Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis.

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer1-3. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.

Discovery of lipid profiles in plasma-derived extracellular vesicles as biomarkers for breast cancer diagnosis.

Lipids are a major component of extracellular vesicles; however, their significance in tumorigenesis and progression has not been well elucidated. As we previously found that lipid profiles drastically changed in breast tumors upon progression, we hypothesized that lipid profiles of plasma-derived extracellular vesicles could be utilized as breast cancer biomarkers. Here, we adopted modified sucrose cushion ultracentrifugation to isolate plasma-derived extracellular vesicles from breast cancer (n = 105), benign (n = 11), and healthy individuals (n = 43) in two independent cohorts (n = 126 and n = 33) and conducted targeted lipidomic analysis. We established a breast cancer diagnostic model comprising three lipids that showed favorable performance with the area under the receiver operating characteristic curve of 0.759, 0.743, and 0.804 in the training, internal validation, and external test sets, respectively. Moreover, we identified several lipids that could effectively discriminate breast cancer progression and subtypes: phosphatidylethanolamines and phosphatidylserines were relatively higher in Stage III, whereas phosphatidylcholines and sphingomyelins were higher in Stage IV; phosphatidylcholines and ceramides were correspondingly concentrated in HER2-positive patients, while lysophosphatidylcholines and polyunsaturated triglycerides were concentrated in the triple-negative breast cancer subtype. Lipid profiling of plasma-derived extracellular vesicles is a non-invasive and promising approach for diagnosing, staging, and subtyping breast cancer.

Downregulation of Odd-Skipped Related 2, a Novel Regulator of Epithelial-Mesenchymal Transition, Enables Efficient Somatic Cell Reprogramming.

Somatic cell reprogramming proceeds through a series of events to generate induced pluripotent stem cells (iPSCs). The early stage of reprogramming of mouse embryonic fibroblasts is characterized by rapid cell proliferation and morphological changes, which are accompanied by downregulation of mesenchyme-associated genes. However, the functional relevance of their downregulation to reprogramming remains poorly defined. In this study, we have screened transcriptional regulators that are downregulated immediately upon reprogramming, presumably through direct targeting by reprogramming factors. To test if these transcriptional regulators impact reprogramming when expressed continuously, we generated an expression vector that harbors human cytomegalovirus upstream open reading frame 2 (uORF2), which reduces translation to minimize the detrimental effect of an expressed protein. Screening of transcriptional regulators with this expression vector revealed that downregulation of (odd-skipped related 2 [Osr2]) is crucial for efficient reprogramming. Using a cell-based model for epithelial-mesenchymal transition (EMT), we show that Osr2 is a novel EMT regulator that acts through induction of transforming growth factor-ß (TGF-ß) signaling. During reprogramming, Osr2 downregulation not only diminishes TGF-ß signaling but also allows activation of Wnt signaling, thus promoting mesenchymal-epithelial transition (MET) toward acquisition of pluripotency. Our results illuminate the functional significance of Osr2 downregulation in erasing the mesenchymal phenotype at an early stage of somatic cell reprogramming.

The liposome of trehalose dimycolate extracted from M. bovis BCG induces antitumor immunity via the activation of dendritic cells and CD8+ T cells.

Intravesical Bovis bacillus Calmette-Guérin (BCG) therapy is the most effective immunotherapy for bladder cancer, but it sometime causes serious side effects because of its inclusion of live bacteria. It is necessary to develop a more active but less toxic immunotherapeutic agent. Trehalose 6,6'-dimycolate (TDM), the most abundant hydrophobic glycolipid of the BCG cell wall, has been reported to show various immunostimulatory activities such as granulomagenesis and adjuvant activity. Here, we developed cationic liposomes incorporating TDM purified from Mycobacterium bovis BCG Connaught, and we investigated the antitumor effect of the cationic liposome TDM (Lip-TDM). Lip-TDM exerted an antitumor effect in bladder cancer, colon cancer, and melanoma-bearing mouse models that was comparable or even superior to that of BCG, with no body weight loss or granuloma formation. The antitumor effect of Lip-TDM disappeared in two types of mice: those with depletion of CD8+ T cells, and those with knockout of macrophage-inducible C-type lectin (Mincle) which recognize TDM. Lip-TDM treatment enhanced the maturation and migration of dendritic cells in the tumor microenvironment in a Mincle-dependent manner. Our results elucidate mechanisms that underlie Lip-TDM treatment and suggest that Lip-TDM has potential as a safe and effective treatment for various cancers.

pJRES Binning Algorithm (JBA): a new method to facilitate the recovery of metabolic information from pJRES 1H NMR spectra.

MOTIVATION: Data processing is a key bottleneck for 1H NMR-based metabolic profiling of complex biological mixtures, such as biofluids. These spectra typically contain several thousands of signals, corresponding to possibly few hundreds of metabolites. A number of binning-based methods have been proposed to reduce the dimensionality of 1 D 1H NMR datasets, including statistical recoupling of variables (SRV). Here, we introduce a new binning method, named JBA ("pJRES Binning Algorithm"), which aims to extend the applicability of SRV to pJRES spectra. RESULTS: The performance of JBA is comprehensively evaluated using 617 plasma 1H NMR spectra from the FGENTCARD cohort. The results presented here show that JBA exhibits higher sensitivity than SRV to detect peaks from low-abundance metabolites. In addition, JBA allows a more efficient removal of spectral variables corresponding to pure electronic noise, and this has a positive impact on multivariate model building. AVAILABILITY AND IMPLEMENTATION: The algorithm is implemented using the MWASTools R/Bioconductor package. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Extracellular vesicles as trans-genomic agents: Emerging roles in disease and evolution.

The composition of genetic material in extracellular vesicles (EV) has sparked interest particularly in the potential for horizontal gene transfer by EV. Although the RNA content of EV has been studied extensively, few reports have examined the DNA content of EV. It is still unclear how DNA is packaged inside EV, and whether they are functional in recipient cells. In this review, we describe the biological significance of genetic material in EV and their possible impacts in recipient cells, with focus on DNA from cancer cell-derived EV and the potential roles they may play in the cancer microenvironment. Another important feature of the genetic content of EV is the presence of retrotransposon elements. In this review, we discuss the possibility of an EV-mediated mechanism for the dispersal of retrotransposon elements, and their potential involvement in the development of genetically influenced diseases. In addition to this, we discuss the potential involvement of EV in the transfer of genetic material across species, and their possible impacts in modulating genome evolution.

J-Resolved 1H NMR 1D-Projections for Large-Scale Metabolic Phenotyping Studies: Application to Blood Plasma Analysis.

1H nuclear magnetic resonance (NMR) spectroscopy-based metabolic phenotyping is now widely used for large-scale epidemiological applications. To minimize signal overlap present in 1D 1H NMR spectra, we have investigated the use of 2D J-resolved (JRES) 1H NMR spectroscopy for large-scale phenotyping studies. In particular, we have evaluated the use of the 1D projections of the 2D JRES spectra (pJRES), which provide single peaks for each of the J-coupled multiplets, using 705 human plasma samples from the FGENTCARD cohort. On the basis of the assessment of several objective analytical criteria (spectral dispersion, attenuation of macromolecular signals, cross-spectral correlation with GC-MS metabolites, analytical reproducibility and biomarker discovery potential), we concluded that the pJRES approach exhibits suitable properties for implementation in large-scale molecular epidemiology workflows.

Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono-hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R(2) > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co-existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.

Selective detection of complementarity-determining regions of monoclonal antibody by limiting protease access to the substrate: nano-surface and molecular-orientation limited proteolysis.

We report a novel method for the selective proteolysis by limiting protease access to the substrate, which we have named nano-surface and molecular-orientation limited (nSMOL) proteolysis. We focus on the identification of the Fab and quantitation of antibodies. This method successfully performed limited proteolysis on the Fab.

Identification and quantification of amyloid beta-related peptides in human plasma using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Proteolytic processing of the amyloid precursor protein (APP) by ß-secretase and γ-secretase leads to the generation and deposition of amyloid ß (Aß) in Alzheimer's disease (AD). N-terminally or C-terminally truncated Aß variants have been found in human cerebrospinal fluid and cultured cell media using immunoprecipitation and mass spectrometry. Unfortunately, the profile of plasma Aß variants has not been revealed due to the difficulty of isolating Aß from plasma. We present here for the first time studies of Aß and related peptides in human plasma. Twenty-two Aß-related peptides including novel peptides truncated before the ß-secretase site were detected in human plasma and 20 of the peptides were identified by tandem mass spectrometry. Using an internal standard, we developed a quantitative assay for the Aß-related peptides and demonstrated plasma dilution linearity and the precision required for their quantitation. The present method should enhance the understanding of APP processing and clearance in AD progression.

The declining insulinogenic index correlates with inflammation and metabolic dysregulation in non-obese individuals assessed by blood gene expression.

AIMS: Diabetes onset is difficult to predict. Since decreased insulinogenic index (IGI) is observed in prediabetes, and blood gene expression correlates with insulin secretion, candidate biomarkers can be identified. METHODS: We collected blood from 96 participants (54 males, 42 females) in 2008 (age: 52.5 years) and 2016 for clinical and gene expression analyses. IGI was derived from values of insulin and glucose at fasting and at 30 min post-OGTT. Two subgroups were identified based on IGI variation: "Minor change in IGI" group with absolute value variation between -0.05 and +0.05, and "Decrease in IGI" group with a variation between -20 and -0.05. RESULTS: Following the comparison of "Minor change in IGI" and "Decrease in IGI" groups at time 0 (2008), we identified 77 genes correlating with declining IGI, related to response to lipid, carbohydrate, and hormone metabolism, response to stress and DNA metabolic processes. Over the eight years, genes correlating to declining IGI were related to inflammation, metabolic and hormonal dysregulation. Individuals with minor change in IGI, instead, featured homeostatic and regenerative responses. CONCLUSIONS: By blood gene expression analysis of non-obese individuals, we identified potential gene biomarkers correlating to declining IGI, associated to a pathophysiology of inflammation and metabolic dysregulation.

Plasma low-molecular-weight proteome profiling identified neuropeptide-Y as a prostate cancer biomarker polypeptide.

In prostate cancer diagnosis, PSA test has greatly contributed to the early detection of prostate cancer; however, expanding overdiagnosis and unnecessary biopsies have emerged as serious issues. To explore plasma biomarkers complementing the specificity of PSA test, we developed a unique proteomic technology QUEST-MS (Quick Enrichment of Small Targets for Mass Spectrometry). The QUEST-MS method based on 96-well formatted sequential reversed-phase chromatography allowing efficient enrichment of <20 kDa proteins quickly and reproducibly. Plasma from 24 healthy controls, 19 benign prostate hypertrophy patients, and 73 prostate cancer patients were purified with QUEST-MS and analyzed by LC/MS/MS. Among 153 057 nonredundant peptides, 189 peptides showed prostate cancer specific detection pattern, which included a neurotransmitter polypeptide neuropeptide-Y (NPY). We further validated the screening results by targeted multiple reaction monitoring technology using independent sample set (n = 110). The ROC curve analysis revealed that logistic regression-based combination of NPY, and PSA showed 81.5% sensitivity and 82.2% specificity for prostate cancer diagnosis. Thus QUEST-MS technology allowed comprehensive and high-throughput profiling of plasma polypeptides and had potential to effectively uncover very low abundant tumor-derived small molecules, such as neurotransmitters, peptide hormones, or cytokines.

High-resolution imaging mass spectrometry reveals detailed spatial distribution of phosphatidylinositols in human breast cancer.

High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is an emerging application for lipid research that provides a comprehensive and detailed spatial distribution of ionized molecules. Recent lipidomic approach has identified several phospholipids and phosphatidylinositols (PIs) are accumulated in breast cancer tissues and are therefore novel biomarker candidates. Because their distribution and significance remain unclear, we investigated the precise spatial distribution of PIs in human breast cancer tissues using high-resolution MALDI IMS. We evaluated tissues from nine human breast cancers and one normal mammary gland by negative ion MALDI IMS at a resolution of 10 µm. We detected 10 PIs with different fatty acid compositions, and their proportions were remarkably variable in the malignant epithelial regions. High-resolution imaging enabled us to discriminate cancer cell clusters from the adjacent stromal tissue within epithelial regions; moreover, this technique revealed that several PIs were specifically localized to cancer cell clusters. These PIs were heterogeneously distributed within cancer cell clusters, allowing us to identify two different populations of cancer cells that predominantly expressed either PI(18:0/18:1) or PI(18:0/20:3). Tracing the expression level of PIs during cancer cell progression suggested that the latter population is associated with the invasion. Our study documents a novel model for phospholipid analysis of breast cancer tissues by using high-resolution MALDI IMS and identifies candidate PIs that can describe a specific phenotype of cancer cells.

Multi epitope-targeting immunoprecipitation using F(ab') fragments with high affinity and specificity for the enhanced detection of a peptide with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Human plasma has been frequently studied using mass spectrometry for new biomarker discovery, although detection of low-abundance biological molecules can be challenging due to sample complexity and dynamic protein concentration ranges of plasma proteins. While immunoprecipitation coupled with mass spectrometric analysis is an essential method for overcoming this difficulty, its sensitivity can be insufficient to detect clinically relevant circulating biomarkers because of limited antibody affinity or specificity. To increase antibody affinity, we developed a strategy using a F(ab') fragment coupled to polyethylene glycol. We produced hetero-F(ab')-(PEG)24 beads composed of two monoclonal antiamyloid ß antibodies (6E10 and 4G8) that are specific for different epitopes of amyloid ß and assessed the detection limit of amyloid ß(1-28)-spiked human plasma. In human plasma, the detection limit of amyloid ß(1-28) was 6.14 pM, which was 25- to 50-fold more sensitive than single IgG-protein G beads. In addition, an introduction of polyethylene glycol as a linker reduced nonspecific binding, leading to highly specific MS detection. Finally, the present IP method enabled the detection of endogenous amyloid ß(1-40) in 250 µL of human plasma with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This technique provides a powerful approach for enhancing the sensitivity and specificity of immunoprecipitation (IP)-MS for detection of low-abundance peptides in plasma and has the potential to accelerate MS-based clinical applications.

Structural characterization of pyridylaminated oligosaccharides derived from neutral glycosphingolipids by high-sensitivity capillary electrophoresis-mass spectrometry.

High-sensitivity capillary electrophoresis-electrospray ionization quadrupole ion trap time-of-flight mass spectrometry (CE-ESI-QIT-TOF MS) was developed to structurally characterize four kinds of pyridylaminated (PA) oligosaccharides, i.e., lactose (Lac)-PA, globotriose (Gb3)-PA, globotetraose (Gb4)-PA, and IV(3) αGalNAc-Gb4 (Forssman antigen)-PA, derived from neutral glycosphingolipids. The CE-MS system included the head-column field-amplified sample stacking (HC-FASS) method for effective sample injection into a capillary column in CE, a sheathless interface between CE and a mass spectrometer, and MS and tandem MS (MS(2)) measurements with narrow mass range repeated high-speed switching. The total sensitivity of the developed CE-MS system was about 20,000 times higher than that of the conventional CE-MS system consisting of pressure injection, a sheath-flow interface, and a wide mass range measurement. The MS and MS(2) spectra of the four PA-oligosaccharides at a concentration of 25 amol/µL in mixtures (each 250 amol/10 µL in a tube) clearly showed protonated molecular ions ([M + H](+)) and the fragment ions responsible for the sequential elimination of saccharides. The developed CE-MS system is a powerful method for the structural characterization of glycosphingolipids extracted from very small amounts of biological materials and could be extended to the structural characterization of oligosaccharides derived from glycoproteins.

Structural characterization of neutral glycosphingolipids using high-performance liquid chromatography-electrospray ionization mass spectrometry with a repeated high-speed polarity and MSn switching system.

Four types of neutral glycosphingolipids (LacCer, Gb3Cer, Gb4Cer, and IV3αGalNAc-Gb4Cer; 10 pmol each) were analyzed using high-performance liquid chromatography (HPLC)-electrospray ionization quadrupole ion trap time-of-flight (ESI-QIT-TOF) mass spectrometry (MS) with a repeated high-speed polarity and MSn switching system. This system can provide six types of mass spectra, including positive and negative ion MS, MS2, and MS3 spectra, within 1 s per cycle. Using HPLC with a normal-phase column, information on the molecular weights of major molecular species of four neutral glycosphingolipids was obtained by detecting [M+Na]+ in the positive ion mode mass spectra and [M−H]− in the negative ion mode mass spectra. Sequences of glycosphingolipid oligosaccharide were obtained in the negative ion MS2 spectra. In addition, information on the ceramide structures was clearly obtained in the negative ion MS3 mass spectra. GlcCer molecular species were analyzed by HPLC-ESI-QIT-TOF MS with a reversed-phase column using 1 pmole of GlcCer. The structures of the seven molecular species of GlcCer, namely, d18:1-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:1-C23:0, d18:1-C24:1, and d18:1-C24:0, were characterized using positive ion MS and negative ion MS, MS2, and MS3. The established HPLC-ESI-QIT-TOF MS with MSn switching and a normal phase column has been successfully applied to the structural characterization of LacCer and Gb4Cer in a crude mixture prepared from human erythrocytes.

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid.

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.

Genetic influences on human blood metabolites in the Japanese population.

An increase in ethnic diversity in genetic studies has the potential to provide unprecedented insights into how genetic variations influence human phenotypes. In this study, we conducted a quantitative trait locus (QTL) analysis of 121 metabolites measured using gas chromatography-mass spectrometry with plasma samples from 4,888 Japanese individuals. We found 60 metabolite-gene associations, of which 13 have not been previously reported. Meta-analyses with another Japanese and a European study identified six and two additional unreported loci, respectively. Genetic variants influencing metabolite levels were more enriched in protein-coding regions than in the regulatory regions while being associated with the risk of various diseases. Finally, we identified a signature of strong negative selection for uric acid ( S ˆ  = -1.53, p = 6.2 × 10-18). Our study expanded the knowledge of genetic influences on human blood metabolites, providing valuable insights into their physiological, pathological, and selective properties.

Hyperfructosemia in sleep disordered breathing: metabolome analysis of Nagahama study.

Sleep disordered breathing (SDB), mainly obstructive sleep apnea (OSA), constitutes a major health problem due to the large number of patients. Intermittent hypoxia caused by SDB induces alterations in metabolic function. Nevertheless, metabolites characteristic for SDB are largely unknown. In this study, we performed gas chromatography-mass spectrometry-based targeted metabolome analysis using data from The Nagahama Study (n = 6373). SDB-related metabolites were defined based on their variable importance score in orthogonal partial least squares discriminant analysis and fold changes in normalized peak-intensity levels between moderate-severe SDB patients and participants without SDB. We identified 20 metabolites as SDB-related, and interestingly, these metabolites were frequently included in pathways related to fructose. Multivariate analysis revealed that moderate-severe SDB was a significant factor for increased plasma fructose levels (ß = 0.210, P = 0.006, generalized linear model) even after the adjustment of confounding factors. We further investigated changes in plasma fructose levels after continuous positive airway pressure (CPAP) treatment using samples from patients with OSA (n = 60) diagnosed by polysomnography at Kyoto University Hospital, and found that patients with marked hypoxemia exhibited prominent hyperfructosemia and their plasma fructose levels lowered after CPAP treatment. These data suggest that hyperfructosemia is the abnormality characteristic to SDB, which can be reduced by CPAP treatment.