Photochemical water oxidation by crystalline polymorphs of manganese oxides: structural requirements for catalysis.

Manganese oxides occur naturally as minerals in at least 30 different crystal structures, providing a rigorous test system to explore the significance of atomic positions on the catalytic efficiency of water oxidation. In this study, we chose to systematically compare eight synthetic oxide structures containing Mn(III) and Mn(IV) only, with particular emphasis on the five known structural polymorphs of MnO2. We have adapted literature synthesis methods to obtain pure polymorphs and validated their homogeneity and crystallinity by powder X-ray diffraction and both transmission and scanning electron microscopies. Measurement of water oxidation rate by oxygen evolution in aqueous solution was conducted with dispersed nanoparticulate manganese oxides and a standard ruthenium dye photo-oxidant system. No Ru was absorbed on the catalyst surface as observed by XPS and EDX. The post reaction atomic structure was completely preserved with no amorphization, as observed by HRTEM. Catalytic activities, normalized to surface area (BET), decrease in the series Mn2O3 > Mn3O4 ≫ λ-MnO2, where the latter is derived from spinel LiMn2O4 following partial Li(+) removal. No catalytic activity is observed from LiMn2O4 and four of the MnO2 polymorphs, in contrast to some literature reports with polydispersed manganese oxides and electro-deposited films. Catalytic activity within the eight examined Mn oxides was found exclusively for (distorted) cubic phases, Mn2O3 (bixbyite), Mn3O4 (hausmannite), and λ-MnO2 (spinel), all containing Mn(III) possessing longer Mn-O bonds between edge-sharing MnO6 octahedra. Electronically degenerate Mn(III) has antibonding electronic configuration e(g)(1) which imparts lattice distortions due to the Jahn-Teller effect that are hypothesized to contribute to structural flexibility important for catalytic turnover in water oxidation at the surface.

Complex gas hydrate from the Cascadia margin.

Natural gas hydrates are a potential source of energy and may play a role in climate change and geological hazards. Most natural gas hydrate appears to be in the form of 'structure I', with methane as the trapped guest molecule, although 'structure II' hydrate has also been identified, with guest molecules such as isobutane and propane, as well as lighter hydrocarbons. A third hydrate structure, 'structure H', which is capable of trapping larger guest molecules, has been produced in the laboratory, but it has not been confirmed that it occurs in the natural environment. Here we characterize the structure, gas content and composition, and distribution of guest molecules in a complex natural hydrate sample recovered from Barkley canyon, on the northern Cascadia margin. We show that the sample contains structure H hydrate, and thus provides direct evidence for the natural occurrence of this hydrate structure. The structure H hydrate is intimately associated with structure II hydrate, and the two structures contain more than 13 different hydrocarbon guest molecules. We also demonstrate that the stability field of the complex gas hydrate lies between those of structure II and structure H hydrates, indicating that this form of hydrate is more stable than structure I and may thus potentially be found in a wider pressure-temperature regime than can methane hydrate deposits.

Isolation and optimisation of the oleaginous yeast Sporobolomyces roseus for biosynthesis of 13C isotopically labelled 18-carbon unsaturated fatty acids and trans 18:1 and 18:2 derivatives through synthesis.

An oleaginous and psychrotrophic strain (F38-3) of Sporobolomyces roseus Kluyver & van Niel was isolated from a salt marsh environment in Nova Scotia, Canada following a screening program to select for high producers of 18-carbon unsaturated fatty acids. Fatty acid production was characterised as a function of temperature at 20 g glucose L(-1), and optimal yields were obtained at 14°C, achieving 5.7 g dw biomass and 39.2% total fatty acids by dry weight, with 18:1, 18:2 and 18:3 all-cis fatty acids accounting for 49.4%, 14.3% and 6.7% of total fatty acids (TFA), respectively--the highest reported for this species. Production of 18:3 was inversely correlated to growth temperature, rising from 2% of TFA at 30°C to 8.9% at 6°C. Cultivation of isolate F38-3 on universally (13)C (U-(13)C) labelled glucose and subsequent transesterification and isolation of the fatty acid methyl esters (FAMEs) by preparative chromatography yielded pure, highly (13)C-enriched (>90%) 18:1, 18:2 and 18:3 all-cis FAMEs. The U-(13)C 18:1 FAME was catalytically converted to U-(13)C 18:1 trans-9 and purified to >99.5% purity. The U-(13)C 18:2 was converted by alkaline isomerisation into a 50/50 mixture of 18:2 cis-9, trans-11 and 18:2 trans-10, cis-12 isomers and purified to >95.0% purity. Overall, 10%, by weight, of labelled glucose fed to isolate F38-3 was recovered as fatty acid methyl esters and 7.5% as 18-carbon unsaturated fats, and the final isomerisation reactions resulted in yields of 80% or greater. The ultimate goal of the work is to develop methodologies to produce (13)C-labelled metabolic tracers as tools to study the metabolism of trans fats.

Natural Killer Cells as Key Mediators in Type I Diabetes Immunopathology.

The immunopathology of type I diabetes (T1D) presents a complicated case in part because of the multifactorial origin of this disease. Typically, T1D is thought to occur as a result of autoimmunity toward islets of Langerhans, resulting in the destruction of insulin-producing cells (ß cells) and thus lifelong reliance on exogenous insulin. However, that explanation obscures much of the underlying mechanism, and the actual precipitating events along with the associated actors (latent viral infection, diverse immune cell types and their roles) are not completely understood. Notably, there is a malfunctioning in the regulation of cytotoxic CD8+ T cells that target endocrine cells through antigen-mediated attack. Further examination has revealed the likelihood of an imbalance in distinct subpopulations of tolerogenic and cytotoxic natural killer (NK) cells that may be the catalyst of adaptive immune system malfunction. The contributions of components outside the immune system, including environmental factors such as chronic viral infection also need more consideration, and much of the recent literature investigating the origins of this disease have focused on these factors. In this review, the details of the immunopathology of T1D regarding NK cell disfunction is discussed, along with how those mechanisms stand within the context of general autoimmune disorders. Finally, the rarer cases of latent autoimmune, COVID-19 (viral), and immune checkpoint inhibitor (ICI) induced diabetes are discussed as their exceptional pathology offers insight into the evolution of the disease as a whole.

The Importance of Proper Oxygenation in 3D Culture.

Cell culture typically employs inexpensive, disposable plasticware, and standard humidified CO2/room air incubators (5% CO2, ∼20% oxygen). These methods have historically proven adequate for the maintenance of viability, function, and proliferation of many cell types, but with broad variation in culture practices. With technological advances it is becoming increasingly clear that cell culture is not a "one size fits all" procedure. Recently, there is a shift toward comprehension of the individual physiological niches of cultured cells. As scale-up production of single cell and 3D aggregates for therapeutic applications has expanded, researchers have focused on understanding the role of many environmental metabolites/forces on cell function and viability. Oxygen, due to its role in cell processes and the requirement for adequate supply to maintain critical energy generation, is one such metabolite gaining increased focus. With the advent of improved sensing technologies and computational predictive modeling, it is becoming evident that parameters such as cell seeding density, culture media height, cellular oxygen consumption rate, and aggregate dimensions should be considered for experimental reproducibility. In this review, we will examine the role of oxygen in 3D cell culture with particular emphasis on primary islets of Langerhans and stem cell-derived insulin-producing SC-ß cells, both known for their high metabolic demands. We will implement finite element modeling (FEM) to simulate historical and current culture methods in referenced manuscripts and innovations focusing on oxygen distribution. Our group and others have shown that oxygen plays a key role in proliferation, differentiation, and function of these 3D aggregates. Their culture in plastic consistently results in core regions of hypoxia/anoxia exacerbated by increased media height, aggregate dimensions, and oxygen consumption rates. Static gas permeable systems ameliorate this problem. The use of rotational culture and other dynamic culture systems also have advantages in terms of oxygen supply but come with the caveat that these endocrine aggregates are also exquisitely sensitive to mechanical perturbation. As recent work demonstrates, there is a strong rationale for the use of alternate in vitro systems to maintain physio-normal environments for cell growth and function for better phenotypic approximation of in vivo counterparts.

Optical sensor arrays designed for guided manufacture of perfluorocarbon nanoemulsions with a non-synthetic stabilizer.

Hydrophobic drugs are incorporated into oil-in-water nanoemulsions (OIW) either as new formulations or repurposed for intravenous delivery. Typically, these are manufactured through stepwise processes of sonication or high-pressure homogenization (HPH). The guiding criteria for most nanoemulsion manufacture are the size and homogeneity/polydispersity of the drug-laden particles with strict requirements for clinical injectables. To date, most formulation optimization is done through trial and error with stepwise sampling during processing utilizing dynamic light scattering (DLS), light obscuration sensing (LOS) or laser particle tracking (LPT) to assess manufacturing progress. The objective of this work was to develop and implement an in-line optical turbidity/nephelometry sensor array for the longitudinal in-process monitoring of nanoemulsion manufacture. A further objective was the use of this sensor array to rapidly optimize the manufacture of a sub-120 nm oxygen carrying perfluorocarbon nanoemulsion with a non-synthetic stabilizer. During processing, samples were taken for particle size measurement and further characterization. There was a significant correlation and agreement between particle size and sensor signal as well as improved process reproducibility through sensor-guided manufacture. Given the cost associated with nanoemulsion development and scale-up manufacture, our sensor arrays could be an invaluable tool for efficient and cost-effective drug development. Sensor-guided manufacturing was used to optimize oxygen-carrying nanoemulsions. These were tested, in vitro, for their ability to improve the viability of encapsulated endocrine clusters (mouse insulinoma, Min6) and to eliminate hypoxia due to oxygen mass transfer limitations. The nanomulsions significantly improved encapsulated cluster viability and reduced hypoxia within the microcapsule environment. STATEMENT OF SIGNIFICANCE: Nanoemulsions are rapidly becoming vehicles for the controlled release delivery of both hydrophilic and hydrophobic drugs given their large surface area for exchange. As work shifts from bench to large scale manufacturing, there is a critical need for technologies that can monitor and accumulate data during processing, particularly regarding the endpoint criteria of particle size and stability. To date, no such technology has been implemented in nanoemulsion manufacture. In this paper we develop and implement an optical sensor array for in-line nanoemulsion process monitoring and then use the array to optimize the development and manufacture of novel reproducible oxygen carrying nanoemulsions lacking synthetic surfactants.

Detection of Zika virus in mouse mammary gland and breast milk.

Clinical reports of Zika Virus (ZIKV) RNA detection in breast milk have been described, but evidence conflicts as to whether this RNA represents infectious virus. We infected post-parturient AG129 murine dams deficient in type I and II interferon receptors with ZIKV. ZIKV RNA was detected in pup stomach milk clots (SMC) as early as 1 day post maternal infection (dpi) and persisted as late as 7 dpi. In mammary tissues, ZIKV replication was demonstrated by immunohistochemistry in multiple cell types including cells morphologically consistent with myoepithelial cells. No mastitis was seen histopathologically. In the SMC and tissues of the nursing pups, no infectious virus was detected via focus forming assay. However, serial passages of fresh milk supernatant yielded infectious virus, and immunohistochemistry showed ZIKV replication protein associated with degraded cells in SMC. These results suggest that breast milk may contain infectious ZIKV. However, breast milk transmission (BMT) does not occur in this mouse strain that is highly sensitive to ZIKV infection. These results suggest a low risk for breast milk transmission of ZIKV, and provide a platform for investigating ZIKV entry into milk and mechanisms which may prevent or permit BMT.

Preparation and characterization of a suite of ephedra-containing standard reference materials.

The National Institute of Standards and Technology, the U.S. Food and Drug Administration, Center for Drug Evaluation and Research and Center for Food Safety and Applied Nutrition, and the National Institutes of Health, Office of Dietary Supplements, are collaborating to produce a series of Standard Reference Materials (SRMs) for dietary supplements. A suite of ephedra materials is the first in the series, and this paper describes the acquisition, preparation, and value assignment of these materials: SRMs 3240 Ephedra sinica Stapf Aerial Parts, 3241 E. sinica Stapf Native Extract, 3242 E. sinica Stapf Commercial Extract, 3243 Ephedra-Containing Solid Oral Dosage Form, and 3244 Ephedra-Containing Protein Powder. Values are assigned for ephedrine alkaloids and toxic elements in all 5 materials. Values are assigned for other analytes (e.g., caffeine, nutrient elements, proximates, etc.) in some of the materials, as appropriate. Materials in this suite of SRMs are intended for use as primary control materials when values are assigned to in-house (secondary) control materials and for validation of analytical methods for the measurement of alkaloids, toxic elements, and, in the case of SRM 3244, nutrients in similar materials.

The role of low-energy electrons in the high-energy radiolysis of condensed CF3I.

The dynamics of electron-induced reactions in condensed trifluoroiodomethane (CF3I) were studied under ultrahigh vacuum conditions. Seven CF3I radiolysis products (C2F6, C2F5I, C2F3I, CF2I2, C2F4I2, CFI3 and C2F3I3) were identified using temperature-programmed desorption experiments conducted after irradiation with 4 eV electrons. Although C2F6 formation at energies above 4 eV is ascribed to electron-induced electronic excitation followed by prompt dissociation of the C-I bond to form [Formula: see text] radicals that dimerize, the formation of the other six radiolysis products at low sub-ionization incident electron energies is attributed to dissociative electron attachment (DEA) because of the observed resonance peaks in the radiolysis product yields as functions of incident electron energy (∼2 to ∼ 7 eV). All seven CF3I electron-induced reaction products were also identified following irradiation with 500 eV electrons. While dissociative electron attachment and/or electron impact excitation may play an important role in the high-energy radiation-induced synthesis of the high-yield product C2F6, a dramatic enhancement of up to ∼ 2 × 10(4) in product yield per electron at 500 eV relative to that at 4 eV for some products suggests, however, that DEA is not the dominant mechanism for the high-energy radiation-induced synthesis of those products.

Biosynthetic production of universally (13)C-labelled polyunsaturated fatty acids as reference materials for natural health product research.

Long-chain polyunsaturated fatty acids (LCPUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have become important natural health products with numerous proven benefits related to brain function and cardiovascular health. Not only are omega-3 fatty acids available in a plethora of dietary supplements, but they are also increasingly being incorporated as triglycerides into conventional foods, including bread, milk, yoghurt and confectionaries. Recently, transgenic oil seed crops and livestock have been developed that enhance omega-3 fatty acid content. This diverse array of matrices presents a difficult analytical challenge and is compounded further by samples generated through clinical research. Stable isotope (13)C-labelled LCPUFA standards offer many advantages as research tools because they may be distinguished from their naturally abundant counterparts by mass spectrometry and directly incorporated as internal standards into analytical procedures. Further, (13)C-labelled LCPUFAs are safe to use as metabolic tracers to study uptake and metabolism in humans. Currently, (13)C-labelled LCPUFAs are expensive, available in limited supply and not in triglyceride form. To resolve these issues, marine heterotrophic microorganisms are being isolated and screened for LCPUFA production with a view to the efficient biosynthetic production of U-(13)C-labelled fatty acids using U-(13)C glucose as a carbon source. Of 37 isolates obtained, most were thraustochytrids, and either DHA or omega-6 docosapentaenoic acid (22:5n-6) were produced as the major LCPUFA. The marine protist Hyalochlorella marina was identified as a novel source of EPA and omega-3 docosapentaenoic acid (22:5n-3). As proof of principle, gram-level production of (13)C-labelled DHA has been achieved with high chemical purity ( >99%) and high (13)C incorporation levels (>90%), as confirmed by NMR and MS analyses. Finally, U-(13)C-DHA was enzymatically re-esterified to glycerol to yield a (13)C-labelled tridocosahexaenoin.

A systematic approach to quantitation of ephedra alkaloids in natural health products.

A method for accurate determination of ephedrine (E) alkaloids in natural health products (NHP) is described. The NIST dietary supplement standard reference materials (SRMs) were selected for these studies. These SRMs comprise ground Ma Huang herb (Ephedra sinica Stapf.), a spray dried extract of the former, and commercial formulations derived from gel caps and a protein drink. The efficiency of sonication-assisted extraction and Soxhlet extraction was studied using both ammonium formate and potassium phosphate in 3% methanol as extraction media. The efficiency of SPE clean-up of the extract deteriorated rapidly when increasing amounts of sample matrix or analyte were processed, because of limited cartridge capacity. Quantitation by the method of additions was required to ensure the highest accuracy using both LC-UV and ES-LC-MS-MS techniques. Whereas the LC-UV method is more convenient and precise, the results are more questionable than ES-LC-MS-MS, because species-specific detection is not possible.

Determination of ephedrine alkaloids in dietary supplement standard reference materials.

A suite of five ephedra-containing dietary supplement Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST) with certified values for ephedrine alkaloids, synephrine, caffeine, and selected toxic trace elements. The materials represent a variety of natural, extracted, and processed sample matrixes that provide different analytical challenges. The constituents have been determined by multiple independent methods with measurements performed by NIST and by three collaborating laboratories. The methods utilized different sample extraction and cleanup steps in addition to different instrumental analytical techniques and approaches to quantification. In addition, food-matrix proximates were determined by National Food Processor Association laboratories for one of the ephedra-containing SRMs. The SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and related botanical materials.

Photochemical alkylation of inorganic selenium in the presence of low molecular weight organic acids.

Using a flow-through photochemical reactor and a low pressure mercury lamp as a UV source, alkyl selenium species are formed from inorganic selenium(IV) in the presence of low molecular weight organic acids (LMW acids). The volatile alkyl Se species were cryogenically trapped and identified by GC-MS and GC-ICP-MS. In the presence of formic, acetic, propionic and malonic acids, inorganic selenium(IV) is converted by UV irradiation to volatile selenium hydride and carbonyl, dimethylselenide and diethylselenide, respectively. Se(IV) was successfully removed from contaminated agricultural drainage waters (California, U.S.A.) using a batch photoreactor system Se. Photochemical alkylation may thus offer a promising means of converting toxic selenium salts, present in contaminated water, to less toxic dimethylselenide. The LMW acids and photochemical alkylation process may also be key to understanding the source of atmospheric selenium and are likely involved in its mobility in the natural anaerobic environment.

Vapor generation by UV irradiation for sample introduction with atomic spectrometry.

Volatile species of the conventional hydride-forming elements (As, Bi, Sb, Se, Sn, Pb, Cd, Te), Hg, transition metals (Ni, Co, Cu, Fe), noble metals (Ag, Au, Rh, Pd, Pt), and nonmetals (I, S) were generated following UV irradiation of their aqueous solutions to which low molecular weight carboxylic acids (formic, acetic, propionic) had been added. Free radicals arising from photodissociation of the latter provide a new and useful alternative to the common methods of chemical/electrochemical vapor generation techniques for the determination of these analytes by atomic spectrometry. Quantitative estimates of the efficiencies of these generation processes were not undertaken, although calculated signal-to-background ratios (>1500 for 5 ng/mL As, Sb, Bi, Se, and Te; 20 for 10 ng/mL Sn, Cu, Rh, Au, Pd, Pt, and Cd; 2400 for 1 ng/mL Hg; and 1000 for Co using ICP-TOF-MS detection) do provide clear evidence of the efficacy of this approach for sample introduction. In the case of Ni and Se, the tetracarbonyl and alkylated selenium compounds have been identified, respectively.

UV vapor generation for determination of selenium by heated quartz tube atomic absorption spectrometry.

A new vapor generation technique utilizing UV irradiation coupled with atomic absorption for the determination of selenium in aqueous solutions is described. In the presence of low molecular weight organic acid solutions, inorganic selenium(IV) is converted by UV irradiation to volatile selenium species, which are then rapidly transported to a heated quartz tube atomizer for detection by atomic absorption spectrometry. Optimum conditions for photochemical vapor generation and interferences from concomitant elements were investigated. Identification of the volatile products using cryotrapping GC/MS analysis revealed that inorganic selenium(IV) is converted to volatile selenium hydride, selenium carbonyl, dimethyl selenide, and diethyl selenide in the presence of formic, acetic, propionic, and malonic acids, respectively. In acetic acid solution, the efficiency of generation was estimated to be 50 +/- 10%. No interference from Ni(2+) and Co(2+) at concentrations of 500 and 100 mg L(-)(1), respectively, was evident. A detection limit of 2.5 microg L(-)(1) and a relative sensitivity of 1.2 microg L(-)(1) (1% absorption) with a precision of 1.2% (RSD, n = 11) at 50 microg L(-)(1) were obtained.

Separation and quantitation of the stereoisomers of ephedra alkaloids in natural health products using flow injection-electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry.

A method is described for the determination of ephedrine (E) and pseudoephedrine (PE) and their metabolites norephedrine (NE), norpseudoephedrine (NPE), methylephedrine (ME), and methylpseudoephedrine (MPE) alkaloids in natural health products by flow injection-electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry (FI-ESI-FAIMS-MS). The determination of the six alkaloids requires the separation of diastereomic pairs of E-PE, NE-NPE, and ME-MPE. FAIMS was able to resolve/separate these isomeric pairs based on their gas-phase ion mobility differences. The FAIMS-based separation and detection approach has been tested on over-the-counter diet pills. Following the extraction of the tablets, either by pressurized fluid extraction developed in-house or with sonication, the ephedra alkaloids were quantified using a modified isotope dilution approach. Detection limits for the alkaloids ranged from 0.1 to 3 ng/mL, and a linear range of at least 2 orders of magnitude was observed for the six analytes. The throughput of the current configuration of the FI-ESI-FAIMS-MS system is 2 min/sample, which is significantly higher than conventional chromatographic approaches. The developed FI-ESI-FAIMS-MS method has been compared with a conventional LC-UV analysis, and good agreement has been found for the major alkaloids.