Targeted mass spectrometry reveals interferon-dependent eicosanoid and fatty acid alterations in chronic myeloid leukaemia

Bioactive lipids are involved in cellular signalling events with links to human disease. Many of these are involved in inflammation under normal and pathological conditions. Despite being attractive molecules from a pharmacological point of view, the detection and quantification of lipids has been a major challenge. Here, we have optimised a liquid chromatography–dynamic multiple reaction monitoring–targeted mass spectrometry (LC-dMRM-MS) approach to profile eicosanoids and fatty acids in biological samples. In particular, by applying this analytic workflow to study a cellular model of chronic myeloid leukaemia (CML), we found that the levels of intra- and extracellular 2-Arachidonoylglycerol (2-AG), intracellular Arachidonic Acid (AA), extracellular Prostaglandin F2α (PGF2α), extracellular 5-Hydroxyeicosatetraenoic acid (5-HETE), extracellular Palmitic acid (PA, C16:0) and extracellular Stearic acid (SA, C18:0), were altered in response to immunomodulation by type I interferon (IFN-I), a currently approved treatment for CML. Our observations indicate changes in eicosanoid and fatty acid metabolism, with potential relevance in the context of cancer inflammation and CML

SARS-CoV-2 mutations affect antigen processing by the proteasome to alter CD8+ T cell responses

Mutations within viral epitopes can result in escape from T cells, but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two SARS-CoV-2 nucleoprotein CD8+ epitopes, we investigated the contribution of these flanking mutations to proteasomal processing and T cell activation. We found decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103 N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on proteasomal processing, either contributing to T cell escape or enhancement that may be exploited for future vaccine design

Ketoester methacrylate resin, secondary amine clean-up in the presence of primary amines

A ketoester resin was developed as the basis for a selective scavenger for primary amines in the presence of secondary amines. The utility of the scavenger was demonstrated with a range of reductive amination chemistries with both mono- and diamines. The resin's specificity is based on the removal of the primary amines via their enamines.<br/

Synthesis of resins via multiparallel suspension polymerization

A system for the multiparallel synthesis of polymer resins for solid-phase synthesis is presented, accelerating the preparation of new resins using standard suspension polymerization protocols. The system was evaluated by preparing a range of polystyrene resins with well-defined characteristics

Optimizing 2D gas chromatography mass spectrometry for robust tissue, serum and urine metabolite profiling

Two-dimensional gas chromatography mass spectrometry (GCxGC-MS) is utilized to an increasing extent in biomedical metabolomics. Here, we established and adapted metabolite extraction and derivatization protocols for cell/tissue biopsy, serum and urine samples according to their individual properties. GCxGC-MS analysis revealed detection of ~600 molecular features from which 165 were characterized representing different classes such as amino acids, fatty acids, lipids, carbohydrates, nucleotides and small polar components of glycolysis and the Krebs cycle using electron impact (EI) spectrum matching and validation using external standard compounds. Advantages of two-dimensional gas chromatography based resolution were demonstrated by optimizing gradient length and separation through modulation between the first and second column, leading to a marked increase in metabolite identification due to improved separation as exemplified for lactate versus pyruvate, talopyranose versus methyl palmitate and inosine versus docosahexaenoic acid. Our results demonstrate that GCxGC-MS represents a robust metabolomics platform for discovery and targeted studies that can be used with samples derived from the clinic

Overexpression of CLC-3 is regulated by XRCC5 and is a poor prognostic biomarker for gastric cancer

Abstract Background Recently, many potential prognostic biomarkers for gastric cancer (GC) have been identified, but the prognosis of advanced GC patients remains poor. Chloride channels are promising cancer biomarkers, and their family member chloride channel-3 (CLC-3) is involved in multiple biological behaviors. However, whether CLC-3 is a prognostic biomarker for GC patients is rarely reported. The molecular mechanisms by which CLC-3 is regulated in GC are unclear. Methods The expression of CLC-3 and XRCC5 in human specimens was analyzed using immunohistochemistry. The primary biological functions and pathways related to CLC-3 were enriched by RNA sequencing. A 5′-biotin-labeled DNA probe with a promoter region between − 248 and + 226 was synthesized to pull down CLC-3 promoter-binding proteins. Functional studies were detected by MTS, clone formation, wound scratch, transwell, and xenograft mice model. Mechanistic studies were investigated by streptavidin-agarose-mediated DNA pull-down, mass spectrometry, ChIP, dual-luciferase reporter assay system, Co-IP, and immunofluorescence. Results The results showed that CLC-3 was overexpressed in human GC tissues and that overexpression of CLC-3 was a poor prognostic biomarker for GC patients (P = 0.012). Furthermore, higher expression of CLC-3 was correlated with deeper tumor invasion (P = 0.006) and increased lymph node metastasis (P = 0.016), and knockdown of CLC-3 inhibited cell proliferation and migration in vitro. In addition, X-ray repair cross-complementing 5 (XRCC5) was identified as a CLC-3 promoter-binding protein, and both CLC-3 (HR 1.671; 95% CI 1.012–2.758; P = 0.045) and XRCC5 (HR 1.795; 95% CI 1.076–2.994; P = 0.025) were prognostic factors of overall survival in GC patients. The in vitro and in vivo results showed that the expression and function of CLC-3 were inhibited after XRCC5 knockdown, and the inhibition effects were rescued by CLC-3 overexpression. Meanwhile, the expression and function of CLC-3 were promoted after XRCC5 overexpression, and the promotion effects were reversed by the CLC-3 knockdown. The mechanistic study revealed that knockdown of XRCC5 suppressed the binding of XRCC5 to the CLC-3 promoter and subsequent promoter activity, thus regulating CLC-3 expression at the transcriptional level by interacting with PARP1. Conclusions Our findings indicate that overexpression of CLC-3 is regulated by XRCC5 and is a poor prognostic biomarker for gastric cancer. Double targeting CLC-3 and XRCC5 may provide the promising therapeutic potential for GC treatment

Synthesis of resins via multiparallel suspension polymerization

A system for the multiparallel synthesis of polymer resins for solid-phase synthesis is presented, accelerating the preparation of new resins using standard suspension polymerization protocols. The system was evaluated by preparing a range of polystyrene resins with well-defined characteristics

Optimizing 2D gas chromatography mass spectrometry for robust tissue, serum and urine metabolite profiling

Two-dimensional gas chromatography mass spectrometry (GCxGC-MS) is utilized to an increasing extent in biomedical metabolomics. Here, we established and adapted metabolite extraction and derivatization protocols for cell/tissue biopsy, serum and urine samples according to their individual properties. GCxGC-MS analysis revealed detection of ~600 molecular features from which 165 were characterized representing different classes such as amino acids, fatty acids, lipids, carbohydrates, nucleotides and small polar components of glycolysis and the Krebs cycle using electron impact (EI) spectrum matching and validation using external standard compounds. Advantages of two-dimensional gas chromatography based resolution were demonstrated by optimizing gradient length and separation through modulation between the first and second column, leading to a marked increase in metabolite identification due to improved separation as exemplified for lactate versus pyruvate, talopyranose versus methyl palmitate and inosine versus docosahexaenoic acid. Our results demonstrate that GCxGC-MS represents a robust metabolomics platform for discovery and targeted studies that can be used with samples derived from the clinic

Biallelic FBXW7 knockout induces AKAP8-mediated DNA damage in neighbouring wildtype cells

10.1038/s41420-023-01494-yCell Death Discovery9