Gut microbiota as a trigger of accelerated directional adaptive evolution. Acquisition of herbivory in the context of extracellular vesicles, microRNAs and inter-kingdom crosstalk

According to a traditional view, the specific diet in vertebrates is one of the key factors structuring the composition of the gut microbiota. In this interpretation, the microbiota assumes a subordinate position, where the larger host shapes, through evolution and its fitness, the taxonomical composition of the hosted microbiota. The present contribution shows how the evolution of herbivory, framed within the new concept of holobiont, the possibility of inter-kingdom crosstalk and its epigenetic effects, could pave the way to a completely reversed interpretation: instead of being passively shaped, the microbiota can mold and shape the general host body structure to increase its fitness. Central elements to consider in this context are the inter-kingdom crosstalk, the possibility of transporting RNAs through nanovesicles in feces from parents to offspring, and the activation of epigenetic processes passed on vertically from generation to generation. The new hypothesis is that the gut microbiota could play a great role in the macroevolutionary dynamics of herbivorous vertebrates, causing directly through host-microbiota dialog of epigenetic nature (i.e., methylation, histone acetylation, etc.), major changes in the organisms phenotype. The vertical exchange of the same microbial communities from parents to offspring, the interaction of these microbes with fairly uniform genotypes, and the socially restricted groups where these processes take place, could all explain the reasons why herbivory has appeared several time (and independently) during the evolution of vertebrates. The new interpretation could also represent a key factor in understanding the convergent evolution of analogous body structures in very distant lineages

Digital detection of exosomes by interferometric imaging

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio

Lysosomal Delivery of Bioactive Proteins to Living Human Cells via Engineered Exosomes

Exosomes are naturally secreted nanovesicles derived from mammalian cells that are used for intercellular communication in vivo. As a result, they can potentially be used for intracellular delivery of therapeutics for disease treatment. We have developed an exosome pseudotyping approach using vesicular stomatitis virus glycoprotein (VSVG) to produce protein chimeras that optimize production of modified exosomes containing protein therapeutics and facilitate effective delivery to their target cells. To the VSVG transmembrane scaffold, we have fused both fluorescent and luminescent reporters for exosome tracking/visualization and quantification of activity respectively. Through our design, we have shown the biogenesis of VSVG modified exosomes from transfected producer cells through fluorescence imaging and the production of a VSVG-based stable cell line. In addition, we have characterized isolated engineered exosomes and shown that they exhibited the correct size, distribution, and molecular markers, while retaining the bioactivity of their protein cargo. Furthermore, we show that our engineered exosomes and their protein cargo are internalized by multiple cell lines into the endosomal and lysosomal compartments of those cells. Lastly, these modified exosomes can confer their bioactive cargo, either a luminescent reporter or puromycin resistance into these target cells. In summary, this study presents a novel approach to exosome engineering to enhance therapeutic protein loading and delivery, and more importantly, shows the delivery of modified exosomes to intracellular lysosomal compartments. This aspect leads to the assumption that in future studies, these engineered exosomes can be used as a vehicle for delivery of therapeutic proteins for treatment of lysosomal storage diseases

Self-Assembling Peptide Detergents Stabilize Isolated Photosystem Ion a Dry Surface for an Extended Time

We used a class of designed peptide detergents to stabilize photosystem I (PS-I) upon extended drying under N(2) on a gold-coated-Ni-NTA glass surface. PS-I is a chlorophyll-containing membrane protein complex that is the primary reducer of ferredoxin and the electron acceptor of plastocyanin. We isolated the complex from the thylakoids of spinach chloroplasts using a chemical detergent. The chlorophyll molecules associated with the PS-I complex provide an intrinsic steady-state emission spectrum between 650 and 800 nm at −196.15 °C that reflects the organization of the pigment-protein interactions. In the absence of detergents, a large blue shift of the fluorescence maxima from approximately 735 nm to approximately 685 nm indicates a disruption in light-harvesting subunit organization, thus revealing chlorophyll−protein interactions. The commonly used membrane protein-stabilizing detergents, N-dodecyl-β-D-maltoside and N-octyl-β-D-glucoside, only partially stabilized the approximately 735-nm complex with approximately 685-nm spectroscopic shift. However, prior to drying, addition of the peptide detergent acetyl- AAAAAAK at increasing concentration significantly stabilized the PS-I complex. Moreover, in the presence of acetyl- AAAAAAK, the PS-I complex is stable in a dried form at room temperature for at least 3 wk. Another peptide detergent, acetyl-VVVVVVD, also stabilized the complex but to a lesser extent. These observations suggest that the peptide detergents may effectively stabilize membrane proteins in the solid-state. These designed peptide detergents may facilitate the study of diverse types of membrane proteins

Deformable Nanovesicles Synthesized through an Adaptable Microfluidic Platform for Enhanced Localized Transdermal Drug Delivery.

Phospholipid-based deformable nanovesicles (DNVs) that have flexibility in shape offer an adaptable and facile method to encapsulate diverse classes of therapeutics and facilitate localized transdermal delivery while minimizing systemic exposure. Here we report the use of a microfluidic reactor for the synthesis of DNVs and show that alteration of input parameters such as flow speeds as well as molar and flow rate ratios increases entrapment efficiency of drugs and allows fine-tuning of DNV size, elasticity, and surface charge. To determine the ability of DNV-encapsulated drug to be delivered transdermally to a local site, we synthesized, characterized, and tested DNVs carrying the fluorescently labeled hydrophilic bisphosphonate drug AF-647 zoledronate (AF647-Zol). AF647-Zol DNVs were lyophilized, resuspended, and applied topically as a paste to the calvarial skin of mice. High-resolution fluorescent imaging and confocal microscopy revealed significant increase of encapsulated payload delivery to the target tissue-cranial bone-by DNVs as compared to nondeformable nanovesicles (NVs) or aqueous drug solutions. Interestingly, NV delivery was not superior to aqueous drug solution. Our studies show that microfluidic reactor-synthesized DNVs can be produced in good yield, with high encapsulation efficiency, reproducibility, and stability after storage, and represent a useful vehicle for localized transdermal drug delivery

Nanoparticles for drug delivery across the blood-brain barrier: a cell culture study

Background: Efficient drug delivery across the blood-brain barriers (BBB) is a central problem in pharmaceutical treatment of neurological diseases. Most pharmaceutical drug candidates including hydrophilic molecules, biopharmaceuticals, and efflux transporter ligands have a low permeability across barriers. To solve this unmet therapeutical need vesicular or solid nanoparticle drug delivery systems targeting physiological transporters of the BBB hold a great promise. Curcumin extracted from the plant turmeric possesses anti-oxidative, anti-inflammatory and neuroprotective properties and is a potential treatment for different cerebral diseases. However, the clinical application of this natural compound is hampered by its poor water solubility and absorption, rapid metabolism and systemic elimination resulting in low bioavailability. Nanosized, biocompatible and biodegradable vesicles containing Evans blue-albumin as a model molecule and a hydrophobic therapeutic biomolecule, curcumin were prepared and characterized. In addition, fluorescent solid nanoparticles were also examined. The aim of our study was to test the cellular toxicity and penetration of nanovesicles loaded with albumin or curcumin and fluorescent nanoparticles all containing ligands for transporters on culture models of the BBB. Methods: The nanovesicles and fluorescent solid nanoparticles were labelled with different ligands, biotin, a glucose analogue and glutathione. Primary rat and human hCMEC/D3 brain endothelial cells were used as in vitro model systems of the BBB. The cellular toxicity of the nanoparticles was measured by real-time cell microelectric sensing (RTCA-SP, ACEA Biosciences) and MTT assay. The permeability tests were performed on triple co-culture BBB model and hCMEC/D3 cells using Transwell inserts. Brain endothelial uptake of nanoparticles was quantified by fluorescent spectroscopy and visualized by confocal microscopy. Results: No toxicity for loaded or unloaded nanovesicles or solid nanoparticles was found by MTT assay and impedance measurements. The loading of curcumin into liposomes significantly decreased its toxicity for brain endothelial cells at high concentrations. The presence of a glucose analogue in nanovesicles increased the uptake of the model molecule to cultured brain endothelial cells. The brain endothelial uptake of both loaded nanovesicles and solid nanoparticles could be followed by confocal microscopy. Conclusion: Our data indicate that encapsulation of lipophilic or macromolecular drugs into nanovesicles may decrease cellular toxicity and increase uptake and transport at the BBB, however the type of the targeting ligand and its coupling to the nanoparticle may be crucial for efficacy

Human dopamine receptor nanovesicles for gate-potential modulators in high-performance field-effect transistor biosensors

The development of molecular detection that allows rapid responses with high sensitivity and selectivity remains challenging. Herein, we demonstrate the strategy of novel bio-nanotechnology to successfully fabricate high-performance dopamine (DA) biosensor using DA Receptor-containing uniform-particle-shaped Nanovesicles-immobilized Carboxylated poly(3,4-ethylenedioxythiophene) (CPEDOT) NTs (DRNCNs). DA molecules are commonly associated with serious diseases, such as Parkinson's and Alzheimer's diseases. For the first time, nanovesicles containing a human DA receptor D1 (hDRD1) were successfully constructed from HEK-293 cells, stably expressing hDRD1. The nanovesicles containing hDRD1 as gate-potential modulator on the conducting polymer (CP) nanomaterial transistors provided high-performance responses to DA molecule owing to their uniform, monodispersive morphologies and outstanding discrimination ability. Specifically, the DRNCNs were integrated into a liquid-ion gated field-effect transistor (FET) system via immobilization and attachment processes, leading to high sensitivity and excellent selectivity toward DA in liquid state. Unprecedentedly, the minimum detectable level (MDL) from the field-induced DA responses was as low as 10 pM in real- time, which is 10 times more sensitive than that of previously reported CP based-DA biosensors. Moreover, the FET-type DRNCN biosensor had a rapid response time (<1 s) and showed excellent selectivity in human serum

Optimization of a multiple water-in-oil-in-water nanoemulsion encasing bacteriophages for inhalational antibiotherapy

Infectious bacterial diseases still remain the main cause of human premature deaths, especially in developing countries. The emergence and spread of pathogenic bacteria resistant to many chemical antibiotics (multidrugresistant strains) have created the need for the development of novel therapeutic agents. Bacteriophages have proven to be an interesting and effective alternative in the management of persistent bacterial infections where conventional chemical antibiotherapies fail. The lethality and specificity of bacteriophages for specific bacteria, their ability to replicate within bacterial hosts and safety of these human-friendly viruses makes them highly lethal antibacterial agents, besides being efficient and relatively cost-effective. Group A streptococci (GAS) are serious human pathogens that cause infections ranging from mild pharyngitis, tonsillitis, to chronic rheumatic heart disease and, in some cases, severe streptococcal toxic shock syndrome and necrotizing fasciitis. The frequency and severity of GAS infections has been increasing over the last decades, which has promoted extensive research on the improvement of naturally occurring antimicrobials as alternatives to their conventional chemical counterparts. In this research effort, development and optimization of a biotechnological process for the inhalational administration of a bacteriophage was pursued, using strategies of nanoencapsulation within lipid nanovesicles. This method of targeting may have a high potential for the treatment of bacterial infections of the respiratory tract, caused mainly by Streptococcus pyogenes. As a proof-of-concept for the nanoencapsulation strategy, and since there is not yet available a strictly lytic bacteriophage cocktail for Streptococcus pyogenes, a well-defined and characterized bacteriophage was utilized, viz. bacteriophage T4. Water-in-oil-in-water (W/O/W) multiple emulsions are nanosystems in which dispersions of small water droplets within larger oil droplets are themselves dispersed in a continuous aqueous phase. Due to their compartimentalized internal structure, multiple emulsions present important advantages over simple O/W emulsions for encapsulation of biomolecules, such as the ability to carry both polar and non-polar molecules, and a better control over releasing of therapeutic molecules. Bacteriophage T4 was accordingly entrapped within W/O/W multiple nanoemulsions, aiming at mimicking the multifunctional design of biology, optimized with several lipid matrices, poloxamers and stabilizing layer compositions. Physicochemical characterization of the optimized bacteriophage-encasing nanovesicle formulations encompassed determination of particle (hydrodynamic) size, size distribution and particle charge (Zeta potential), via Dynamic Light Scattering analysis, surface morphology via Cryo-SEM, and thermal analysis via DSC, whereas antimicrobial activity of the nanoemulsions produced were evaluated via the “spot-test” using appropriate bacterial cultures