ObjectiveThe multi-systemic inflammation as a result of COVID-19 can persevere long after the initial symptoms of the illness have subsided. These effects are referred to as Long-COVID. Our research focused on the contribution of the Spike protein S1 subunit of SARS-CoV-2 (Spike S1) on the lung inflammation mediated by NLRP3 inflammasome machinery and the cytokine releases, interleukin 6 (IL-6), IL-1beta, and IL-18, in lung epithelial cells. This study has attempted to identify the naturally- occurring agents that act against inflammation-related long-COVID. The seed meal of Perilla frutescens (P. frutescens), which contains two major dietary polyphenols (rosmarinic acid and luteolin), has been reported to exhibit anti-inflammation activities. Therefore, we have established the ethyl acetate fraction of P. frutescens seed meal (PFEA) and determined its anti-inflammatory effects on Spike S1 exposure in A549 lung cells.MethodsPFEA was established using solvent-partitioned extraction. Rosmarinic acid (Ra) and luteolin (Lu) in PFEA were identified using the HPLC technique. The inhibitory effects of PFEA and its active compounds against Spike S1-induced inflammatory response in A549 cells were determined by RT-PCR and ELISA. The mechanistic study of anti-inflammatory properties of PFEA and Lu were determined using western blot technique.ResultsPFEA was found to contain Ra (388.70 ± 11.12 mg/g extract) and Lu (248.82 ± 12.34 mg/g extract) as its major polyphenols. Accordingly, A549 lung cells were pre-treated with PFEA (12.5-100 μg/mL) and its two major compounds (2.5-20 μg/mL) prior to the Spike S1 exposure at 100 ng/mL. PFEA dose-dependently exhibited anti-inflammatory properties upon Spike S1-exposed A549 cells through IL-6, IL-1β, IL-18, and NLRP3 gene suppressions, as well as IL-6, IL-1β, and IL-18 cytokine releases with statistical significance (p < 0.05). Importantly, Lu possesses superior anti-inflammatory properties when compared with Ra (p < 0.01). Mechanistically, PFEA and Lu effectively attenuated a Spike S1-induced inflammatory response through downregulation of the JAK1/STAT3-inflammasome-dependent inflammatory pathway as evidenced by the downregulation of NLRP3, ASC, and cleaved-caspase-1 of the NLRP3 inflammasome components and by modulating the phosphorylation of JAK1 and STAT3 proteins (p < 0.05).ConclusionThe findings suggested that luteolin and PFEA can modulate the signaling cascades that regulate Spike S1-induced lung inflammation during the incidence of Long-COVID. Consequently, luteolin and P. frutescens may be introduced as potential candidates in the preventive therapeutic strategy for inflammation-related post-acute sequelae of COVID-19.

Yarrowia is a yeast genus that has been used as a model oleaginous taxon for a wide array of studies. However, information regarding metabolite changes within Yarrowia spp. under different environmental conditions is still limited. Among various factors affecting Yarrowia metabolism, nitrogen-limiting conditions have a profound effect on the metabolic state of yeast. In this study, a time-course LC-MS/MS-based metabolome analysis of Y. lipolytica was performed to determine the optimal cultivation time and carbon-to-nitrogen ratio for studying the effects of nitrogen-limiting conditions on Yarrowia; we found that cultivation time of 36 h and carbon-to-nitrogen ratio of 4:1 and 5:0 was suitable for studying the effects of nitrogen-limiting conditions on Yarrowia and these conditions were applied to six strains of Yarrowia. These six strains of Yarrowia showed similar responses to nitrogen-limiting conditions; however, each strain had a unique metabolomic profile. Purine and pyrimidine metabolism were the most highly affected biological pathways in nitrogen-limiting conditions, indicating that these conditions affect energy availability within cells. This stress leads to a shift in cells to the utilization of a less ATP-dependent biological pathway. This information will be beneficial for the development of Yarrowia strains for further scientific and industrial applications.

Purpose: Cell-free DNA analysis is a powerful tool for non-invasively predicting patient outcomes. We analyzed the size distribution of cfDNA and assessed its prognostic and diagnostic values in an osteosarcoma cohort. Experimental Design: The fragment size distribution and level of cfDNA were analyzed in 15 healthy donors and 50 osteosarcoma patients using automated capillary electrophoresis. The prognostic performance of cfDNA size analysis was assessed using univariate and multivariable analyses. By performing whole-genome sequencing of matched cfDNA and osteosarcoma tissue samples, we investigated the correlation between the size and mutation profiles of cfDNA and the mutation concordance between cfDNA and paired tissue tumors. Results: The size of cfDNA fragments in osteosarcoma patients was significantly shorter than in healthy donors, with the integrative analysis of size distribution and level of cfDNA achieving a high specificity and sensitivity of 100%. The short cfDNA fragment (150 bp cut-off) was an independent prognostic predictor in this osteosarcoma cohort [HR=9.03; 95% CI=1.13-72.20); p=0.038]. Shortened cfDNA fragments were found to be a major source of mutations. Enrichment of cfDNA fragments with less than or equal to 150 bp by in-silico size selection remarkedly improved the detection of copy number variation (CNV) signals up to 2.3-fold when compared to total cfDNA, with a higher concordance rate with matched osteosarcoma tissue. Conclusions: This finding demonstrated the potential of cfDNA size profiling in the stratification of poor prognostic patients with osteosarcoma. The short fragments of cfDNA are a promising source for boosting the detection of significant mutations in osteosarcoma.

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Methyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

<p>Supplementary figure 1. To identify peak of cfDNA fragments, (A) the scheme electropherograms illustrated the identification of cfDNA fragments using cutoff of ≤150 bp, samples with at least one detected cfDNA peak shorter than or equal to 150 bp are considered positive, whereas samples with all cfDNA peaks longer than 150 bp are considered negative. (B) The electropherograms represented the distribution of all detectable peak of cfDNA found in plasma samples from osteosarcoma patients. The table demonstrated the detectable sub-peaks and main peaks in each sample.</p>

<p>Supplementary figure 2. Gel image analysis demonstrated the distribution of cfDNA fragment size in plasma from (A) healthy donors (n=15) and (B) osteosarcoma (n=50). (C) Comparison of cfDNA fragment size distribution pattern between healthy control, patient with osteosarcoma stage IIb, and osteosarcoma stage III. All results were analyzed by QIAxcel screengel software.</p>

<p>Supplementary figure 3. The comparison of cfDNA fragment size distribution pattern between two techniques analysis; automated capillary electrophoresis and genome wide sequencing of sample (A) OS07 (B) OS08, and (C) OS10. For automated capillary electrophoresis, the distribution of cfDNA fragment was analyzed and presented as electropherogram by QIAxcel screengel software. While all read of insert size obtained from whole-genome sequencing was plotted as histogram using Picard software.</p>