Analysis of Self-Assembled Micelle Inhibitory RNA (SAMiRNA) Drug Using Ion-Pairing Reversed-Phase Liquid Chromatography Combined with Mass Spectrometry.

Small interfering RNA (siRNA) is known for its ability to silence the expression of specific genes, demonstrating its promising potential as a therapeutic approach. Self-assembled micelle inhibitory RNA (SAMiRNA) is an oligonucleotide duplex developed to overcome the in vivo delivery limitations of siRNA. SAMiRNA has hydrophilic and hydrophobic groups at both ends of a sense strand, forming a spherical nanostructure that enhances the in vivo delivery efficiency. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the most commonly used method for the analysis of oligonucleotides. Since SAMiRNA is heavily chemically modified, the behavior of SAMiRNA in IP-RPLC combined with mass spectrometry (MS) is anticipated to differ from that of the conventional siRNA drug. The current investigation using IP-RPLC-MS revealed that a distinct duplex peak along with two minor separate strands of antisense and sense was observed at column temperatures below 35 °C in the IP-RPLC system with a 100 mM ammonium bicarbonate buffer system. At column temperatures higher than 35 °C, however, two fully denatured single strands were observed. The mass spectrum from the chromatographic peak of the SAMiRNA duplex contained signals from the duplex, the antisense, and the sense, probably due to duplex denaturation during the MS ionization process. The current comprehensive analysis results will make a substantial contribution to the future application of IP-RPLC-MS in the analysis of SAMiRNA.

Evaluation of the effect of dimethyl fumarate on human bone marrow-derived mesenchymal stem cells using bottom-up proteomics.

Mesenchymal stem cells (MSCs) have potential as a viable treatment option in the field of regenerative medicine, but MSC-based therapy needs to be more efficient. Preconditioning is a method to improve MSC-based therapy, and dimethyl fumarate (DMF) - an agent that can enhance the antioxidative capacity of cells - can be considered for preconditioning of MSCs. In this study, we treated bone marrow-derived MSCs with DMF and evaluated their proteome using bottom-up proteomics. The MSCs were exposed to 10 µM DMF for 24 h, followed by lysis with an SDS solution, digestion with trypsin using an s-trap column, and analysis using nanoLC-MS/MS, which identified 2262 proteins with confidence. Bioinformatic analysis of the identified proteins revealed 47 upregulated proteins and 81 downregulated proteins upon DMF treatment. Pathway enrichment analysis suggested a possible decrease in autophagy and a decrease in the activity of the TCA cycle, while indicating a potential increase in proliferation and antioxidant activity in DMF-treated MSCs compared to untreated MSCs. Our findings suggest that DMF can enhance the proliferation of MSCs and increase their stability, and that preconditioning could improve the therapeutic efficacy of MSCs for the treatment of regenerative diseases.

Size-exclusion chromatography for the characterization of urinary extracellular vesicles.

In recent years, extracellular vesicles (EVs) have gained attention for their potential as biomarkers for the early diagnosis and treatment of various diseases. Traditionally, EV isolation has relied exclusively on ultracentrifugation. However, alternative enrichment methods such as size-exclusion chromatography (SEC) and polyethylene glycol-based precipitation have been introduced. This study utilized SEC as a characterization tool to assess the efficiency of EV isolation. Urinary EVs isolated from human urine using centrifugation (40,000 × g) were analyzed using an SEC column with a pore size of 1000 Å, an inner diameter of 7.8 mm, and a length of 300 mm. The EVs were detected sequentially using UV (280 nm) and fluorescence (λex/em = 550 nm/565 nm); the EVs were observed at approximately 6 min, while the proteins were observed at approximately 12 min. The repeated centrifugation enrichment steps resulted in an increase in EV peaks and a decrease in protein peaks. SEC analysis of the enriched EV samples confirmed that a four-cycle repetition of centrifugation is necessary for successful EV enrichment and removal of non-EV proteins from 40 mL of human urine.

Controlled growth of redox polymer network on single enzyme molecule for stable and sensitive enzyme electrode.

The electrochemical applications of enzymes are often hampered by poor enzyme stability and low electron conductivity. In this work, a novel enzyme nanogel based on atom transfer radical polymerization (ATRP) has been developed for highly sensitive detection of glucose based on ferrocene (Fc) embedded in crosslinked polymer network nanogel. Enzyme surfaces are successively modified with Br initiator, and then in situ atom transfer radical polymerization (ATRP) was performed to build up crosslinked polyacrylamide network. The resulting single enzyme nanogel (ATRP-SEG) is uniform in size fairly. ATRP-SEG reveals bi-phasic inactivation, and the half-life of stable ATRP-SEG after 18-day incubation at 50 °C is 47 days, which is 197 times longer than that of free Gox (5.7 h). By introducing a ferrocene (Fc) containing redox polymer, poly(acrylamide-co-vinylferrocene), the half-life of Fc-ATRP-SEG after 18-day incubation at 50 °C is 49 days. Fc-ATRP-SEG is used for preparation of glucose-sensing electrode, and the sensitivity of Fc-ATRP-SEG electrode is 111 µA cm-2 mM-1, which is 366 and 1270 times higher than those of free GOx (0.303 µA cm-2 mM-1) and ATRP-SEG (0.0874 µA cm-2 mM-1), respectively. Fc-ATRP-SEG electrode maintained 90% of initial current density under 4 °C storage condition and repetitive usages every day for 7 days. Even the electrode repeatedly used in continuous harsh condition (250 rpm, room temperature), the current density maintained 96% after 12 h incubation at operational condition.

Electro-biocatalytic production of formate from carbon dioxide using an oxygen-stable whole cell biocatalyst.

The use of biocatalysts to convert CO2 into useful chemicals is a promising alternative to chemical conversion. In this study, the electro-biocatalytic conversion of CO2 to formate was attempted with a whole cell biocatalyst. Eight species of Methylobacteria were tested for CO2 reduction, and one of them, Methylobacterium extorquens AM1, exhibited an exceptionally higher capability to synthesize formate from CO2 by supplying electrons with electrodes, which produced formate concentrations of up to 60mM. The oxygen stability of the biocatalyst was investigated, and the results indicated that the whole cell catalyst still exhibited CO2 reduction activity even after being exposed to oxygen gas. From the results, we could demonstrate the electro-biocatalytic conversion of CO2 to formate using an obligate aerobe, M. extorquens AM1, as a whole cell biocatalyst without providing extra cofactors or hydrogen gas. This electro-biocatalytic process suggests a promising approach toward feasible way of CO2 conversion to formate.