A hierarchical assembly pathway directs the unique subunit arrangement of TRiC/CCT.

How the essential eukaryotic chaperonin TRiC/CCT assembles from eight distinct subunits into a unique double-ring architecture remains undefined. We show TRiC assembly involves a hierarchical pathway that segregates subunits with distinct functional properties until holocomplex (HC) completion. A stable, likely early intermediate arises from small oligomers containing CCT2, CCT4, CCT5, and CCT7, contiguous subunits that constitute the negatively charged hemisphere of the TRiC chamber, which has weak affinity for unfolded actin. The remaining subunits CCT8, CCT1, CCT3, and CCT6, which comprise the positively charged chamber hemisphere that binds unfolded actin more strongly, join the ring individually. Unincorporated late-assembling subunits are highly labile in cells, which prevents their accumulation and premature substrate binding. Recapitulation of assembly in a recombinant system demonstrates that the subunits in each hemisphere readily form stable, noncanonical TRiC-like HCs with aberrant functional properties. Thus, regulation of TRiC assembly along a biochemical axis disfavors the formation of stable alternative chaperonin complexes.

Rab27a GTPase and its effector Myosin Va are host factors required for efficient Oropouche virus cell egress.

Oropouche fever, a debilitating illness common in South America, is caused by Oropouche virus (OROV), an arbovirus. OROV belongs to the Peribunyaviridae family, a large group of RNA viruses. Little is known about the biology of Peribunyaviridae in host cells, especially assembly and egress processes. Our research reveals that the small GTPase Rab27a mediates intracellular transport of OROV induced compartments and viral release from infected cells. We show that Rab27a interacts with OROV glycoproteins and colocalizes with OROV during late phases of the infection cycle. Moreover, Rab27a activity is required for OROV trafficking to the cell periphery and efficient release of infectious particles. Consistently, depleting Rab27a's downstream effector, Myosin Va, or inhibiting actin polymerization also hinders OROV compartments targeting to the cell periphery and infectious viral particle egress. These data indicate that OROV hijacks Rab27a activity for intracellular transport and cell externalization. Understanding these crucial mechanisms of OROV's replication cycle may offer potential targets for therapeutic interventions and aid in controlling the spread of Oropouche fever.

Oropouche Virus Glycoprotein Topology and Cellular Requirements for Glycoprotein Secretion.

Oropouche virus (OROV; genus Orthobunyavirus) is the etiological agent of Oropouche fever, a debilitating febrile illness common in South America. We used recombinant expression of the OROV M polyprotein, which encodes the surface glycoproteins Gn and Gc plus the nonstructural protein NSm, to probe the cellular determinants for OROV assembly and budding. Gn and Gc self-assemble and are secreted independently of NSm. Mature OROV Gn has two predicted transmembrane domains that are crucial for glycoprotein translocation to the Golgi complex and glycoprotein secretion, and unlike related orthobunyaviruses, both transmembrane domains are retained during Gn maturation. Disruption of Golgi function using the drugs brefeldin A and monensin inhibits glycoprotein secretion. Infection studies have previously shown that the cellular endosomal sorting complexes required for transport (ESCRT) machinery is recruited to Golgi membranes during OROV assembly and that ESCRT activity is required for virus secretion. A dominant-negative form of the ESCRT-associated ATPase VPS4 significantly reduces recombinant OROV glycoprotein secretion and blocks virus release from infected cells, and VPS4 partly colocalizes with OROV glycoproteins and membranes costained with Golgi markers. Furthermore, immunoprecipitation and fluorescence microscopy experiments demonstrate that OROV glycoproteins interact with the ESCRT-III component CHMP6, with overexpression of a dominant-negative form of CHMP6 significantly reducing OROV glycoprotein secretion. Taken together, our data highlight differences in M polyprotein processing across orthobunyaviruses, indicate that Golgi and ESCRT function are required for glycoprotein secretion, and identify CHMP6 as an ESCRT-III component that interacts with OROV glycoproteins. IMPORTANCE Oropouche virus causes Oropouche fever, a debilitating illness common in South America that is characterized by high fever, headache, myalgia, and vomiting. The tripartite genome of this zoonotic virus is capable of reassortment, and there have been multiple epidemics of Oropouche fever in South America over the last 50 years, making Oropouche virus infection a significant threat to public health. However, the molecular characteristics of this arbovirus are poorly understood. We developed a recombinant protein expression system to investigate the cellular determinants of OROV glycoprotein maturation and secretion. We show that the proteolytic processing of the M polypeptide, which encodes the surface glycoproteins (Gn and Gc) plus a nonstructural protein (NSm), differs between OROV and its close relative Bunyamwera virus. Furthermore, we demonstrate that OROV M glycoprotein secretion requires the cellular endosomal sorting complexes required for transport (ESCRT) membrane-remodeling machinery and identify that the OROV glycoproteins interact with the ESCRT protein CHMP6.

ESCRT machinery components are required for Orthobunyavirus particle production in Golgi compartments.

Peribunyaviridae is a large family of RNA viruses with several members that cause mild to severe diseases in humans and livestock. Despite their importance in public heath very little is known about the host cell factors hijacked by these viruses to support assembly and cell egress. Here we show that assembly of Oropouche virus, a member of the genus Orthobunyavirus that causes a frequent arboviral infection in South America countries, involves budding of virus particles toward the lumen of Golgi cisternae. As viral replication progresses, these Golgi subcompartments become enlarged and physically separated from Golgi stacks, forming Oropouche viral factory (Vfs) units. At the ultrastructural level, these virally modified Golgi cisternae acquire an MVB appearance, and while they lack typical early and late endosome markers, they become enriched in endosomal complex required for transport (ESCRT) proteins that are involved in MVB biogenesis. Further microscopy and viral replication analysis showed that functional ESCRT machinery is required for efficient Vf morphogenesis and production of infectious OROV particles. Taken together, our results indicate that OROV attracts ESCRT machinery components to Golgi cisternae to mediate membrane remodeling events required for viral assembly and budding at these compartments. This represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.

Design, synthesis, antileishmanial, and antifungal biological evaluation of novel 3,5-disubstituted isoxazole compounds based on 5-nitrofuran scaffolds.

Nineteen 3,5-disubstituted-isoxazole analogs were synthesized based on nitrofuran scaffolds, by a [3 + 2] cycloaddition reaction between terminal acetylenes and 5-nitrofuran chloro-oxime. The compounds were obtained in moderate to very good yields (45-91%). The antileishmanial activity was assayed against the promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. Alkylchlorinated compounds 14p-r were active on both the promastigote and amastigote forms, with emphasis on compound 14p, which showed strong activity against the amastigote form (IC50 = 0.6 µM and selectivity index [SI] = 5.2). In the alkyl series, compound 14o stands out with an IC50 = 8.5 µM and SI = 8.0 on the amastigote form. In the aromatic series, the most active compounds were those containing electron-donor groups, such as trimethoxy isoxazole 14g (IC50 = 1.2 µM and SI = 20.2); compound 14h, with IC50 = 7.0 µM and SI = 6.1; and compound 14j containing the 4-SCH3 group, with IC50 = 5.7 µM and SI = 10.2. In addition, the antifungal activity of 19 nitrofuran isoxazoles was evaluated against five strains of Candida (C. albicans, C. parapsilosis, C. krusei, C. tropicalis, and C. glabrata). Eleven isoxazole derivatives were active against C. parapsilosis, and compound 14o was found to be the most active (minimal inhibitory concentration [MIC] = 3.4 µM) for this strain. Compound 14p was active against all the strains tested, with an MIC = 17.5 µM for C. glabrata, lower than that of the fluconazole used as the reference drug.

The polyproline-motif of S6K2: eIF5A translational dependence and importance for protein-protein interactions.

Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2∆Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.

Evaluation of laser therapy and alpha-lipoic acid for the treatment of burning mouth syndrome: a randomized clinical trial.

The aim of this study was to evaluate the efficacy of low-level laser therapy (LLLT) and alpha-lipoic acid (ALA) in the treatment of burning mouth syndrome (BMS) and secondary oral burning (SOB) by unstimulated sialometry, symptom assessment, and measurement of salivary TNF-α levels. Forty-four patients were randomized into four treatment groups: BMS/laser (n = 10), BMS/ALA (n = 5), SOB/laser (n = 15), and SOB/ALA (n = 14). The control group consisted of eight healthy female subjects. Unstimulated salivary flow was measured before and after treatment, and the collected saliva was stored at - 20 °C for the analysis of TNF-α. Symptoms were evaluated before and after treatment using a pain visual analog scale. Most patients were women (81.8%) during menopause (72.2%). LLLT and ALA were efficient in increasing salivary flow only in BMS but provided symptom relief in both conditions. TNF-α levels did not differ between patients with BMS and SOB or between those patients and the control group. No differences were observed in posttreatment TNF-α levels in either condition. The results of this study suggest that LLLT and ALA are efficient therapies in reducing burning mouth symptoms, with LLLT being more efficient than ALA.

Mapping surface residues of eIF5A that are important for binding to the ribosome using alanine scanning mutagenesis.

The translation elongation factor eIF5A is conserved through evolution and is necessary to rescue the ribosome during translation elongation of polyproline-containing proteins. Although the site of eIF5A binding to the ribosome is known, no systematic analysis has been performed so far to determine the important residues on the surface of eIF5A required for ribosome binding. In this study, we used clustered charged-to-alanine mutagenesis and structural modeling to address this question. We generated four new mutants of yeast eIF5A: tif51A-4, tif51A-6, tif51A-7 and tif51A-11, and complementation analysis revealed that tif51A-4 and tif51A-7 could not sustain cell growth in a strain lacking wild-type eIF5A. Moreover, the allele tif51A-4 also displayed negative dominance over wild-type eIF5A. Both in vivo GST-pulldowns and in vitro fluorescence anisotropy demonstrated that eIF5A from mutant tif51A-7 exhibited an importantly reduced affinity for the ribosome, implicating the charged residues in cluster 7 as determinant features on the eIF5A surface for contacting the ribosome. Notably, modified eIF5A from mutant tif51A-4, despite exhibiting the most severe growth phenotype, did not abolish ribosome interactions as with mutant tif51A-7. Taking into account the modeling eIF5A + 80S + P-tRNA complex, our data suggest that interactions of eIF5A with ribosomal protein L1 are more important to stabilize the interaction with the ribosome as a whole than the contacts with P-tRNA. Finally, the ability of eIF5A from tif51A-4 to bind to the ribosome while potentially blocking physical interaction with P-tRNA could explain its dominant negative phenotype.

The future point-of-care detection of disease and its data capture and handling.

Point-of-care detection is a widely studied area that attracts effort and interest from a large number of fields and companies. However, there is also increased interest from the general public in this type of device, which has driven enormous changes in the design and conception of these developments and the way data is handled. Therefore, future point-of-care detection has to include communication with front-end technology, such as smartphones and networks, automation of manufacture, and the incorporation of concepts like the Internet of Things (IoT) and cloud computing. Three key examples, based on different sensing technology, are analyzed in detail on the basis of these items to highlight a route for the future design and development of point-of-care detection devices and their data capture and handling.

Comparative Analysis of Psychological, Hormonal, and Genetic Factors Between Burning Mouth Syndrome and Secondary Oral Burning.

OBJECTIVE: The objective of this study was to evaluate the association between psychological, hormonal, and genetic factors with the development of burning mouth syndrome (BMS) and secondary oral burning (SOB) in order to provide a better characterization and classification of these conditions. DESIGN: Cross sectional study. SETTING: Patients with complaints of mouth burning registered at the Oral Diagnostic Service of the Federal University of Rio Grande do Norte between 2000 and 2013. SUBJECTS: The sample consisted of 163 subjects divided into a group of patients with BMS (n = 64) and a group of subjects with SOB (n = 99). METHODS: The following variables were analyzed: passive and stimulated saliva flow, stress levels and phase, depression, anxiety, serum cortisol and dehydroepiandrosterone (DHEA) levels, and the presence of polymorphisms in the interleukin 6 (IL-6) gene. RESULTS: The results showed significant differences in the presence of xerostomia (p = 0.01), hyposalivation at rest (p < 0.001) and symptoms of depression (p = 0.033) between the two groups, which were more prevalent in the BMS group. DHEA levels were lower in the BMS group (p = 0.003) and were sensitive and specific for the diagnosis of this condition. Genetic analysis revealed no significant association between the polymorphisms analyzed and the development of BMS. CONCLUSION: These results suggest a possible role of depression, as well as of reduced DHEA levels, as associated factors for development of BMS.

Influence of Dentin Deproteinization on Bonding Degradation: 1-year Results.

INTRODUCTION: The decalcification protocol of dentin surface with sodium hypochlorite removes the exposed collagen fibrils and could improve the longevity of the bonding interface. This study evaluated the influence of collagen removal with 10% sodium hypochlorite (10% NaOCl) on the longitudinal shear bond strength (SBS) of adhesives to dentin. MATERIALS AND METHODS: Seventy-two extracted human molars were sectioned and the buccal and lingual surfaces were flattened and acid etched with 37% phosphoric acid for 15 seconds. The specimens were divided into six groups (n = 12 teeth - 24 sections), according to adhesive and collagen removal protocol: group 1: UNO Dentastic; group 2: Prime and Bond NT; group 3: Single Bond; group 4: 10% NaOCl + UNO Dentastic; group 5: 10% NaOCl + Prime and Bond NT; group 6: 10% NaOCl + Single Bond. Composite Z100 buildup was prepared, and the SBS test was assessed after 24 hours and 1 year. Data were submitted to three-way analysis of variance (ANOVA) and Tukey tests (p < 0.05). RESULTS: The mean values (MPa) were for 24 hours: G1: 22.45B; G2: 7.90DE; G3: 12.56CD; G4: 19.85BC; G5: 33.73A; G6: 20.77B; and for 1 year: G1: 2.43E; G2: 2.26E; G3: 4.3DE; G4: 18.79BC; G5: 26.49AB; G6: 22.73B. CONCLUSION: Dentin deproteinization treatment with 10% NaOCI influenced the SBS compared with conventional treatment. The negative influence on SBS detected for conventional groups at 1-year interval was not detected for deproteinized groups. CLINICAL RELEVANCE: The longevity of hybrid layer is critical due to the hydrolysis process at the adhesive interface over time. The use of 10% NaOCl deproteinization protocol might improve the longevity of bonding in adhesive restorations.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

Profiling Germinal Center-like B Cell Responses to Conjugate Vaccines Using Synthetic Immune Organoids.

Glycoengineered bacteria have emerged as a cost-effective platform for rapid and controllable biosynthesis of designer conjugate vaccines. However, little is known about the engagement of such conjugates with naïve B cells to induce the formation of germinal centers (GC), a subanatomical microenvironment that converts naïve B cells into antibody-secreting plasma cells. Using a three-dimensional biomaterials-based B-cell follicular organoid system, we demonstrate that conjugates triggered robust expression of hallmark GC markers, B cell receptor clustering, intracellular signaling, and somatic hypermutation. These responses depended on the relative immunogenicity of the conjugate and correlated with the humoral response in vivo. The occurrence of these mechanisms was exploited for the discovery of high-affinity antibodies against components of the conjugate on a time scale that was significantly shorter than for typical animal immunization-based workflows. Collectively, these findings highlight the potential of synthetic organoids for rapidly predicting conjugate vaccine efficacy as well as expediting antigen-specific antibody discovery.

SaLT&PepPr is an interface-predicting language model for designing peptide-guided protein degraders.

Protein-protein interactions (PPIs) are critical for biological processes and predicting the sites of these interactions is useful for both computational and experimental applications. We present a Structure-agnostic Language Transformer and Peptide Prioritization (SaLT&PepPr) pipeline to predict interaction interfaces from a protein sequence alone for the subsequent generation of peptidic binding motifs. Our model fine-tunes the ESM-2 protein language model (pLM) with a per-position prediction task to identify PPI sites using data from the PDB, and prioritizes motifs which are most likely to be involved within inter-chain binding. By only using amino acid sequence as input, our model is competitive with structural homology-based methods, but exhibits reduced performance compared with deep learning models that input both structural and sequence features. Inspired by our previous results using co-crystals to engineer target-binding "guide" peptides, we curate PPI databases to identify partners for subsequent peptide derivation. Fusing guide peptides to an E3 ubiquitin ligase domain, we demonstrate degradation of endogenous ß-catenin, 4E-BP2, and TRIM8, and highlight the nanomolar binding affinity, low off-targeting propensity, and function-altering capability of our best-performing degraders in cancer cells. In total, our study suggests that prioritizing binders from natural interactions via pLMs can enable programmable protein targeting and modulation.

Morphological, biochemical, physiological and molecular aspects of the response of Fusobacterium nucleatum exposed to subinhibitory concentrations of antimicrobials.

Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of ß-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host-bacteria relationship.

Cotranslational Mechanisms of Protein Biogenesis and Complex Assembly in Eukaryotes.

The formation of protein complexes is crucial to most biological functions. The cellular mechanisms governing protein complex biogenesis are not yet well understood, but some principles of cotranslational and posttranslational assembly are beginning to emerge. In bacteria, this process is favored by operons encoding subunits of protein complexes. Eukaryotic cells do not have polycistronic mRNAs, raising the question of how they orchestrate the encounter of unassembled subunits. Here we review the constraints and mechanisms governing eukaryotic co- and posttranslational protein folding and assembly, including the influence of elongation rate on nascent chain targeting, folding, and chaperone interactions. Recent evidence shows that mRNAs encoding subunits of oligomeric assemblies can undergo localized translation and form cytoplasmic condensates that might facilitate the assembly of protein complexes. Understanding the interplay between localized mRNA translation and cotranslational proteostasis will be critical to defining protein complex assembly in vivo.

Congo Red Decolorization Using Textile Filters and Laccase-Based Nanocomposites in Continuous Flow Bioreactors.

Removal of azo and diazo dye content from textile industry wastewaters is crucial due to their environmental impact. Here, we report on the use of the fungal laccase from Pycnoporus sanguineus CS43 immobilized on silica nanoparticles and entrapped in textile-based filters for the degradation of Congo Red. Laccase immobilization and synthesis of the nanocomposites were carried out by two different methods, one in the presence of acetone and the second using water as solvent. This led to a change in the hydrophobicity of the obtained biofilters. Successful preparation of the nanocomposites was confirmed via FTIR spectroscopy. Changes in the secondary structure of the enzyme were inspected through the second derivative of the FTIR spectra. Six different types of filter were fabricated and tested in a continuous flow bioreactor in terms of their decolorization capabilities of Congo Red. The results indicate removal efficiencies that approached 40% for enzymes immobilized on the more hydrophobic supports. Backscattered electron (BSE) images of the different filters were obtained before and after the decolorization process. Percentage of decolorization and activity loss were determined as a function of time until a plateau in decolorization activity was reached. Experimental data was used to recreate the decolorization process in COMSOL Multiphysics® (Stockholm, Sweden). These simulations were used to determine the proper combination of parameters to maximize decolorization. Our findings suggest that the treatment of textile-based filters with immobilized laccase in conjunction with hydrophobic nanocomposites provides a suitable avenue to achieve more efficient laccase dye decolorization (39%) than that obtained with similar filters treated only with free laccase (8%). Filters treated with silica-based nanocomposites and immobilized laccases showed an increase in their decolorization capability, probably due to changes in their wetting phenomena.

Enhanced Catalytic Dye Decolorization by Microencapsulation of Laccase from P. Sanguineus CS43 in Natural and Synthetic Polymers.

Polymeric microcapsules with the fungal laccase from Pycnoporus sanguineus CS43 may represent an attractive avenue for the removal or degradation of dyes from wastewaters. Microcapsules of alginate/chitosan (9.23 ± 0.12 µm) and poly(styrenesulfonate) (PSS) (9.25 ± 0.35 µm) were synthesized and subsequently tested for catalytic activity in the decolorization of the diazo dye Congo Red. Successful encapsulation into the materials was verified via confocal microscopy of labeled enzyme molecules. Laccase activity was measured as a function of time and the initial reaction rates were recovered for each preparation, showing up to sevenfold increase with respect to free laccase. The ability of substrates to diffuse through the pores of the microcapsules was evaluated with the aid of fluorescent dyes and confocal microscopy. pH and thermal stability were also measured for encapsulates, showing catalytic activity for pH values as low as 4 and temperatures of about 80 °C. Scanning electron microscope (SEM) analyses demonstrated the ability of PSS capsules to avoid accumulation of byproducts and, therefore, superior catalytic performance. This was corroborated by the direct observation of substrates diffusing in and out of the materials. Compared with our PSS preparation, alginate/chitosan microcapsules studied by others degrade 2.6 times more dye, albeit with a 135-fold increase in units of enzyme per mg of dye. Similarly, poly(vinyl) alcohol microcapsules from degrade 1.7 times more dye, despite an eightfold increase in units of enzyme per mg of dye. This could be potentially beneficial from the economic viewpoint as a significantly lower amount of enzyme might be needed for the same decolorization level achieved with similar encapsulated systems.

Multifunctional magnetite nanoparticles to enable delivery of siRNA for the potential treatment of Alzheimer's.

Therapeutic drugs for Alzheimer's disease have been extensively studied due to its recurrence and abundance among neurodegenerative diseases. It is thought that the accumulation of amyloid precursor protein (APP) products, a consequence of an up-regulation of the ß-site APP-cleaving enzyme 1 (BACE1), is the main triggering mechanism during the early stages of the disease. This study aims to explore the ability of a multifunctional conjugate based on magnetite nanoparticles for the cellular delivery of siRNA against the expression of the BACE1 gene. We immobilized the siRNA strand on PEGylated magnetite nanoparticles and investigated the effects on biocompatibility and efficacy of the conjugation. Similarly, we co-immobilized the translocating protein OmpA on PEGylated nanoparticles to enhance cellular uptake and endosomal escape. BACE1 suppression was statistically significant in HFF-1 cells, without any presence of a cytotoxic effect. The delivery of the nanoconjugate was achieved through endocytosis pathways, where endosome formation was likely escaped due to the proton-sponge effect characteristic of PEGylated nanoparticles or mainly by direct translocation in the case of OmpA/PEGylated nanoparticles.

Magnetite-OmpA Nanobioconjugates as Cell-Penetrating Vehicles with Endosomal Escape Abilities.

Outer membrane protein A (OmpA) has been extensively studied in Gram-negative bacteria due to its relevance in the adhesion of pathogens to host cells and its surfactant capabilities. It consists of a hydrophobic ß-barrel domain and a hydrophilic periplasmic domain, that confers OmpA an amphiphilic structure. This study aims to elucidate the capacity of Escherichia coli OmpA to translocate liposomal membranes and serve as a potential cell-penetrating vehicle. We immobilized OmpA on magnetite nanoparticles and investigated the possible functional changes exhibited by OmpA after immobilization. Liposomal intake was addressed using egg lecithin liposomes as a model, where magnetite-OmpA nanobioconjugates were able to translocate the liposomal membrane and caused a disruptive effect when subjected to a magnetic field. Nanobioconjugates showed both low cytotoxicity and hemolytic tendency. Additional interactions within the intracellular space led to altered viability results via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confocal microscopy images revealed that immobilized nanoparticles effectively enter the cytoplasm of THP-1 and Vero cells by different routes, and, subsequently, some escape endosomes, lysosomes, and other intracellular compartments with relatively high efficiencies. This was demonstrated by co-localization analyses with LysoTracker green that showed Pearson correlations of about 80 and 28%.