Wonwoong Lee

Electromagnetized gold nanoparticles mediate direct lineage reprogramming into induced dopamine neurons in vivo for Parkinson's disease therapy

Electromagnetic fields (EMF) are physical energy fields generated by electrically charged objects, and specific ranges of EMF can influence numerous biological processes, which include the control of cell fate and plasticity. In this study, we show that electromagnetized gold nanoparticles (AuNPs) in the presence of specific EMF conditions facilitate an efficient direct lineage reprogramming to induced dopamine neurons in vitro and in vivo. Remarkably, electromagnetic stimulation leads to a specific activation of the histone acetyltransferase Brd2, which results in histone H3K27 acetylation and a robust activation of neuron-specific genes. In vivo dopaminergic neuron reprogramming by EMF stimulation of AuNPs efficiently and non-invasively alleviated symptoms in mouse Parkinsons disease models. This study provides a proof of principle for EMF-based in vivo lineage conversion as a potentially viable and safe therapeutic strategy for the treatment of neurodegenerative disorders.

Structural determination of glucosylceramides isolated from marine sponge by fast atom bombardment collision‐induced dissociation linked scan at constant B/E

Five glucosylceramides (GlcCers) were isolated by reversed phase high-performance liquid chromatography from the MeOH extracts of a marine sponge, Haliclona (Reniera) sp., collected from the coast of Ulleung Island, Korea, and analyzed by fast atom bombardment mass spectrometry (FAB-MS) in positive-ion mode. FAB-mass spectra of these compounds included protonated molecules [M + H](+) and abundant sodiated molecules [M + Na](+) from a mixture of m-NBA and NaI. The structures of these GlcCers, which were similar, were elucidated by FAB-linked scan at constant B/E. To find diagnostic ions for their characterization, the GlcCers were analyzed by collision-induced dissociation (CID) linked scan at constant B/E. The CID-linked scan at constant B/E of [M + H](+) and [M + Na](+) precursor ions resulted in the formation of numerous characteristic product ions via a series of dissociative processes. The product ions formed by charge-remote fragmentation provided important information for the characterization of the fatty N-acyl chain moiety and the sphingoid base, commonly referred to as the long-chain base. The product ions at m/z 203 and 502 were diagnostic for the presence of a sodiated sugar ring and beta-D-glucosylsphinganine, respectively. For further confirmation of the structure of the fatty N-acyl chain moiety in each GlcCer, fatty acid methyl esters were obtained from the five GlcCers by methanolysis and analyzed by FAB-MS in positive-ion mode. On the basis of these dissociation patterns, the structures of the five GlcCers from marine sponge were elucidated. In addition, the accurate mass measurement was performed to obtain the elemental composition of the GlcCers isolated from marine sponge.

Metabolic profiling of tyrosine, tryptophan, and glutamate in human urine using gas chromatography–tandem mass spectrometry combined with single SPE cleanup

The tyrosine, tryptophan, and glutamate metabolic pathways play key roles on pathological state of neuronal functions and the change of their levels in biological systems reflects the progress degree of neuronal diseases. Comprehensive profiling of these metabolites is important to find new biomarkers for diagnosis or prognosis of various neuronal diseases. However, the overall profiling analysis of various neurochemicals in biological sample is confronted with several limitations due to their low concentration and physicochemical properties and the coexistence of matrices. We developed an efficient and feasible method using gas chromatography-tandem mass spectrometry (GC-MS/MS). Wide-bore mixed cation exchange (MCX) SPE process enables a rapid and effective cleanup of 20 neurochemicals even including acidic and basic neurochemicals in a single SPE cartridge by using different composition of eluents. Selective derivatization of various types of metabolites was applied to achieve highly chromatographic separation and sensitive mass detection. Appropriate selection of precursor and product transition ions used in multiple reaction-monitoring (MRM) mode based on the MS/MS fragmentations of the derivatized neurochemicals could be significantly minimized the matrix effects and enhanced the reliability of quantification results. The developed method was validated in terms of linearity, limits of detection, precision, accuracy, and matrix effects. The intra- and inter-assay analytical variations were less than 10%. The overall linearity for all of the targets was excellent (R2≥0.996). The detection limits ranged between 0.38 and 8.13ng/mL for the acidic neurochemicals and between 0.02 and 11.1ng/mL for the basic neurochemicals. The developed protocol will be expected to be a promising tool for the understanding of the pathological state and diagnosis of various neuronal diseases.

Reliable screening and confirmation of 156 multi-class illegal adulterants in dietary supplements based on extracted common ion chromatograms by ultra-high-performance liquid chromatography-quadrupole/time of flight-mass spectrometry

An analytical method for the reliable screening and confirmation of 156 illegal drugs (58 erectile dysfunction drugs, 49 synthetic steroids, 26 anabolic steroids, and 23 anti-histamine drugs) in supplementary diets using ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) was developed. Various types of supplements (liquid, capsule, powder, pill and tablet) with complicated matrices were pretreated by simple liquid-liquid extraction. The wide scope of 156 target compounds was effectively determined within 15min in the positive ion mode, detecting the compounds at a sub-ppb level. Their MS/MS spectra were preferentially investigated to find diagnostic common ions according to the structural similarity of diverse adulterants. For the rapid screening of multiple classes of the target adulterants, extracted common ion chromatograms (ECICs) based on specific fragments of similar molecular moieties were attempted. A database including the elemental compositions, retention times, and MS/MS spectra was built for the confirmation of adulterants. The established method was validated in terms of the linearity, limits of detection (LOD), precision, and accuracy. The linear correlation coefficient and limit of detection ranged from 0.9880 to 1 and from 0.02 to 16.04ng/mL, respectively. The precision and accuracy of intra- and inter-day experiments for the spiked samples at the range of 0.2 and 16.0ng/mL were from 0.16 to 13.50% and 0.19-11.48%, respectively, with relative standard deviation. Mean recoveries ranged from 81.6 to 124.7%, and relative standard deviation was less than 9.20%. The screening and confirmation method demonstrated the usefulness of UHPLC-Q/TOF-MS combined with ECICs as a promising approach for the analysis of multi-class adulterants. Finally, the established method was successfully applied for the monitoring of several types of dietary supplements in routine analysis.

Amorphous silica nanoparticle-induced perturbation of cholesterol homeostasis as a function of surface area highlights safe-by-design implementation: an integrated multi-OMICS analysis

To close the knowledge gap between the wide application of amorphous silica nanoparticles (aSiNPs) and their health impact, the present study endeavored to investigate the molecular mechanisms involved in aSiNPs-mediated hepatotoxicity with a systems toxicology approach and how it is related to the physico–chemical properties of aSiNPs. To this end, we used four types of aSiNPs with different surface areas: aSiNP-116 (surface area: 116 m2 g−1), aSiNP-189 (surface area: 189 m2 g−1), aSiNP-26 (surface area: 26 m2 g−1) and aSiNP-8 (surface area: 8.3 m2 g−1); we also used the human hepatoma (HepG2) cell line as a model system. We applied multi-OMICS (DNA microarray based transcriptomics and GC-MS based lipidomics) followed by bioinformatics analysis in aSiNP-116 treated HepG2 cells. The perturbations of steroid-cholesterol biosynthesis were revealed by KEGG (with significantly altered genes) and IMPaLA (with integrated significantly altered genes and metabolites) pathway analysis. Furthermore, in corroboration with in silico analysis, the biochemical tests exhibited a concentration dependent increase in total cholesterol levels due to aSiNP-116 treatment. In a subsequent step, the hypothesis derived for aSiNP-116 was further tested for cells exposed to other aSiNPs (aSiNP-189, aSiNP-26 and aSiNP-8) with GC-MS based lipidomics as well as biochemical tests. The alterations in cholesterol biosynthesis were found to be directly proportional with the surface area of the aSiNPs, i.e., the larger the surface area, the higher the cholesterol level. Taken together, perturbation of cholesterol biosynthesis as a function of surface area was found to be a principal mode-of-action of aSiNPs exposure, which necessitates a safe-by-design approach for its biological applications.

Comprehensive chemical profiling of Pinellia species tuber and processed Pinellia tuber by gas chromatography–mass spectrometry and liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry

A comprehensive profiling method was established for the determination of various chemicals in Pinellia (P.) ternata and pedatisecta species. The profiling method comprises a fast ultrasonic extraction with various solvents, followed by GC-MS and LC-APCI-MS analysis. A total of 73 polar components as trimethylsilyl (TMS) derivatives were detected in methanol extract by GC-MS. The main components of the P. species were profiled as several kinds of fatty acids, amino acids, nucleic acids, carbohydrates, and phenolic compounds. The hexane extract was analyzed by LC-APCI-MS for the lipid profiling. A total of 35 lipid constituents [fatty acids and their esters, mono-, di-, and tri-acylglycerols] and four phytosterols were observed and tentatively characterized by LC-APCI-MS/MS. Among the phytochemicals detected in the hexane extract, triacylglycerols (TAGs) as the major component were identified by LC-APCI-MS and MS/MS. Based on the identified components, a significant difference in the chemical compositions of P. species tuber and processed P. ternata was found that the complete disappearance of TAGs and a considerable decrement of sucrose were observed in processed P. ternata. Furthermore, the degradation mechanism for TAGs in the presence of alum solution is suggested to occur during the processing P. ternata. Malic acid was found to be a characteristic compound for the classification of P. ternata and pedatisecta with different geographic origins. Based on the validated GC/MS method, twenty-four P. ternata, processed P. ternata and P. pedatisecta samples were profiled to measure the overall abundance of specific groups of compound and to identify diagnostic compounds. In addition, principal component analysis (PCA) on the GC/MS profiling data revealed a clear classification of P. species samples. In this study, the full chemical complement was for the first time reported for quality evaluation of P. species. The method can be usefully applied for phytochemical analysis of related herbal medicines.

Analytical trend of perfluorinated compounds in environmental and biota samples

Perfluorinated compounds have characteristics of resistance to heat, acidic, basic conditions and also resist water, oil, grease, pollutant. Futhermore they are used by various industrial material, nowadays, they produced in large scale for indutrial and commercial areas. However, they also resist metabolizing and degrading in environmental system (plant, animal, even human body). Moreover, in animal`s bodies, PFCs can be accumulated in organ (eg; liver) and lead to liver cell necrosis even oncogenesis. Perfluorinated compounds are newly registered as new persistent organic pollutants (POPs) on Stockholm convention in 2009. Therefore necessity for analytical methodology for determination of PFCs in various environmental samples is even more increased. This study discussed sample preparation and instrumental conditions for the analysis of PFCs in environmental and biota samples.

Profiling of a wide range of neurochemicals in human urine by very-high-performance liquid chromatography-tandem mass spectrometry combined with in situ selective derivatization

Development of a reliable analytical method of neurochemicals in biological fluids is important to discover potential biomarkers for the diagnosis, treatment and prognosis of neurological disorders. However, neurochemical profiling of biological samples is challenging because of highly different polarities between basic and acidic neurochemicals, low physiological levels, and high matrix interference in biological samples. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method combined with in situ selective derivatization for comprehensive profiling of 20 neurochemicals in urine was developed for a wide range of neurochemicals. In situ selective derivatization greatly improved the peak capacity on a reversed-phase C18 column and sensitive mass detection in LC-ESI-MS/MS-positive ion mode due to reduction of the distinct physicochemical properties between acidic and basic neurochemicals. The MS/MS spectra of neurochemicals exhibited specific ions, such as losses of amine, methanol, or methyl formate molecules from protonated molecules, enabling selection of appropriate multiple reaction monitoring (MRM) ions for selective and sensitive detection. The developed method was validated in terms of linearity, limit of detection (LOD) and limit of quantification (LOQ), precision, accuracy, and recovery. The correlation coefficients (R2) of calibration curves were above 0.9961. The ranges of LODs and LOQs were 0.1-3.6ng/mL and 0.3-12.0ng/mL, respectively. The overall precision and accuracy were 0.52-16.74% and 82.26-118.17%, respectively. The method was successfully applied to simultaneously profile the metabolic pathways of tyrosine, tryptophan, and glutamate in Parkinsons disease patient urine (PD, n=21) and control urine (n=10). Significant differences (P≤0.01) between two groups in the activity of phenylethanolamine N-methyltransferase (PNMT) and alcohol dehydrogenase (ADH) were observed. In conclusion, this method provides reliable quantification of a wide range of neurochemicals in human urine and would be helpful for finding biomarkers related to specific neuronal diseases.

An in situ extraction and derivatization method for rapid analysis of short-chain fatty acids in rat fecal samples by gas chromatography tandem mass spectrometry

Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and changes in their levels in a biological system reflect the progress of various diseases. However, analysis of SCFAs in biological samples is limited due to their easy loss during sample workup, their low concentrations, and the coexistence of matrices. In this study, we developed a feasible method for the analysis of SCFAs in rat fecal samples using gas chromatography-tandem mass spectrometry (GC-MS/MS) combined with an in situ extraction and derivatization method. More specifically, in situ extraction and derivatization were applied for the determination of SCFAs using tetrabutylammonium hydrogen sulfate (TBAHS) and pentafluorobenzyl bromide (PFBBr). TBAHS concentration and pH values, as well as the temperature and time of the derivatization reaction were effectively optimized. Moreover, the proposed process reduced the analysis time and limited the loss of SCFAs during the sample workup. It also improved their peak shapes and resulted in longer GC retentions and high sensitivity in MS detection. Use of GC-MS/MS could offer high sensitivity and selectivity for SCFA-PFB derivatives in complicated matrix samples by selecting a multiple-ion reaction monitoring (MRM) transition ion at m/z 181 that corresponds to the [PFB]+ ion. The established method was also validated in terms of linearity, limits of detection (LODs) and quantification (LOQs), and matrix effects. It was proved to be linear (r2 > 0.997), with lower LODs of 5–24 ng mL−1, and lower LOQs of 0.05–0.1 μg mL−1 for all SCFAs. The proposed method will be useful for the determination of SCFAs in rat fecal samples and for the clinical diagnosis of various diseases.