Development of a microchip capillary electrophoresis method for determination of the purity and integrity of mRNA in lipid nanoparticle vaccines

Messenger RNA (mRNA)-based vaccines are advantageous because they can be relatively quicker and more cost efficient to manufacture compared to other traditional vaccine products. Lipid nanoparticles have three common purposes: delivery, self-adjuvanting properties, and mRNA protection. Faster vaccine development requires an efficient and fast assay to monitor mRNA purity and integrity. Microchip CE is known to be a robust technology that is capable of rapid separation. Here, we describe the development and optimization of a purity and integrity assay for mRNA-based vaccines encapsulated in lipid nanoparticles using commercial microchip-based separation. The analytical parameters of the optimized assay were assessed and the method is a stability indicating assay.     

Development of ultra-high-performance supercritical fluid chromatography-mass spectrometry assays to analyze potential biomarkers in sweat

Liquid chromatography-mass spectrometry methods were required to afford the rapid separation and detection of purines and small organic acids. These compounds are found in sweat and sebum and are potential biomarkers for the early detection of pressures sores. Two ultra-high-performance supercritical fluid chromatography-mass spectrometry assays have been successfully developed for both classes of compounds. Separation for purines was achieved using a gradient of supercritical carbon dioxide and methanol with a 1-aminoanthracene sub 2 μm particle size column followed by positive ion electrospray ionization. Separation for organic acids was achieved using a gradient of supercritical carbon dioxide and methanol (50 mM ammonium acetate 2% water) with a Diol sub 2 μm particle size column followed by negative ion electrospray ionization. Calibration curves were created in the absence of internal standards and R² values > 0.96 were achieved using single ion monitoring methods for the protonated purines and the deprotonated acids. The two new assays afford rapid analytical methods for the separation and detection of potential biomarkers in human sweat leading to the early detection and prevention of pressure sores.     

A guide for the meaningful surface analysis of wood by XPS and ToF-SIMS

The surface analysis of wood and wood products is becoming increasingly important for reasons ranging from the investigation of molecular constituents through to the optimization of industrial processes. As with any natural product, wood analysis is not straightforward, and this review aims to provide guidance for the successful surface analysis of wood by XPS and ToF-SIMS. Through example experiments, three themes are addressed relevant to obtaining meaningful results: considerations related to heterogeneity in the composition of wood (e.g., growth rings); the impact of the chemical removal of minor wood components known as extractives, and whether such a process is necessary; and the potential for misleading or erroneous results as a result of contamination occurring during sample preparation. In addition to discussing successful sample preparation approaches, the important role to be played by MVA in surface analysis is emphasized, particularly in the analysis of ToF-SIMS data. Examples of ToF-SIMS/MVA are provided that highlight the identification of contamination in sample preparation, the quantification of wood composition in terms of cellulose and lignin, and the indication of age of softwood samples. Through consideration of the complexities that influence wood surface analysis, the design and interpretation of consequential experiments become easier and more accurate.     

Purification,Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.     

Determination of trace elements in organic material by inductively coupled plasma-mass spectrometry

Summary For purposes of trace element analysis of organic materials a combined analytical procedure is tested. It consists of a pressure decomposition by nitric acid at 220° C in quartz vessels. The measurements are performed in the diluted digests by inductively coupled plasma/mass spectrometry. Analytical figures of merit are given.Dedicated to Prof. Dr. Karl Winsauer on the occasion of his 60th birthday.     

MEASUREMENT OF CELL LYSIS BY LIGHT SCATTERING

Abstract— A method is presented which is capable of continuously monitoring the degree of hemolysis in erythrocyte suspensions too dilute to be monitored by conventional light transmission techniques. Scattered light is used to non-destructively assess hemolysis in sparse monolayers which are particularly well suited to many photohemolytic studies. The small angle scattering (<10°), measured here, shows a transient decline as cells settle in a culture dish and then is constant if no lysis occurs. Lysis is indicated by a decrease in scattered light to < 20% of initial intensity when lysis is complete. The light used to monitor lysis is restricted to wavelengths longer than 700 nm which is outside the absorption band of many. photosensitizers of current interest, and is a wavelength range at which light scattering is relatively independent of changes in cell volume. In photohemolytic studies with phloxine B lysis values from light scattering are shown to correlate well with lysis values from hemoglobin release. An apparatus is described which is capable of periodically measuring lysis in eight suspensions without intervention by the experimenter.     

Übergangsmetall-methylen-komplexe: LVI. Organoruthenium-komplexe mit offenkettigen und carbocyclischen alkyliden-brücken: Synthese,konstitution, isomerie,protonierung und thermolytischer abbau

The eighteen new μ-alkylidene ruthenium complexes 5a–r and 5t are very easily and cleanly obtained along the diazoalkane or the hydrazone routes that involve treatment of the dinuclear, metal-metal doubly bonded precursor compound [(η⁵-C₅H₅)Ru(μ-NO)]₂ (3) either with the diazoalkanes oxidizing agent (e.g., MnO₂), with the respective hydrazones. Similarly, sulfur dioxide adds cleanly to the RuRu double bond of 3, thus giving the complex (μ-SO₂)[(η⁵-C₅H₅)Ru(NO)]₂ (5s). Regardless of the nature of the carbene bridge ligands, the dimetallacyclopropanes exhibit, in contrast to their iron analogues, exclusively terminal nitrosyl ligands. cis/trans-Isomerism with predominating amounts of the trans-isomers is observed for the derivatives that display unsymmetrically substituted carbene bridges.Treatment of the μ-methylene- and μ-ethylidene complexes (μ-CH₂)[(η⁵-C₅H₅)Ru(NO)]₂ (5a) and (μ-CHCH₃)[(η⁵-C tetrafluoroboric acid or trifluoromethanesulfonic acid in diethyl ether yields, at ambient temperature, quantitatively the ionic complexes 6a,b and 7a,b, respectively, which were shown by ¹H NMR spectroscopy to contain metal-metal bridging hydrogen functionalities. The reaction of hydrogen bromide with 5a under the same conditions gives the neutral bromo(methyl) complex 6d. This latter compound results from the isolable ionic intermediate of composition [(μ-CH₂)(μ-H){(η⁵C₅H₅)Ru(NO)}₂]⁺Br^? (6c), which reaction stems from the nucleophilicity of the halide ion present in 6c.     

“Dibutylmagnesium”, a convenient reagent for the synthesis of useful organic magnesium reagents MgA2 including cyclopentadienyls,aryloxides, and amides. Preparation of Zr(C5H5)Cl3. X-ray structure of [Mg{μ-N(SiMe)3C6H4N}(SiMe3)-o(OEt2)]2

n-Heptane-soluble “di-butylmagnesium” (I) (a commercially available material, prepared by addition of LiBu^s to MgBuⁿCl, and subsequent addition of ca. 5% MgOct₂ⁿ) has been found to be a useful starting material for obtaining numerous organic magnesium compounds. This is illustrated by its reaction with a number of protic compounds HA to give in good yields Mg(C₅H₅)₂, Mg(C₅H₄Me)₂, or the new compounds MgA₂: IV (A = C₅H₄SiMe₃), V [A = C₅H₃(SiMe₃)₂], VII (A = OC₆H₂Bu₂^t-2,6-Me-4), and X [A₂ = N(SiMe₃)C₆H₄N(SiMe₃)-o(OEt₂)]. The value of such compounds MgA₂ as mild ligand transfer reagents is illustrated by the synthesis of Zr(C₅H₃X₂)Cl₃ (X = H or SiMe₃). Compound X was isolated from OEt₂ solution as the crystalline dimer

with two o-$\text{N}$(SiMe₃)C₆H₄$\text{N}$(SiMe₃) ligands bridging two magnesium atoms and a terminal OEt₂ ligand completing a distorted tetrahedral environment around each Mg. Some key parameters are: MgN_t 1.997(7), MgN_b 2.083(8), MgO 2.041(7) Å; OMgN_t 112.1(3), OMgN_b 119.7(3), and N_tMgN_b 118.5(3)°.     

Transport and kinetics at microheterogeneous electrodes: Part V. The methylviologen/platinum system

Results are reported for the reaction of methylviologen radical cation, MV⁺ with platinum colloidal particles, studied by stopped flow spectrophotometry. The rate of the reaction depends on the gaseous pretreatment of the particles. For particles reduced by hydrogen, the kinetics are usually first order with respect to MV⁺. The reaction is also first order in the concentration of platinum, and is inhibited in a first order manner by the product MV²⁺. This inhibition suggests that MV²⁺ is adsorbed on the particle surfaces, and this has been confirmed by ac, ring—disc electrode studies on macroscopic platinum electrodes. At high concentrations of MV⁺ some deviation from first order kinetics is observed. These results are all explained by a kinetic model in which either the desorption of MV²⁺ or the adsorption of MV⁺ is the rate limiting process. The rate of consumption of MV⁺ on an oxidised surface is an order of magnitude faster than that on the reduced surface. Ring—disc studies show that this is because the MV⁺ is not producing H₂ but is reducing the surface oxide. The results are shown to fit a simple model which takes into account this titration of the oxide layer. The model also explains why the rate on partially oxidised surfaces will appear to have an order greater than one in [Pt].