Optimized quantification of fatty acids in complex media using advanced analytical techniques.

RATIONALE: Various medium formulations contain essential fatty acids at concentrations ranging from 10 to 100 mg/L. Accurate and precise lipid measurement in media is crucial for monitoring media quality and conducting studies on lipids in the context of cell culture. This study employed two-dimensional gas chromatography (GC × GC) analyses to offer enhanced resolution, sensitivity, and separation performance compared to GC. METHODS: Quantification of fatty acid methyl esters (FAMEs) in a medium was conducted using GC × GC combined with a high-resolution mass spectrometer and flame ionization detector, considering potential interference from nonionic surfactant Tween 80, which was precipitated and removed by optimizing the concentration of cobalt thiocyanate (CTA) solution during pretreatment. This advanced analytical approach enabled identification of cis and trans isomers of identical molecular weights and determination of the location and number of double bonds in the same carbon number structure. RESULTS: Our analysis identified 36 FAMEs within the C6-C24 region, and a 5% CTA solution was optimal for efficient removal of Tween 80 during lipid extraction. Additionally, this advanced method minimized FAME contamination and loss during pretreatment, thereby significantly reducing the sample volume required to detect trace levels of FAMEs. This improvement led to a fatty acid recovery rate of 106% while maintaining the average relative standard deviation for the target FAMEs of about 3%. CONCLUSIONS: Our research paves the way for future investigation into medium quality control and the role of fatty acids in cell culture. This offers the possibility for economical and effective trace quantification of fatty acids in complex media.

In-depth proteome analysis of brain tissue from Ewsr1 knockout mouse by multiplexed isobaric tandem mass tag labeling.

EWS RNA binding protein 1 (EWSR1) is a multifunctional protein whose epigenetic signatures contribute to the pathogenesis of various human diseases, such as neurodegenerative disorders, skin development, and tumorigenic processes. However, the specific cellular functions and physiological characteristics of EWSR1 remain unclear. In this study, we used quantitative mass spectrometry-based proteomics with tandem mass tag labeling to investigate the global proteome changes in brain tissue in Ewsr1 knockout and wild-type mice. From 9115 identified proteins, we selected 118 differentially expressed proteins, which is common to three quantitative data processing strategies including only protein level normalizations and spectrum-protein level normalization. Bioinformatics analysis of these common differentially expressed proteins revealed that proteins up-regulated in Ewsr1 knockout mouse are mostly related to the positive regulation of bone remodeling and inflammatory response. The down-regulated proteins were associated with the regulation of neurotransmitter levels or amino acid metabolic processes. Collectively, these findings provide insight into the physiological function and pathogenesis of EWSR1 on protein level. Better understanding of EWSR1 and its protein interactions will advance the field of clinical research into neuronal disorders. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD026994.

Digital Therapeutics Product Repositioning via Multiplex Link Prediction Based on Genomic, Chemical, and Technological Information.

Digital therapeutics (DTx) companies are making efforts to expand their consumer base in the growing market. To this end, many of them reposition their existing products to identify new target diseases. In this study, we provide a link prediction framework for DTx product repositioning on the basis of a multiplex disorder network by integrating multiple data sources, such as associated genes between disorders, shared drugs, and patents of treatment protocols. To capture the disorders' latent features, both random-walk-based and deep-learning-based graph embedding methods are applied to transform the graph structure into vectors. Consequently, new indications are suggested for DTx products based on the cosine similarity between original and candidate disorders. Our framework was applied to five psychiatric DTx products to determine new target disorders that have the highest treatment potential for each product. Therefore, the study results are expected to assist DTx firms in entering the novel target market with low risk within a short period. Moreover, the applicability of DTx products to a wider variety of disorders may increase access to overall patient groups and gradually improve public health.

Oil spill fingerprint of low sulfur fuel oil in South Korea.

A low sulfur fuel oil (LSFO) spill accident occurred in South Korea on December 17, 2019, before the introduction of the International Maritime Organization (IMO) sulfur limit. In this study, chromatograms, percentage weathering plots (PW-plots), and diagnostic ratios (DRs) of LSFOs collected in different areas during in the early spillage were compared for oil spill fingerprint. The source oil was conformed as LSFO according to physical properties and spill oils, like the source oil, show high n-alkanes and low benzo[b]naphto[1,2-d]thiophene (BNT) distribution. In the PW-plots, spill oils exhibited a decreasing trend with the reduction of low-molecular-weight compounds, which were affected by evaporation. The relative difference in the DRs was below 14%, indicating that the source and spill oils matched, excluding the ratios consisting of evaporated compounds. These results showed that spill oils confirmed as LSFO were evaporated during the initial spillage stage, and matched to the source oil.

Multiplex PCR analysis of virulence genes and their influence on antibiotic resistance in Enterococcus spp. isolated from broiler chicken.

Enterococcus spp. are opportunistic pathogens that cause lameness in broiler chickens, resulting in serious economic losses worldwide. Virulence of Enterococcus spp. is associated with several putative virulence genes including fsr, efm, esp, cylA, cad1, ace, gelE, and asa1. In this study, multiplex polymerase chain reaction (PCR) for the simultaneous detection of these virulence genes in Enterococcus spp. was developed, and detection limits for E. faecium, E. faecalis, and E. hirae were 64.0 pg/µL, 320.0 pg/µL, and 1.6 ng/µL DNA, respectively. Among 80 Enterococcus isolates tested, efm and cad1 were detected in all 26 E. faecium samples, and only cad1 was observed in E. hirae. Additionally, the presence of virulence genes in 25 E. faecalis isolates were 100% for cad1, 88.0% for gelE, 64.0% for fsr, 44.0% for asa1, 16.0% for cylA, and 4.0% for esp. No virulence genes were found in E. gallinarum isolates. A total of 49 isolates were resistant to tigecycline and to at least 2 different classes of antibiotics. The most prevalent resistance was to ciprofloxacin (73.5%), quinupristin/dalfopristin (55.1%), and tetracycline (49.0%). No strains were resistant to vancomycin or linezolid. This is the first multiplex PCR assay to simultaneously detect eight virulence genes in Enterococcus spp., and the method provides diagnostic value for accurate, rapid, and convenient detection of virulence genes. Additionally, we report the prevalence of virulence genes and antimicrobial resistance in Enterococcus isolates from commercial broiler chickens suffering lameness.