Dried blood spot (DBS) sampling is a method with advantages over conventional blood sampling in relation to collection, cost, storage, and transportation. Such advantages have led to its wide use in ...newborn screening (NBS). Although target analysis of various biomolecules is conducted in NBS, protein quantification‐based NBS is still in its infancy. Thus, it is important to clarify how many proteins could be quantitatively detected in DBS samples using advanced liquid chromatography–mass spectrometry (LC–MS/MS) technologies; a catalogue of proteins detectable in DBSs by LC–MS/MS will enable us to judge which causative proteins in genetic diseases can be monitored at the protein level in NBS. In this review, we outline conventional proteome analyses of DBSs with a distinction between target and nontarget approaches. Additionally, we discuss the future perspectives for proteome analysis of DBSs in NBS of genetic diseases.
Eosinophils are multifaceted immune cells with diverse functions that enhance allergic inflammation. Cysteinyl leukotrienes (cys-LTs), mainly synthesized in eosinophils, are a class of inflammatory ...lipid mediators produced via multiple enzymatic reactions from arachidonic acid. Multiple clinical studies have reported dysregulated fatty acid metabolism in severe asthma and aspirin-exacerbated respiratory diseases. Therefore, understanding the mechanism responsible for this metabolic abnormality has attracted a lot of attention. In eosinophils, various stimuli (including cytokines, chemokines, and pathogen-derived factors) prime and/or induce leukotriene generation and secretion. Cell–cell interactions with component cells (endothelial cells, epithelial cells, fibroblasts) also enhance this machinery to augment allergic responses. Nasal polyp-derived eosinophils from patients with eosinophilic rhinosinusitis present a characteristic fatty acid metabolism with selectively higher production of leukotriene D4. Interestingly, type 2 cytokines and microbiome components might be responsible for this metabolic change with altered enzyme expression. Here, we review the regulation of fatty acid metabolism, especially cys-LT metabolism, in human eosinophils toward allergic inflammatory status.
Data-independent acquisition (DIA)-mass spectrometry (MS)-based proteomic analysis overtop the existing data-dependent acquisition (DDA)-MS-based proteomic analysis to enable deep proteome coverage ...and precise relative quantitative analysis in single-shot liquid chromatography (LC)-MS/MS. However, DIA-MS-based proteomic analysis has not yet been optimized in terms of system robustness and throughput, particularly for its practical applications. We established a single-shot LC-MS/MS system with an MS measurement time of 90 min for a highly sensitive and deep proteomic analysis by optimizing the conditions of DIA and nanoLC. We identified 7020 and 4068 proteins from 200 ng and 10 ng, respectively, of tryptic floating human embryonic kidney cells 293 (HEK293F) cell digest by performing the constructed LC-MS method with a protein sequence database search. The numbers of identified proteins from 200 ng and 10 ng of tryptic HEK293F increased to 8509 and 5706, respectively, by searching the chromatogram library created by gas-phase fractionated DIA. Moreover, DIA protein quantification was highly reproducible, with median coefficients of variation of 4.3% in eight replicate analyses. We could demonstrate the power of this system by applying the proteomic analysis to detect subtle changes in protein profiles between cerebrums in germ-free and specific pathogen-free mice, which successfully showed that >40 proteins were differentially produced between the cerebrums in the presence or absence of bacteria.
The balance between bacterial colonization and its containment in the intestine is indispensable for the symbiotic relationship between humans and their bacteria. One component to maintain ...homeostasis at the mucosal surfaces is immunoglobulin A (IgA), the most abundant immunoglobulin in mammals
. Several studies have revealed important characteristics of poly-reactive IgA
, which is produced naturally without commensal bacteria. Considering the dynamic changes within the gut environment, however, it remains uncertain how the commensal-reactive IgA pool is shaped and how such IgA affects the microbial community. Here we show that acetate-one of the major gut microbial metabolites-not only increases the production of IgA in the colon, but also alters the capacity of the IgA pool to bind to specific microorganisms including Enterobacterales. Induction of commensal-reactive IgA and changes in the IgA repertoire by acetate were observed in mice monocolonized with Escherichia coli, which belongs to Enterobacterales, but not with the major commensal Bacteroides thetaiotaomicron, which suggests that acetate directs selective IgA binding to certain microorganisms. Mechanistically, acetate orchestrated the interactions between epithelial and immune cells, induced microbially stimulated CD4 T cells to support T-cell-dependent IgA production and, as a consequence, altered the localization of these bacteria within the colon. Collectively, we identified a role for gut microbial metabolites in the regulation of differential IgA production to maintain mucosal homeostasis.
The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. ...Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of
in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.
MicroRNAs (miRNAs) are incorporated into miRNP complexes and regulate protein expression post-transcriptionally through binding to 3'-untranslated regions of target mRNAs. Here we describe a ...recapitulation of let-7 miRNA-mediated translational repression in a cell-free system, which was established with extracts prepared from HEK293F cells overexpressing miRNA pathway components. In this system, both the cap and poly(A) tail are required for the translational repression, and let-7 directs the deadenylation of target mRNAs. Our work suggests that let-7 miRNPs containing Argonaute and GW182 impair the synergistic enhancement of translation by the 5'-cap and 3'-poly(A) tail, resulting in translational repression.
Allergic asthma is an inflammatory disease characterized by lung eosinophilia controlled by type 2 cytokines. Cysteine proteases are potent triggers of allergic inflammation by causing barrier ...disruption in lung epithelial cells inducing the elevation of interleukin-5 (IL-5) and IL-13 from natural helper (NH) cells, a member of ILC2s, which leads to lung eosinophilia. In this study, we found that basophils play a crucial role in NH cell-mediated eosinophilic inflammation induced by protease allergens. Conditional deletion of basophils caused a resolution of the papain-induced eosinophilia and mucus production. Resolution of eosinophilia was also observed in mice lacking IL-4 specifically in basophils, indicating that basophil-derived IL-4 enhanced expression of the chemokine CCL11, as well as IL-5, IL-9, and IL-13 in NH cells, thus attracting eosinophils. These results demonstrate that IL-4 from basophils has an important role in the NH-derived cytokine and chemokine expression, subsequently leading to protease allergen-induced airway inflammation.
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•Basophils are essential for protease allergen-induced airway responses•Basophil-derived IL-4 controls ILC2-mediated eosinophilic inflammation•IL-4 is necessary for the expression of CCL11, IL-9, and IL-13 from NH cells•Interaction of basophils and ILC2 controls the IL-33 mediated allergic responses
Improving the reproducibility of proteome analysis systems presents a challenge; therefore, in this study, we developed a new insertion spray ionization (InSpIon) system wherein an InSpIon tube was ...designed with a spray tip inserted into the tube. This system stabilized the spray and subsequently improved the reproducibility of the analysis.
Dried blood spots (DBS) are widely used for screening molecular profiles, including enzymatic activity. However, hydrophilic proteins present in large amounts in blood inhibit detection of other ...proteins in DBS by liquid chromatography-mass spectrometry (LC-MS/MS) without preenrichment. Sodium carbonate precipitation (SCP) can concentrate hydrophobic proteins from DBS and effectively remove soluble hydrophilic proteins. Furthermore, SCP combination with data-independent acquisition (DIA) for quantitative LC-MS/MS enhanced the proteome analysis sensitivity and quantification limits. In this protocol, we have described in detail a simple preenrichment method using SCP and a deep proteome analysis method for LC-MS/MS data using DIA.
Abstract
Autoinflammatory disease is an ‘inborn error of immunity’, resulting in systemic inflammation. Cryopyrin-associated periodic syndrome (CAPS) is a prototypical autoinflammatory disease caused ...by gain-of-function mutations in the NLRP3 (NLR family pyrin domain containing 3) gene; these mutations activate the NLRP3 inflammasome, resulting in overproduction of IL-1β. The first case of CAPS caused by somatic NLRP3 mosaicism was reported in 2005 after identification of variant small peaks by Sanger sequencing. An international collaborative study revealed that the majority of mutation-negative CAPS cases are due to low-level NLRP3 mosaicism, suggesting that central nervous system involvement in somatic mosaicism patients is milder than in genotype-matched heterozygous patients. Recent advances in next-generation sequencing have expanded the number of NLRP3 somatic mosaicism cases and identified a new entity called ‘late-onset CAPS with myeloid-specific NLRP3 mosaicism’; however, no mosaic-specific clinical features have been identified/confirmed yet. With respect to NLRP3 mosaicism in CAPS, a prospective longitudinal study on the variant genotype, its allele frequency and its tissue distribution (along with a comprehensive clinical phenotype) would provide better understanding of NLRP3 mosaicism, resulting in more appropriate patient care and genetic counseling.
Mosaicism in CAPS
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