Extracellular vesicles of the probiotic bacteria E. coli O83 activate innate immunity and prevent allergy in mice.

BACKGROUND: E. coli O83 (Colinfant Newborn) is a Gram-negative (G-) probiotic bacterium used in the clinic. When administered orally, it reduces allergic sensitisation but not allergic asthma. Intranasal administration offers a non-invasive and convenient delivery method. This route bypasses the gastrointestinal tract and provides direct access to the airways, which are the target of asthma prevention. G- bacteria such as E. coli O83 release outer membrane vesicles (OMVs) to communicate with the environment. Here we investigate whether intranasally administered E. coli O83 OMVs (EcO83-OMVs) can reduce allergic airway inflammation in mice. METHODS: EcO83-OMVs were isolated by ultracentrifugation and characterised their number, morphology (shape and size), composition (proteins and lipopolysaccharide; LPS), recognition by innate receptors (using transfected HEK293 cells) and immunomodulatory potential (in naïve splenocytes and bone marrow-derived dendritic cells; BMDCs). Their allergy-preventive effect was investigated in a mouse model of ovalbumin-induced allergic airway inflammation. RESULTS: EcO83-OMVs are spherical nanoparticles with a size of about 110 nm. They contain LPS and protein cargo. We identified a total of 1120 proteins, 136 of which were enriched in OMVs compared to parent bacteria. Proteins from the flagellum dominated. OMVs activated the pattern recognition receptors TLR2/4/5 as well as NOD1 and NOD2. EcO83-OMVs induced the production of pro- and anti-inflammatory cytokines in splenocytes and BMDCs. Intranasal administration of EcO83-OMVs inhibited airway hyperresponsiveness, and decreased airway eosinophilia, Th2 cytokine production and mucus secretion. CONCLUSIONS: We demonstrate for the first time that intranasally administered OMVs from probiotic G- bacteria have an anti-allergic effect. Our study highlights the advantages of OMVs as a safe platform for the prophylactic treatment of allergy. Video Abstract.

Characterization of the Inflammatory Response Evoked by Bacterial Membrane Vesicles in Intestinal Cells Reveals an RIPK2-Dependent Activation by Enterotoxigenic Escherichia coli Vesicles.

Although the immunomodulatory potency of bacterial membrane vesicles (MVs) is widely acknowledged, their interactions with host cells and the underlying signaling pathways have not been well studied. Herein, we provide a comparative analysis of the proinflammatory cytokine profile secreted by human intestinal epithelial cells exposed to MVs derived from 32 gut bacteria. In general, outer membrane vesicles (OMVs) from Gram-negative bacteria induced a stronger proinflammatory response than MVs from Gram-positive bacteria. However, the quality and quantity of cytokine induction varied between MVs from different species, highlighting their unique immunomodulatory properties. OMVs from enterotoxigenic Escherichia coli (ETEC) were among those showing the strongest proinflammatory potency. In depth analyses revealed that the immunomodulatory activity of ETEC OMVs relies on a so far unprecedented two-step mechanism, including their internalization into host cells followed by intracellular recognition. First, OMVs are efficiently taken up by intestinal epithelial cells, which mainly depends on caveolin-mediated endocytosis as well as the presence of the outer membrane porins OmpA and OmpF on the MVs. Second, lipopolysaccharide (LPS) delivered by OMVs is intracellularly recognized by novel caspase- and RIPK2-dependent pathways. This recognition likely occurs via detection of the lipid A moiety as ETEC OMVs with underacylated LPS exhibited reduced proinflammatory potency but similar uptake dynamics compared to OMVs derived from wild-type (WT) ETEC. Intracellular recognition of ETEC OMVs in intestinal epithelial cells is pivotal for the proinflammatory response as inhibition of OMV uptake also abolished cytokine induction. The study signifies the importance of OMV internalization by host cells to exercise their immunomodulatory activities. IMPORTANCE The release of membrane vesicles from the bacterial cell surface is highly conserved among most bacterial species, including outer membrane vesicles (OMVs) from Gram-negative bacteria as well as vesicles liberated from the cytoplasmic membrane of Gram-positive bacteria. It is becoming increasingly evident that these multifactorial spheres, carrying membranous, periplasmic, and even cytosolic content, contribute to intra- and interspecies communication. In particular, gut microbiota and the host engage in a myriad of immunogenic and metabolic interactions. This study highlights the individual immunomodulatory activities of bacterial membrane vesicles from different enteric species and provides new mechanistic insights into the recognition of ETEC OMVs by human intestinal epithelial cells.

The selective heating effect of microwave irradiation on a binary mixture of water and polyethylene oxide: a molecular dynamics simulation approach.

In this study, we investigate the molecular mechanisms of a microwave-driven selective heating process by performing molecular dynamics simulations for three different systems including pure water, pure polyethylene oxide (PEO), and water-PEO mixed systems in the presence of a microwave with two different intensities of electric field such as 0.001 V Å-1 and 0.01 V Å-1 at a frequency of 100 GHz. First, from performing molecular dynamics simulations of CO and CO2 in the presence of the microwave, it is confirmed that the molecular dipole moment is responsible for the rotational motion induced by the oscillating electric field. Second, by analyzing the MD simulations of the pure water system, we discover that the dipole moment of water exhibits a time lag with respect to the microwave. During the heating process, however, the temperature, kinetic, and potential energies increase synchronously with the oscillating electric field of the microwave, showing that the heating of the water system is caused by the molecular reaction of water to the microwave. Comparing the water-PEO mixed system to the pure water and pure PEO systems, the water-PEO mixed system has a higher heating rate than the pure PEO system but a lower heating rate than the pure water system. Therefore, we conclude that heating the water-PEO mixed system is driven by water molecules selectively activated by microwave irradiation. We also calculate the diffusion coefficients of water molecules and PEO chains by describing their mean square displacements, demonstrating that the diffusion coefficients are increased in the presence of microwaves for both water and PEO in pure and mixed systems. Lastly, during the microwave heating process, the structures of the water-PEO mixed system are altered as a function of the intensity of electric field, which is mainly driven by the response of water molecules.

Life cycle assessment and techno-economic analysis of nitrogen recovery by ammonia air-stripping from wastewater treatment.

Wastewater treatment plants (WWTPs) with anaerobic digestion of biosolids produce an ammonia-rich sidestream out of which nitrogen can be recovered through air stripping. Recovered ammonia can be used to produce ammonium sulfate (AS) for agricultural use, enabling the circular return of nitrogen as fertilizer to the food system. We investigate the cost and life cycle environmental impact of recovering ammonia from the sidestream of WWTPs for conversion to AS and compare it to AS production from the Haber Bosch process. We perform life cycle assessment (LCA) to investigate the environmental impact of AS fertilizer production by air-stripping ammonia from WWTP sidestreams at varying sidestream nitrogen concentrations. Techno-economic analysis (TEA) is performed to assess the break-even selling price of sidestream AS production at a WWTP in the City of Philadelphia. Greenhouse gas emissions for air-stripping technology range between 0.2 and 0.5 kg CO2e/kg AS, about six times lower than the hydrocarbon-based Haber-Bosch process, estimated at 2.5 kg CO2e/kg AS. Further reduction of greenhouse gas emissions is feasible by replacing fossil-based energy use in air-stripping process (82-98 % of net energy demand) with renewable sources. Also, a significant reduction in mineral depletion and improvement in human and ecosystem health are observed for the air-stripping approach. Furthermore, the break-even selling price for installing sidestream-based AS production at the Philadelphia's WWTP, considering capital and operating costs, is estimated at $0.046/kg AS (100 %), which is 92 % lower than the 2014 estimate of AS's average selling price at farms in the United States. We conclude that even with varying ammonia concentrations and high sidestream volume, air-stripping technology offers an environmentally and economically favorable option for implementing nitrogen recovery and simultaneous production of AS at WWTPs.

Outer Membrane Vesicles of Vibrio cholerae Protect and Deliver Active Cholera Toxin to Host Cells via Porin-Dependent Uptake.

Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.

Outer Membrane Vesiculation Facilitates Surface Exchange and In Vivo Adaptation of Vibrio cholerae.

Gram-negative bacteria release outer membrane vesicles into the external milieu to deliver effector molecules that alter the host and facilitate virulence. Vesicle formation is driven by phospholipid accumulation in the outer membrane and regulated by the phospholipid transporter VacJ/Yrb. We use the facultative human pathogen Vibrio cholerae to show that VacJ/Yrb is silenced early during mammalian infection, which stimulates vesiculation that expedites bacterial surface exchange and adaptation to the host environment. Hypervesiculating strains rapidly alter their bacterial membrane composition and exhibit enhanced intestinal colonization fitness. This adaptation is exemplified by faster accumulation of glycine-modified lipopolysaccharide (LPS) and depletion of outer membrane porin OmpT, which confers resistance to host-derived antimicrobial peptides and bile, respectively. The competitive advantage of hypervesiculation is lost upon pre-adaptation to bile and antimicrobial peptides, indicating the importance of these adaptive processes. Thus, bacteria use outer membrane vesiculation to exchange cell surface components, thereby increasing survival during mammalian infection.

Polypropylene Carbonate-Based Adaptive Buffer Layer for Stable Interfaces of Solid Polymer Lithium Metal Batteries.

Solid polymer electrolytes (SPEs) have the potential to enhance the safety and energy density of lithium batteries. However, poor interfacial contact between the lithium metal anode and SPE leads to high interfacial resistance and low specific capacity of the battery. In this work, we present a novel strategy to improve this solid-solid interface problem and maintain good interfacial contact during battery cycling by introducing an adaptive buffer layer (ABL) between the Li metal anode and SPE. The ABL consists of low molecular-weight polypropylene carbonate , poly(ethylene oxide) (PEO), and lithium salt. Rheological experiments indicate that ABL is viscoelastic and that it flows with a higher viscosity compared to PEO-only SPE. ABL also has higher ionic conductivity than PEO-only SPE. In the presence of ABL, the interface resistance of the Li/ABL/SPE/LiFePO4 battery only increased 20% after 150 cycles, whereas that of the battery without ABL increased by 117%. In addition, because ABL makes a good solid-solid interface contact between the Li metal anode and SPE, the battery with ABL delivered an initial discharge specific capacity of >110 mA·h/g, which is nearly twice that of the battery without ABL, which is 60 mA·h/g. Moreover, ABL is able to maintain electrode-electrolyte interfacial contact during battery cycling, which stabilizes the battery Coulombic efficiency.

Phototriggered Depolymerization of Flexible Poly(phthalaldehyde) Substrates by Integrated Organic Light-Emitting Diodes.

We demonstrate phototriggered depolymerization of a low ceiling temperature ( Tc) polymer, poly(phthalaldehyde) (PPHA), via internal light emission from integrated organic light-emitting diodes (OLEDs) fabricated directly on flexible PPHA substrates with silver nanowire electrodes. The depolymerization of the PPHA substrates is triggered by absorption of the OLED emission by a sensitizer that activates a photoacid generator via energetically favorable electron transfer. We confirm with Fourier-transform infrared spectroscopy that the photon doses delivered by the integrated OLED are sufficient to depolymerize the PPHA substrates. We determine this critical dosage by measuring the operating lifetimes of the OLEDs whose failure is believed to be due to significant mechanical softening during the liquefaction of decomposed phthalaldehyde monomers.

Isolation of Outer Membrane Vesicles Including Their Quantitative and Qualitative Analyses.

Outer membrane vesicles (OMVs) are naturally secreted from the bacterial cell surface and therefore localized in the cell-free supernatant of bacterial cultures. Here we describe methods for crude and density gradient-purified OMV isolation and protocols for control analyses for protein profiling (SDS-PAGE), detection of indicator proteins (immunoblot analysis), lipid profiling (lipid extraction and LC-MS analysis), vesicle size determination (NanoSight), rough estimation of biomass (TrayCell™), as well as quantifications of defined OMV components, e.g., proteins (Bradford) and LPS (Purpald).

A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria.

Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.

Liquid-phase exfoliation of graphene in organic solvents with addition of naphthalene.

We report a facile method for the production of graphene sheets through liquid-phase exfoliation of graphite in organic solvents with addition of naphthalene. The production yield of graphene is significantly increased with the addition of naphthalene in most solvents tested in this work. Naphthalene serves as a "molecular wedge" to intercalate into the edge of graphite, which plays a key role during sonication and significantly improves the production yield of graphene. The graphene concentration of the dispersion in 1-methyl-2-pyrrolidinone is as high as 0.15 mg/mL (after sonication for 90 min), which allows this method to easily produce films and composites for a range of applications.

Low-dielectric constant insulators for future integrated circuits and packages.

Future integrated circuits and packages will require extraordinary dielectric materials for interconnects to allow transistor advances to be translated into system-level advances. Exceedingly low-permittivity and low-loss materials are required at every level of the electronic system, from chip-level insulators to packages and printed wiring boards. In this review, the requirements and goals for future insulators are discussed followed by a summary of current state-of-the-art materials and technical approaches. Much work needs to be done for insulating materials and structures to meet future needs.

Characterization of anion exchange ionomers in hybrid polymer electrolyte fuel cells.

Anion exchange ionomers (AEI) synthesized here were characterized by use of a novel fuel cell configuration. The new analysis method involves assembling the AEI electrode of interest as the cathode in a hybrid, acid/alkaline, fuel cell configuration. The hybrid cell includes a conventional proton conducting anode/membrane half-cell along with the anionic conductor of interest at the cathode. Electrochemical impedance spectroscopy and voltammetry were used to evaluate the performance of the hybrid AEI-containing fuel cell with H2 and O2. In particular, the AEI electrode response in impedance spectroscopy was easily identified because the contributions from other components are largely minimized in the presented hybrid cell configuration. The properties of ionomers used in the AEI electrode were shown to have a substantial effect on the electrode performance. Low catalyst utilization, due to high water uptake and low conductivity, was identified as the major causes of poor performance in AEI electrodes.

Cation electrochemical stability in chloroaluminate ionic liquids.

The electrochemical stability of 10 organic cations, which can be used in ionic liquids (IL), was investigated as solutes in acetonitrile (ACN). The stability of three of the salts, BenMe2EtNCl (salt III), 1-butyl-2-methyl pyrrolidium chloride (salt VI), and its structural isomer, BuMe2ProNCl (salt VII), were also compared in chloroaluminate ILs. The chloroaluminate ILs of salts VI and VII are investigated for the first time. The NaCl-neutralized ILs of salts VI and VII have melting points of 43.2 and 3.7 degrees C, respectively. The benzyl-substituted cation, salt III, was more easily reduced in ACN or as the neutral chloroaluminate IL than the alkyl-substituted cation, salt VII, due to the better leaving ability of the benzyl group. Mass spectroscopy measurements before and after electrolysis on the benzyl-substituted solutions confirmed that reduction involves the loss of an alkyl group. In ACN, salt VI was found to be the most difficult to reduce (1 mA/cm2 at -2.09 V) due to its cyclic structure. However, in the chloroaluminate IL, the pyrrolidinium cation was more easily reduced than salt III or its isomer, salt VII, resulting in an insoluble black deposit. This is consistent with the mass spectrometry data, which do not show formation of low-molecular-weight products, as in the reduction of salts III and VII. The IL of salt VII was the most stable in the presence of sodium. Sodium ions could be reduced and reoxidized with a maximum Coulombic efficiency of 94.1% versus 87.2% for salt VI. Reduction of the pyrrolidinium cation produces insoluble products, most likely through opening of the cyclic ring, and an inferior medium for sodium ion reduction compared to the benzyl- and butyl-substituted cations, even though reduction of the cation occurs at a more negative potential in acetonitrile.

Clinical evaluation of the Keeler Pulsair 3000 non-contact tonometer.

BACKGROUND: The Keeler Pulsair 3000 is a recently introduced non-contact tonometer that is especially useful for children and those for whom sitting at a slit-lamp or table-mounted unit would be difficult. In this project, intraocular pressures (IOPs) measured by Keeler Pulsair 3000 and Goldmann tonometers were compared to assess validity and reliability of the Pulsair 3000 data. METHODS: Two Pulsair 3000 IOPs (each the mean of four individual air-puff readings) and two Goldmann IOPs were measured for each eye of 113 subjects. Subjects were also asked which measurement technique they preferred. RESULTS: IOPs ranged from 9 to 28 mmHg. Correlations between the two Goldmann IOPs measured for the right and left eyes were 0.98 and 0.97, respectively. These values are higher than correlations between Pulsair and Goldmann measurements (0.86 to 0.91). Pulsair 3000 IOPs were slightly above Goldmann values for pressures of less than 15 mmHg and slightly below for IOPs greater than 15 mmHg. Extrapolation to a Goldmann IOP of 30 mmHg suggests the Pulsair 3000 would read about 6% (1.7 mmHg) too low at this IOP. Eight eyes (7%) had differences between Pulsair 3000 and Goldmann IOP readings of 5.0 mmHg or more. Single outlier pressures accounted for these differences in three out of the eight eyes. CONCLUSIONS: In the range of 10 to 24 mmHg, the Pulsair 3000 tonometer produced IOP readings that corresponded well with Goldmann values for most eyes and was preferred by the majority of subjects who indicated a preference. The Pulsair 3000 is relatively easy to use by technicians and has numerous special applications in optometric practice (e.g. measuring IOPs for pediatric patients and those with compromised corneas).