h-1 versus 0.55 g L-1 h-1). Overall, the deletion of ZRT3 emerges as a promising strategy to increase strain robustness in LE industrial production.

Plasmalemmal V-ATPase as a Potential Biomarker for Lactoferrin-Based Anticancer Therapy.

Lactoferrin (Lf) is a milk-derived protein with well-recognized potential as a therapeutic agent against a wide variety of cancers. This natural protein exhibits health-promoting effects and has several interesting features, including its selectivity towards cancer cells, good tolerability in humans, worldwide availability, and holding a generally recognized as safe (GRAS) status. To prompt the rational clinical application of this promising anticancer compound, previous works aimed to unveil the molecular mechanisms underlying its selective anticancer activity, where plasmalemmal V-ATPase was identified as an Lf target in cancer cells. V-ATPase is a proton pump critical for cellular homeostasis that migrates to the plasma membrane of highly metastatic cancer cells contributing to the acidity of the tumor microenvironment. Cancer cells were found to be susceptible to Lf only when this proton pump is present at the plasma membrane. Plasmalemmal V-ATPase can thus be an excellent biomarker for driving treatment decisions and forecasting clinical outcomes of Lf-based anticancer strategies. Future research endeavors should thus seek to validate this biomarker by thorough preclinical and clinical studies, as well as to develop effective methods for its detection under clinical settings.

A review on lactoferrin as a proton pump inhibitor.

Lactoferrin (Lf) is a versatile natural milk-derived protein that exhibits multiple interesting biological activities. Since it is safe for human administration and currently manufactured using low cost and well-established large-scale processes, the Lf scientific community has been devoted at dissecting its mechanisms of action towards its more rational and efficient use for various applications. Emerging literature has identified proton pumping ATPases as molecular targets of Lf in different cellular models linked to distinct activities of this natural protein. Information on this subject has not been systematically analysed before, hence herein we review the current state of art on the effect of Lf on proton pumping ATPases. Though structurally different, we propose that Lf holds a proton pump inhibitor (PPI)-like activity based on the functional resemblance with the classical inhibitors of the stomach H+/K+-ATPase. The downstream events and outcomes of the PPI-like activity of Lf, as well as its impact for the development of improved Lf applications are also discussed.

The milk-derived lactoferrin inhibits V-ATPase activity by targeting its V1 domain.

Lactoferrin (Lf), a bioactive milk protein, exhibits strong anticancer and antifungal activities. The search for Lf targets and mechanisms of action is of utmost importance to enhance its effective applications. A common feature among Lf-treated cancer and fungal cells is the inhibition of a proton pump called V-ATPase. Lf-driven V-ATPase inhibition leads to cytosolic acidification, ultimately causing cell death of cancer and fungal cells. Given that a detailed elucidation of how Lf and V-ATPase interact is still missing, herein we aimed to fill this gap by employing a five-stage computational approach. Molecular dynamics simulations of both proteins were performed to obtain a robust sampling of their conformational landscape, followed by clustering, which allowed retrieving representative structures, to then perform protein-protein docking. Subsequently, molecular dynamics simulations of the docked complexes and free binding energy calculations were carried out to evaluate the dynamic binding process and build a final ranking based on the binding affinities. Detailed atomist analysis of the top ranked complexes clearly indicates that Lf binds to the V1 cytosolic domain of V-ATPase. Particularly, our data suggest that Lf binds to the interfaces between A/B subunits, where the ATP hydrolysis occurs, thus inhibiting this process. The free energy decomposition analysis further identified key binding residues that will certainly aid in the rational design of follow-up experimental studies, hence bridging computational and experimental biochemistry.

Regulation of Cell Death Induced by Acetic Acid in Yeasts.

Acetic acid has long been considered a molecule of great interest in the yeast research field. It is mostly recognized as a by-product of alcoholic fermentation or as a product of the metabolism of acetic and lactic acid bacteria, as well as of lignocellulosic biomass pretreatment. High acetic acid levels are commonly associated with arrested fermentations or with utilization as vinegar in the food industry. Due to its obvious interest to industrial processes, research on the mechanisms underlying the impact of acetic acid in yeast cells has been increasing. In the past twenty years, a plethora of studies have addressed the intricate cascade of molecular events involved in cell death induced by acetic acid, which is now considered a model in the yeast regulated cell death field. As such, understanding how acetic acid modulates cellular functions brought about important knowledge on modulable targets not only in biotechnology but also in biomedicine. Here, we performed a comprehensive literature review to compile information from published studies performed with lethal concentrations of acetic acid, which shed light on regulated cell death mechanisms. We present an historical retrospective of research on this topic, first providing an overview of the cell death process induced by acetic acid, including functional and structural alterations, followed by an in-depth description of its pharmacological and genetic regulation. As the mechanistic understanding of regulated cell death is crucial both to design improved biomedical strategies and to develop more robust and resilient yeast strains for industrial applications, acetic acid-induced cell death remains a fruitful and open field of study.

Lactoferrin perturbs lipid rafts and requires integrity of Pma1p-lipid rafts association to exert its antifungal activity against Saccharomyces cerevisiae.

Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs ergosterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma1-10 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi.

Emerging insights on the role of V-ATPase in human diseases: Therapeutic challenges and opportunities.

The control of the intracellular pH is vital for the survival of all organisms. Membrane transporters, both at the plasma and intracellular membranes, are key players in maintaining a finely tuned pH balance between intra- and extracellular spaces, and therefore in cellular homeostasis. V-ATPase is a housekeeping ATP-driven proton pump highly conserved among prokaryotes and eukaryotes. This proton pump, which exhibits a complex multisubunit structure based on cell type-specific isoforms, is essential for pH regulation and for a multitude of ubiquitous and specialized functions. Thus, it is not surprising that V-ATPase aberrant overexpression, mislocalization, and mutations in V-ATPase subunit-encoding genes have been associated with several human diseases. However, the ubiquitous expression of this transporter and the high toxicity driven by its off-target inhibition, renders V-ATPase-directed therapies very challenging and increases the need for selective strategies. Here we review emerging evidence linking V-ATPase and both inherited and acquired human diseases, explore the therapeutic challenges and opportunities envisaged from recent data, and advance future research avenues. We highlight the importance of V-ATPases with unique subunit isoform molecular signatures and disease-associated isoforms to design selective V-ATPase-directed therapies. We also discuss the rational design of drug development pipelines and cutting-edge methodological approaches toward V-ATPase-centered drug discovery. Diseases like cancer, osteoporosis, and even fungal infections can benefit from V-ATPase-directed therapies.

Selection of a new peptide homing SK-BR-3 breast cancer cells.

Breast cancer diagnosis remains a challenge, mostly due to its heterogeneity. This reality translates in delayed treatments, increasing treatment aggressiveness and lower chances of overall survival. The conventional detection techniques, although becoming increasingly sophisticated each year, still lack the ability to provide reliable conclusions without being time consuming, expensive, and uncomfortable for the patients. The identification of novel biomarkers for breast cancer research is therefore of utmost relevance for an early diagnosis. Moreover, breast cancer-specific peptide moieties can be used to develop novel targeted drug delivery systems. In this work, we used phage display to identify a novel peptide with specificity to the SK-BR-3 breast cancer cell line. Cytometry assays confirmed its specificity, while bioinformatics and docking studies predicted the potential biomarkers at the SK-BR-3 cells' surface. These findings can be potentially useful in the clinical context, contributing to more specific and targeted therapeutic solutions against HER2-positive breast cancer subtypes.

Resveratrol-Loaded Lipid Nanocarriers Are Internalized By Endocytosis in Yeast.

Different positive pharmacological effects have been attributed to the natural product resveratrol (RSV), including antioxidant, antiaging, and cancer chemopreventive properties. However, its low bioavailability and rapid metabolic degradation has led to the suspicion that many of the biological activities of this compound observed in vitro may not be attainable in humans. To improve its bioavailability and pharmacokinetic profile, attempts have been made to encapsulate RSV into lipid-based nanocarrier systems. Here, the dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) liposomal system (1:2) loaded with RSV revealed appropriate characteristics for drug release purposes: reduced size for cellular uptake (157 ± 23 nm), stability up to 80 days, positive surface charge (ζ ≈ +40 mV), and a controlled biphasic release of RSV from the lipid nanocarriers over a period of almost 50 h at pH 5.0 and 7.4. Moreover, the encapsulation efficiency of the nanocarrier ranged from 70% to 92% and its RSV loading capacity from 9% to 14%, when [RSV] was between 100 and 200 µM. The partition coefficient ( Kp) of RSV between lipid and aqueous phase was log Kp = 3.37 ± 0.10, suggesting moderate to high lipophilicity of this natural compound and reinforcing the lipid nanocarriers' suitability for RSV incorporation. The thermodynamic parameters of RSV partitioning in the lipid nanocarriers at 37 °C (Δ H = 43.76 ± 5.68 kJ mol-1; Δ S = 0.20 ± 0.005 kJ mol-1; and Δ G = -18.46 ± 3.48 kJ mol-1) reflected the spontaneity of the process and the establishment of hydrophobic interactions. The cellular uptake mechanism of the RSV-loaded nanocarriers labeled with the lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was studied in the eukaryotic model system Saccharomyces cerevisiae. Thirty minutes after incubation, yeast cells readily internalized nanocarriers and the spots of blue fluorescence of DPH clustered around the central vacuole in lipid droplets colocalized with the green fluorescence of the lipophilic endocytosis probe FM1-43. Subsequent studies with the endocytosis defective yeast deletion mutant ( end3Δ) and with the endocytosis inhibitor methyl-ß-cyclodextrin supported the involvement of an endocytic pathway. This novel nanotechnology approach opens good perspectives for medical applications.

HS.Register - An Audit-Trail Tool to Respond to the General Data Protection Regulation (GDPR).

Introduction The new General Data Protection Regulation (GDPR) compels health care institutions and their software providers to properly document all personal data processing and provide clear evidence that their systems are inline with the GDPR. All applications involved in personal data processing should therefore produce meaningful event logs that can later be used for the effective auditing of complex processes. Aim This paper aims to describe and evaluate HS.Register, a system created to collect and securely manage at scale audit logs and data produced by a large number of systems. Methods HS.Register creates a single audit log by collecting and aggregating all kinds of meaningful event logs and data (e.g. ActiveDirectory, syslog, log4j, web server logs, REST, SOAP and HL7 messages). It also includes specially built dashboards for easy auditing and monitoring of complex processes, crossing different systems in an integrated way, as well as providing tools for helping on the auditing and on the diagnostics of difficult problems, using a simple web application. HS.Register is currently installed at five large Portuguese Hospitals and is composed of the following open-source components: HAproxy, RabbitMQ, Elasticsearch, Logstash and Kibana. Results HS.Register currently collects and analyses an average of 93 million events per week and it is being used to document and audit HL7 communications. Discussion Auditing tools like HS.Register are likely to become mandatory in the near future to allow for traceability and detailed auditing for GDPR compliance.