Development of a highly efficient solubilization method for mass spectrometric analysis of phospholipids in living single cells.

Phospholipids are vital constituents of the cell membrane and aid in signal transduction. Phospholipid profiles vary distinctively with the cell type. Notably, specific phospholipid molecules are present in significantly higher or lower concentrations in cancer cells versus normal cells. In this study, live single-cell mass spectrometry (MS) was developed for analyzing phospholipids at the single-cell level. This method facilitates rapid molecular analysis of cells under microscopic observation. For nanoelectrospray ionization, phospholipids were extracted from single cells isolated in a glass capillary through a high-efficiency process. Cell-derived phosphatidylcholines were detected with high sensitivity when trehalose C14 was added as a solubilizing reagent. Trehalose C14 can solubilize cells at low concentrations owing to its low critical micelle concentration, and exerts minimal matrix effects (such as suppressing ionization and causing peak overlap) in the MS analysis of cellular molecules. Analyses of phospholipids in Raji and HEV0070 cells using the developed method revealed specific peaks of phosphatidylcholine and sphingomyelin in the respective cells. The developed technique not only affords phospholipid profiles at the single-cell level, but also holds promise for identifying biomarkers associated with various diseases, particularly cancer.

Selective analysis of intracellular UDP-GlcNAc and UDP-GalNAc by hydrophilic interaction liquid chromatography-mass spectrometry.

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is one of the major nucleotide sugars in living organisms and serves as the key donor substrate for the post-translational modification of protein O-GlcNAcylation. It undergoes interconversion to its epimer uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc), which acts as a sugar donor initiating mucin-type O-linked glycosylation. The intracellular levels of the two differ between the cell lines and largely fluctuate in response to metabolic perturbations, and recent studies have focused on the details of their biosynthesis or turnover. However, due to their similar chemical properties, sufficient resolution for the two epimers required non-volatile mobile phases that cannot be applied directly to a mass spectrometer. In this study, to implement simple liquid chromatography-mass spectrometry for UDP-GlcNAc and UDP-GalNAc, we optimized a condition of hydrophilic interaction liquid chromatography-mass spectrometry. We found that the use of ammonium hydroxide and an amide column with an optimized water-acetonitrile ratio, flow rate, and column temperature, provided complete separation of the two. The method allowed the analysis of intracellular levels, a stable isotope-labeled target, and patterns of product ion spectra in a single run with fewer sample preparation steps. The new method can be widely used for mass spectrometric analysis of UDP-GlcNAc and UDP-GalNAc.

Cyclic-di-AMP confers an invasive phenotype on Escherichia coli through elongation of flagellin filaments.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) is isolated from patients with Crohn's disease (CD). AIEC can invade the intestinal epithelium, suggesting that it is involved in the development and pathogenesis of CD. However, the mechanism by which AIEC acquired the invasive phenotype remains unknown. RESULTS: This study was designed to examine the mechanisms of AIEC invasiveness. We found that the flagellin (fliC) expression in AIEC was two-fold higher than that in non-AIEC strains, and this overexpression induced the formation of long-filament flagellin. Deletion of fliC in the AIEC LF82 strain resulted in the disappearance of flagellar filaments and attenuated the motility and invasive ability of the bacterium, suggesting that the formation of long filament flagellin induced by increased fliC expression is required by AIEC to invade the intestinal epithelium. In AIEC and non-AIEC K12 strains cultured in the presence of cyclic-di-AMP (c-di-AMP), the expression of fliC was enhanced, and flagellar filaments were elongated. Stimulation with c-di-AMP enhanced the bacterial motility and ability to invade epithelial cells, even in the non-AIEC K12 strain. CONCLUSIONS: Our findings show that c-di-AMP confers an AIEC-like phenotype on non-AIEC strains by enhancing the expression of fliC. The results should be useful for understanding the pathogenesis of CD.

Development of a liquid chromatography-based versatile bioanalysis for bevacizumab based on pretreatment combining aptamer affinity purification and centrifugal ultrafiltration concentration.

We report on the development of a versatile and accurate bioanalytical method for bevacizumab using a pretreatment method combining affinity purification with anti-idiotypic DNA aptamers and centrifugal ultrafiltration concentration, followed by liquid chromatography (LC)-fluorescence analysis. An affinity purification method using Sepharose beads as an affinity support removed immunoglobulin G and a large amount of coexisting substances in the serum sample. Purified bevacizumab was separated as a single peak by conventional LC and detected fluorometrically, showing good linearity (R2 = 0.999) in the range of 5-200 µg/mL, sufficient to analyze bevacizumab concentrations in the blood of bevacizumab-treated patients. By combining this purification method with a concentration method using a centrifugal filtration device that inhibits non-specific adsorption of bevacizumab, the quantitative range was reduced by a factor of 10 while showing good linearity (R2 = 0.999) in the 0.5-20 µg/mL range. The developed analytical method is expected to be used not only for general bioanalysis of therapeutic mAbs in clinical settings, but also for next-generation antibody drugs that show drug efficacy at low concentrations and for analysis of trace samples.

Charged chiral derivatization for enantioselective imaging of D-,L-2-hydroxyglutaric acid using ion mobility spectrometry/mass spectrometry.

A newly synthesized charged chiral tag-enabled enantioselective imaging of D-,L-2-hydroxyglutaric acid, which are independently associated with the regulation of DNA methylation. The tag-conjugated diastereomers were ionized efficiently through MALDI, separated by ion mobility spectrometry, and further separated from other molecules in mass spectrometry. On-tissue chiral derivatization using the tag facilitated the visualization of different distributions of the two isomers in the mouse testis.

Metabolites for monitoring symptoms and predicting remission in patients with depression who received electroconvulsive therapy: a pilot study.

The lack of biomarkers to monitor and predict the efficacy of electroconvulsive therapy (ECT) has hindered its optimal use. To establish metabolomic markers for monitoring and predicting the treatment efficacy of ECT, we comprehensively evaluated metabolite levels in patients with major depressive disorder (MDD) by performing targeted and non-targeted metabolomic analyses using plasma samples before and after the first, third, and final ECT sessions, and 3-7 days after the final session. We compared the plasma metabolomes of age- and sex-matched healthy controls (HCs). Thirteen hospitalized patients with MDD and their corresponding HCs were included in this study. We observed that patients with MDD exhibited lower levels of amino acids, including gamma-aminobutyric acid (GABA), and metabolites involved in tryptophan metabolism and the kynurenine pathway, and higher levels of cortisol at baseline. Furthermore, we investigated the relationship between metabolite levels and depression severity across seven measurement timepoints along with one correlation analysis and found that amino acids, including GABA and tryptophan catabolites, were significantly correlated with the severity of depression. Despite the exploratory nature of this study due to the limited sample size necessitating further validation, our findings suggest that the blood metabolic profile has potential as a biomarker for ECT.

Tumor Necrosis Factor and Interleukin-1ß Upregulate NRP2 Expression and Promote SARS-CoV-2 Proliferation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) and the auxiliary receptor Neuropilin-1 (NRP1) to enter host cells. NRP1 has another isoform, NRP2, whose function in COVID-19 has seldom been reported. In addition, although patients with severe cases of COVID-19 often exhibit increased levels of proinflammatory cytokines, the relationship between these cytokines and SARS-CoV-2 proliferation remains unknown. The aim of this study is to clarify the roles of proinflammatory cytokines in Neuropilin expressions and in SARS-CoV-2 infection. To identify the expression patterns of NRP under inflamed and noninflamed conditions, next-generation sequencing (RNA-seq), immunohistochemistry, quantitative real-time PCR, and Western blotting were performed using primary cultured fibroblast-like synoviocytes, MH7A (immortalized cell line of human rheumatoid fibroblast-like synoviocytes), immortalized MRC5 (human embryonic lung fibroblast), and synovial tissues. To measure viral proliferative capacity, SARS-CoV-2 infection experiments were also performed. NRP2 was upregulated in inflamed tissues. Cytokine-stimulated human fibroblast cell lines, such as MH7A and immortalized MRC5, revealed that NRP2 expression increased with co-stimulation of tumor necrosis factor α (TNFα) and interleukin-1 beta (IL-1ß) and was suppressed with anti-TNFα antibody alone. TNFα and IL-1ß promoted SARS-CoV-2 proliferation and Spike protein binding. The viral proliferation coincided with the expression of NRP2, which was modulated through plasmid transfections. Our results revealed that proinflammatory cytokines, including TNFα, contribute to NRP2 upregulation and SARS-CoV-2 proliferation in host human cells.

Development of a Nursing Practice Scale for Rheumatoid Arthritis Treatment with Biological Disease-modifying Anti-rheumatic Drugs.

OBJECTIVE: To develop a nursing practice scale for rheumatoid arthritis treatment with biological disease-modifying anti-rheumatic drugs (bDMARDs). METHODS: An anonymous self-administered questionnaire survey was administered to 1826 nurses, 960 of whom were Certified Nurses by Japan Rheumatism Foundation (CNJRFs) and 866 were registered nurses (RNs). Using exploratory factor analysis, criterion validity and known-groups technique, we assessed the reliability and validity of the self-created 19-item Nursing Practice Scale to evaluate the care provided to patients with rheumatoid arthritis receiving bDMARDs based on the nurse's role as clarified from a literature review of relevant studies. RESULTS: A total of 698 (38.4%) responses were collected from 407 CNJRFs and 291 RNs. Exploratory factor analysis was conducted on 18 items to examine three factors: 'nursing to enhance patients' capacity for self-care', 'nursing in which patients participate in decision-making' and 'nursing in which team medical care is promoted'. Cronbach's α was .95. The Spearman coefficient was ρ = .738 for criterion validity. Using the known-groups technique, CNJRFs had higher total scale scores than RNs (p < .05). CONCLUSIONS: The results confirmed the reliability, criterion validity and construct validity of the scale.

N-glycan in the variable region of monoclonal ACPA (CCP-Ab1) promotes the exacerbation of experimental arthritis.

OBJECTIVES: The variable region of most ACPA IgG molecules in the serum of RA patients carries N-glycan (N-glycanV). To analyse the pathogenicity of N-glycanV of ACPAs, we analysed the pathogenicity of a monoclonal ACPA, CCP-Ab1, with or without N-glycanV, which had been isolated from a patient with RA. METHODS: CCP-Ab1 with no N-glycosylation site in the variable region (CCP-Ab1 N-rev) was generated, and antigen binding, the effect on in vitro differentiation of osteoclasts from bone marrow mononuclear cells of autoimmune arthritis-prone SKG mice (the cell size of TRAP+ cells and bone resorption capacity) and the in vivo effect on the onset or exacerbation of autoimmune arthritis in SKG mice were evaluated in comparison with glycosylated CCP-Ab1. RESULTS: Amino acid residues in citrullinated peptide (cfc1), which are essential for binding to CCP-Ab1 N-rev and original CCP-Ab1, were almost identical. The size of TRAP+ cells was significantly larger and osteoclast bone resorption capacity was enhanced in the presence of CCP-Ab1, but not with CCP-Ab1 N-rev. This enhancing activity required the sialic acid of the N-glycan and Fc region of CCP-Ab1. CCP-Ab1, but not CCP-Ab1 N-rev, induced the exacerbation of experimental arthritis in the SKG mouse model. CONCLUSIONS: These data showed that N-glycanV was required for promoting osteoclast differentiation and bone resorption activity in both in vitro and in vivo assays. The present study demonstrated the important role of N-glycanV in the exacerbation of experimental arthritis by ACPAs.

Simultaneous determination of all aminobutyric acids by chiral derivatization and liquid chromatography-tandem mass spectrometry.

Aminobutyric acids include eight structural or stereoisomers that exhibit a wide range of biological activities. Recent evidence on some low abundant isomers have increased the demand for highly selective analysis of all the isomers; however, simultaneous separation of all the aminobutyric acid isomers has not been successful yet, except for a specialized method that uses multiple separation columns and a split of samples. In this study, we developed a new analytical method using chiral derivatization and liquid chromatography-tandem mass spectrometry to separate all the aminobutyric acid isomers in a single separation column. All the diastereomeric derivatives were resolved in a C18 column, and the derivatives showed characteristic fragmentation patterns in tandem mass spectrometry. By using the method, we analyzed the isomers in the Arabidopsis thaliana seeds and revealed the existence of three low abundant isomers, i.e., D-, L-ß-aminoisobutyric acid, and D-ß-aminobutyric acid. The proposed method uses a commercially available chiral derivatizing reagent and a broadly used column; therefore, it can be widely used in biological and food analyses.

Expression of factor XIII originating from synovial fibroblasts and macrophages induced by interleukin-6 signaling.

BACKGROUND: Blood coagulation factor XIII (FXIII) promotes cross-linking between fibrin molecules at the final stage of the blood coagulation cascade. However, its expression in cells or tissues and function, particularly factor XIII subunit B (FXIII-B), remains controversial. Hemorrhagic FXIII deficiency following anti-interleukin-6 (IL-6) receptor antibody treatment has been reported in patients with rheumatoid arthritis (RA). Patients receiving this biologics have reduced FXIII activity when compared to the activity in those treated with other biologics. The relationship between pro-inflammatory cytokines and FXIII expression remains unknown. METHODS: To investigate the expression pattern of FXIII in synovial tissues, immunohistochemistry, RT-qPCR, and western blotting were performed. FXIII-A expressed monocyte-derived macrophages were treated with recombinant IL-6 and anti-IL-6 receptor antibody. RNA sequencing of FXIII-B-overexpressing cells was performed to clarify the function of FXIII-B. RESULTS: The immunohistochemical analysis of synovial tissues revealed that factor XIII subunit A (FXIII-A) was expressed in M2 macrophages, and FXIII-B was expressed in fibroblast-like synoviocytes. IL-6 stimulation upregulated FXIII-A expression in IL-4-induced monocyte-derived macrophages, and the anti-IL-6 receptor antibody suppressed FXIII-A expression. FXIII-B was more abundantly secreted in the supernatant of fibroblast-like synoviocytes compared with that of other cells. RNA sequencing showed that FXIII-B elevated the expression of genes associated with anti-apoptotic molecules and chemokines. CONCLUSIONS: Our findings highlight that synovial tissue is one of the sources of FXIII production. We also have demonstrated IL-6-dependent FXIII-A expression and the novel potential functions of FXIII-B.

Safety and efficacy of filgotinib for Japanese patients with RA and inadequate response to MTX: FINCH 1 52-week results and FINCH 4 48-week results.

OBJECTIVES: To present safety and efficacy of the JAK1 preferential inhibitor filgotinib in Japanese patients with prior inadequate response (IR) to methotrexate (MTX) from a 52-week randomised controlled parent study (PS) and long-term extension (LTE) through June 2020. METHODS: The PS (NCT02889796) randomised MTX-IR patients to filgotinib 200 (FIL200) or 100 mg (FIL100), adalimumab (ADA) 40 mg, or placebo; all took stable background MTX. At week (W) 24, placebo patients were rerandomised to FIL200 or FIL100. The primary endpoint was W12 American College of Rheumatology 20% improvement; safety was assessed by adverse event (AE) reporting. For the LTE (NCT03025308), eligible filgotinib patients continued FIL200/FIL100; ADA patients were rerandomised (blinded) to FIL200 or FIL100; all continued MTX. RESULTS: In all, 114/147 Japanese patients completed the PS, 115 enrolled in LTE, and 103 remained on study in June 2020. In the PS, AEs were consistent with the overall population, and W24 efficacy was maintained or improved through W52, comparable with the overall population. LTE AE incidences were similar between doses; filgotinib efficacy was consistent from baseline to W48 and similar between PS ADA and filgotinib patients. CONCLUSIONS: Among MTX-IR Japanese patients, filgotinib maintained efficacy over 1 year; LTE safety was consistent with the PS.

Integrated safety analysis of filgotinib treatment for rheumatoid arthritis in patients from Japan over a median of 1.5 years.

OBJECTIVE: Characterize safety of the Janus kinase-1 preferential inhibitor filgotinib (FIL) in Japanese patients with moderately to severely active rheumatoid arthritis (RA). METHODS: Data from three Phase 3 trials (NCT02889796, NCT02873936, and NCT02886728) and a long-term extension (NCT03025308) through September 2019 were integrated; patients received ≥1 dose of FIL 200 (FIL200) or 100 mg (FIL100) daily, or placebo (PBO). We calculated exposure-adjusted incidence rates (EAIRs) per 100 patient-years FIL exposure (100PYE) for treatment-emergent adverse events (TEAEs) and adverse events of special interest. RESULTS: Among 3691 total patients and 6080.7 PYE, 229 Japanese patients received FIL for 311.4 PYE (median 1.5, maximum 2.5 years). During the 12-week PBO-controlled period, serious TEAEs and TEAEs leading to study drug disruption were comparable between FIL and PBO. Serious infection rates were 1.9%, 0%, and 2% for FIL200, FIL100, and PBO during the PBO-controlled period; long-term FIL200 and FIL100 EAIRs were 3.8 and 2.1/100PYE. No herpes zoster (HZ) or major adverse cardiovascular events (MACEs) occurred during the PBO-controlled period; long-term FIL200 and FIL100 EAIRs were 3.0 and 2.1/100PYE (HZ) and 0.6 and 0/100PYE (MACE). CONCLUSION: Long-term FIL treatment (median 1.5, maximum 2.5 years exposure) was well tolerated at 100- and 200-mg doses in Japanese patients with RA.

Long-term safety and efficacy of filgotinib treatment for rheumatoid arthritis in Japanese patients naïve to MTX treatment (FINCH 3).

OBJECTIVES: To evaluate the long-term safety and efficacy of filgotinib (FIL) for Japanese patients with rheumatoid arthritis (RA) and limited/no prior methotrexate (MTX) exposure. We present a Japanese population subanalysis of a global randomised-controlled trial at Week 52 and interim long-term extension (LTE) to Week 48 through June 2020. METHODS: Patients were randomised to FIL 200 mg plus MTX, FIL 100 mg plus MTX, FIL 200 mg, or MTX for 52 weeks. At completion, eligible patients could enrol in the LTE. Those receiving FIL continued; those receiving MTX were rerandomised (blinded) to FIL 200 or 100 mg upon discontinuation of MTX. After a 4-week washout period, MTX could be re-added. RESULTS: Adverse event rates at Week 52 and in the LTE to Week 48 were comparable across treatment groups. Week 52 American College of Rheumatology 20% improvement (ACR20) rates were 83% (19/23), 82% (9/11), 75% (9/12), and 76% (19/25) for FIL 200 mg plus MTX, FIL 100 mg plus MTX, FIL 200 mg, and MTX, respectively. Through LTE Week 48, ACR20 rates were maintained. CONCLUSIONS: In the 56 Japanese patients treated with FIL, efficacy was maintained through Week 52 and beyond, with no increases in the incidence of adverse events.

Precolumn derivatization LC/MS method for observation of efficient hydrogen sulfide supply to the kidney via d-cysteine degradation pathway.

d-Cysteine (d-Cys) is metabolized to hydrogen sulfide (H2S) by d-amino acid oxidase (DAO)/3-mercaptopyruvate sulfurtransferase pathway. The pathway is required for H2S supplementation that ameliorates acute kidney injury after the oral administration of d-Cys in mice. However, whether the rate-limiting activity of DAO regulates the tissue-selectivity or the extent of d-Cys degradation and H2S supplementation remains unclear. Here, to analyze the levels of d-Cys and H2S, we use two derivatization methods, a new method with no detectable isomerization of Cys and an established method for H2S. The derivatives were determined by LC/MS using a C18 column. With the methods, we show that inhibition of DAO significantly suppresses the H2S supplementation and d-Cys degradation in the mouse kidney. Additionally, we found that d-Cys is more efficiently metabolized into H2S than l-Cys in the kidney. Our results reveal the utility of the method and support the advantage of d-Cys administration in improving the supply of H2S to the kidneys.

N-terminal peptide fragment constitutes core of amyloid deposition of serum amyloid A: An imaging mass spectrometry study.

Serum amyloid A (SAA) is an acute phase protein, which undergoes structural changes and deposits in the extracellular matrix, causing organ damage. Systemic AA amyloidosis is a relatively common amyloid subtype among the more than 30 amyloid subtypes, but the mechanism of amyloid fibril formation remains unclear. In this study, we investigated the tissue distribution of SAA derived peptides in formalin-fixed paraffin embedded (FFPE) specimens of human myocardium with amyloidosis using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). In the whole SAA protein, four trypsin-digested peptides in the range of SAA2-67 were visualized and the N-terminal peptide; SAA2-15, was selectively localized in the Congo red-positive region. The C-terminal peptides; SAA47-62, SAA48-62, and SAA63-67 were detected not only in the Congo red-positive region but also in the surrounding negative region. Our results demonstrate that the N-terminal SAA2-15 plays a critical role in the formation of AA amyloid fibril, as previously reported. Roles of the C-terminal peptides require further investigation.

Importance of anti-centromere antibodies in the diagnosis of Sjögren's syndrome.

The purpose of the present study was to indicate that patients with anti-centromere antibodies (ACA) also experience ocular/oral dryness like patients with anti-Ro/SSA and/or anti-La/SSB antibodies (anti-SSA/SSB). A total of 80 patients with subjective ocular and/or oral dryness were classified into two groups, namely, anti-SSA/SSB-positive (anti-SSA/SSB [+]) group and ACA-positive (ACA [+]) group. The degree of ocular and oral dryness in ACA (+) patients is similar to that in anti-SSA/SSB (+) patients. On histopathological examination of the labial glands, the area of fibrosis was significantly larger in the ACA (+) group than in the anti-SSA/SSB (+) group. Thus, ACA (+) patients should be examined for Sjögren's syndrome.

N1-methylpseudouridine-incorporated mRNA enhances exogenous protein expression and suppresses immunogenicity in primary human fibroblast-like synoviocytes.

Studies conducted using murine arthritis models have indicated that the use of in vitro-transcribed messenger RNA (IVT mRNA) is an effective therapeutic approach for joint diseases. However, the use of IVT mRNA in human synovial cells has not been widely studied. Recently, the outbreak of the novel coronavirus disease has accelerated the development of innovative mRNA vaccines, such as those containing a modified nucleic acid, N1-methylpseudouridine-5'-triphosphate (m1ψ). IVT mRNA is an attractive tool for biological experiments and drug discovery. To verify the protein expression from IVT mRNA in vitro, primary cultured fibroblast-like synoviocytes (FLS) and MH7A human synovial fibroblast cells were transfected with enhanced green fluorescent protein (EGFP) mRNA with or without m1ψ incorporation. EGFP was detected using western blotting and fluorescence microscopy. A multiplex assay was performed to comprehensively understand IVT mRNA-induced immunogenicity. Gene expression levels were measured using reverse transcription polymerase chain reaction. In both MH7A cells and FLS, cells transfected with EGFP mRNA containing m1ψ generated higher levels of EGFP than those transfected with unmodified EGFP or control mRNAs. The multiplex assay of the FLS culture supernatant and reverse transcription polymerase chain reaction for FLS revealed that both concentration and expression of IL-6, TNF-α, and CXCL10 were upregulated by unmodified EGFP mRNA, whereas they were suppressed by EGFP mRNA with m1ψ. Overall, m1ψ incorporation enhanced protein expression and decreased the expression of cytokines. These findings may contribute to arthritis research.