Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma.

Osteosarcoma is the most common malignant bone cancer in pediatric patients. Patients who respond poorly to chemotherapy experience worse clinical outcomes with a high mortality rate. The major challenge is the lack of effective drugs for these patients. To introduce new drugs for clinical approval, preclinical studies based on in vitro models must demonstrate the potency of the tested drugs, enabling the drugs to enter phase 1 clinical trials. Patient-derived cell culture is a promising testing platform for in vitro studies, as they more accurately recapitulate cancer states and genetic profiles compared to cell lines. In the present study, we established patient-derived osteosarcoma cells (PDC) from a patient who had previously been diagnosed with retinoblastoma. We identified a new variant of a germline mutation in the RB1 gene in the tissue of the patient. The biological effects of this PDC were studied to observe whether the cryopreserved PDC retained a feature of fresh PDC. The cryopreserved PDC preserved the key biological effects, including cell growth, invasive capability, migration, and mineralization, that define the conserved phenotypes compared to fresh PDC. From whole genome sequencing analysis of osteosarcoma tissue and patient-derived cells, we found that cryopreserved PDC was a minor population in the origin tissue and was selectively grown under the culture conditions. The cryopreserved PDC has a high resistance to conventional chemotherapy. This study demonstrated that the established cryopreserved PDC has the aggressive characteristics of osteosarcoma, in particular the chemoresistance phenotype that might be used for further investigation in the chemoresistant mechanism of osteosarcoma. In conclusion, the approach we applied for primary cell culture might be a promising method to generate in vitro models for functional testing of osteosarcoma.

Human Leukocyte Antigen Markers for Distinguishing Pustular Psoriasis and Adult-Onset Immunodeficiency with Pustular Reaction.

Pustular skin diseases, with pustular psoriasis (PP) being the prototype, are immune-mediated diseases characterized by the presence of multiple pustules, resulting from neutrophil accumulation in the layer of epidermis. Sterile skin pustular eruption, like PP, is also observed in 20-30% of patients with adult-onset immunodeficiency syndrome (AOID) and anti-interferon γ autoantibodies (IFN-γ), leading to challenges in classification and diagnosis. While the mechanism underlying this similar phenotype remains unknown, genetic factors in relation to the immune system are suspected of playing an important role. Here, the association between human leukocyte antigen (HLA) genes, which play essential roles in antigen presentation, contributing to immune response, and the presence of skin pustules in AOID and PP was revealed. HLA genotyping of 41 patients from multiple centers in Thailand who presented with multiple sterile skin pustules (17 AOID patients and 24 PP patients) was conducted using a next-generation-sequencing-based approach. In comparison to healthy controls, HLA-B*13:01 (OR = 3.825, 95%CI: 2.08-7.035), C*03:04 (OR = 3.665, 95%CI: 2.102-6.39), and DQB1*05:02 (OR = 2.134, 95%CI: 1.326-3.434) were significantly associated with the group of aforementioned conditions having sterile cutaneous pustules, suggesting a common genetic-related mechanism. We found that DPB1*05:01 (OR = 3.851, p = 0.008) and DRB1*15:02 (OR = 3.195, p = 0.033) have a significant association with pustular reaction in AOID patients, with PP patients used as a control. A variant in the DRB1 gene, rs17885482 (OR = 9.073, p = 0.005), was observed to be a risk factor for PP when using AOID patients who had pustular reactions as a control group. DPB1*05:01 and DRB1*15:02 alleles, as well as the rs17885482 variant in the DRB1 gene, were proposed as novel biomarkers to differentiate PP and AOID patients who first present with multiple sterile skin pustules without known documented underlying conditions.

Gut Microbiota-Mediated Pharmacokinetic Drug-Drug Interactions between Mycophenolic Acid and Trimethoprim-Sulfamethoxazole in Humans.

Mycophenolic acid (MPA) and trimethoprim-sulfamethoxazole (TMP-SMX) are commonly prescribed together in certain groups of patients, including solid organ transplant recipients. However, little is known about the pharmacokinetic drug-drug interactions (DDIs) between these two medications. Therefore, the present study aimed to determine the effects of TMP-SMX on MPA pharmacokinetics in humans and to find out the relationship between MPA pharmacokinetics and gut microbiota alteration. This study enrolled 16 healthy volunteers to take a single oral dose of 1000 mg mycophenolate mofetil (MMF), a prodrug of MPA, administered without and with concurrent use of TMP-SMX (320/1600 mg/day) for five days. The pharmacokinetic parameters of MPA and its glucuronide (MPAG) were measured using high-performance liquid chromatography. The composition of gut microbiota in stool samples was profiled using a 16S rRNA metagenomic sequencing technique during pre- and post-TMP-SMX treatment. Relative abundance, bacterial co-occurrence networks, and correlations between bacterial abundance and pharmacokinetic parameters were investigated. The results showed a significant decrease in systemic MPA exposure when TMP-SMX was coadministered with MMF. Analysis of the gut microbiome revealed altered relative abundance of two enriched genera, namely the genus Bacteroides and Faecalibacterium, following TMP-SMX treatment. The relative abundance of the genera Bacteroides, [Eubacterium] coprostanoligenes group, [Eubacterium] eligens group, and Ruminococcus appeared to be significantly correlated with systemic MPA exposure. Coadministration of TMP-SMX with MMF resulted in a reduction in systemic MPA exposure. The pharmacokinetic DDIs between these two drugs were attributed to the effect of TMP-SMX, a broad-spectrum antibiotic, on gut microbiota-mediated MPA metabolism.

Characterization of Cell-Free DNA Size Distribution in Osteosarcoma Patients.

PURPOSE: Cell-free DNA (cfDNA) analysis is a powerful tool for noninvasively 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 patients with osteosarcoma 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 patients with osteosarcoma 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% confidence interval (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 signals up to 2.3-fold when compared with 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. See related commentary by Weiser et al., p. 2017.

MALDI Mass Spectrometry Imaging Reveals the Existence of an N-Acyl-homoserine Lactone Quorum Sensing System in Pseudomonas putida Biofilms.

Quorum sensing (QS) is generally used to describe the process involving the release and recognition of signaling molecules, such as N-acyl-homoserine lactones, by bacteria to coordinate their response to population density and biofilm development. However, detailed information on the heterogeneity of QS metabolites in biofilms remains largely unknown. Here, we describe the utilization of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to follow the production of specific metabolites, including QS metabolites, during Pseudomonas putida biofilm development. To do so, a method to grow an agar-based biofilm was first established, and MALDI-MSI was used to detect and visualize the distribution of QS metabolites in biofilms at different cultivation times. This study demonstrated that N-acyl-homoserine lactones are homogeneously produced in the early stages of P. putida biofilm formation. In contrast, the spatial distribution of quinolones and pyochelin correlated with the swarming motility of P. putida in mature biofilms. These two metabolites are involved in the production of extracellular polymeric substances and iron chelators. Our study thus contributes to establishing the specific temporal regulation and spatial distribution of N-acyl-homoserine lactone-related metabolites and quinolone and pyochelin in P. putida biofilms.

Luteolin-rich fraction from Perilla frutescens seed meal inhibits spike glycoprotein S1 of SARS-CoV-2-induced NLRP3 inflammasome lung cell inflammation via regulation of JAK1/STAT3 pathway: A potential anti-inflammatory compound against inflammation-induced long-COVID.

Objective: The 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. Methods: PFEA 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. Results: PFEA 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-1ß, IL-18, and NLRP3 gene suppressions, as well as IL-6, IL-1ß, 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). Conclusion: The 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.

Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica.

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.

Metabolomic Analysis of Response to Nitrogen-Limiting Conditions in Yarrowia spp.

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.