The great importance of normalization of LC-MS data for highly-accurate non-targeted metabolomics.

The non-targeted metabolomics analysis of biological samples is very important to understand biological functions and diseases. LC combined with electrospray ionization-based MS has been a powerful tool and widely used for metabolomic analyses. However, the ionization efficiency of electrospray ionization fluctuates for various unexpected reasons such as matrix effects and intraday variations of the instrument performances. To remove these fluctuations, normalization methods have been developed. Such techniques include increasing the sensitivity, separating co-eluting components and normalizing the ionization efficiencies. Normalization techniques allow simultaneously correcting of the ionization efficiencies of the detected metabolite peaks and achieving quantitative non-targeted metabolomics. In this review paper, we focused on these normalization methods for non-targeted metabolomics by LC-MS.

A preliminary report on retrospective dose assessment by FISH translocation assay in FDNPP Nuclear Emergency Worker Study (NEWS).

In Japan, a national project of longitudinal health care and epidemiological research (NEWS) was developed in 2014 to analyse the effects of radiation on human health for workers who responded to the Fukushima Dai-ichi nuclear emergency in 2011. In 2018, peripheral blood for chromosome translocation analysis was collected from 62 workers. Retrospective dose assessment was performed with fluorescence in situ hybridisation translocation (FISH-Tr) assay. The range of estimated doses by FISH-Tr assay was 0-635 mGy, in which 22 workers had estimated doses of more than 189 mGy. Biological dose estimates were five times higher in workers with physically measured total exposure recordings above 70 mGy. It is likely that smoking and medical exposure caused the discrepancy between estimated biological and physical total exposure doses. Thus, there is a possibility that retrospective biodosimetry assessment might over-estimate occupational exposures to workers exposed to chronic radiation during nuclear emergency work.

No increase in translocated chromosomal aberrations, an indicator of ionizing radiation exposure, in childhood thyroid cancer in Fukushima Prefecture.

To investigate the effects of radiation exposure due to the Fukushima nuclear power plant accident, following the disaster Fukushima Prefecture launched thyroid ultrasound examinations of residents who were generally younger than 18 years at the time of the earthquake. As the rate of pediatric thyroid cancer was higher than expected, we conducted biological dose assessment based on the frequency of translocated chromosome (Tr) aberrations using peripheral blood lymphocytes. Tr formation frequency was compared among the thyroid cancer (n = 38, median age 18 years, age range 12-26 years), thyroid-related disease (n = 30, median age 21 years, age range 15-28 years), and healthy controls (n = 31, median age 22 years, age range 20-23 years) groups. Tr aberration frequency was initially significantly higher in the thyroid cancer than in the other two groups; however, differences among the groups disappeared after adjusting for history of CT scan, as 92%, 67%, and 28% of those in the thyroid cancer, thyroid-related disease, and control groups, respectively, had undergone CT previously. Therefore, the significant difference in the initial number of Tr formations is presumably due to radiation exposure from CT. Accordingly, the effects of medical exposure on the chromosomes of children and adolescents should be noted.

In situ molecular analysis of plant tissues by live single-cell mass spectrometry.

We report the development of a rapid, direct molecular analysis of live, single plant cells viewed under a video microscope in their natural environment. A nanoelectrospray tip was used to extract the contents of a single leaf, stem, or petal cell from Pelargonium zonale, and the samples were analyzed on an Orbitrap mass spectrometer by nanoelectrospray ionization. Around a thousand m/z peaks belonging to metabolites and other compounds in each sample were obtained and processed by using statistical tools to find the cell specific molecular peaks. Hybrid high-resolution mass spectrometry analysis was performed to confirm the structure of specific metabolites from the analyzed samples. This method is useful for identifying specific molecules in live single cells from plant tissue and will allow different cell types and stages from different sites in the plant to be compared with morphological observations.

Molecular and functional analysis of cellular phenomena using single-cell mass spectrometry.

Single-cell analysis has attracted attention in many fields of biological studies as a tool to survey the precise mechanisms of cellular and molecular behavior. The development of sensitive mass spectrometry allows the study of molecules in single cells or small regions. Matrix-assisted laser desorption/ionization imaging mass spectrometry and secondary-ion mass spectrometry use in situ ionization of specimens on sample plates to visualize molecular distributions as images from mass spectra. Several single-cell mass spectrometry technologies that initially recover a single cell followed by ionization have been developed. Among them, only nanospray-mediated sampling and ionization named Live Single-cell Mass Spectrometry can be used for real-time analysis. This paper explains that method in detail.

ΔNp63α transcriptionally represses p53 target genes involved in the radiation-induced DNA damage response : ΔNp63α may cause genomic instability in epithelial stem cells.

BACKGROUND: The DNA damage response (DDR) is a mechanism that protects cells against radiation-induced oxidative DNA damage by causing cell cycle arrest and apoptosis. TP63 is a member of the tumour suppressor TP53 gene family, and ΔNp63α, a TP63 splicing variant, is constitutively expressed in the stem cell-containing basal layer of stratified epithelial tissues, including the mammary gland, where it plays a critical role in stemness and tissue development. ΔNp63α has been reported to transcriptionally inhibit the tumour suppression protein p53. This p53-repressive activity may cause genomic instability in epithelial stem cells exposed to radiation. In this study, we analysed the inhibitory effect of ΔNp63α on radiation-induced DDR. METHODS: To elucidate the role of the p53-repressive effect of ΔNp63α in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing ΔNp63α and then performed ectopic and entopic expression experiments using human induced pluripotent stem cells (hiPSCs). After irradiation, the expression of DDR-related genes and proteins in ΔNp63α-expressing and control cells was analysed by RT-qPCR, Western blotting, and flow cytometry. RESULTS: The mRNA/protein expression levels of BAX and p21 were significantly increased in p63-siRNA-treated HMECs (sip63) after X-ray irradiation (4 Gy, 0.7 Gy/min) but not in scramble-siRNA treated HMECs (scr). Transcriptomic analysis showed decreased RNA expression of cell cycle-related genes and increased expression of programmed cell death-related genes in sip63 cells compared to scr cells. Furthermore, flow cytometric analysis revealed an increase in apoptotic cells and a decrease in 5-ethynyl-2´-deoxyuridine uptake in sip63 cells compared to scr cells. On the other hand, both the ectopic and entopic expression of ΔNp63α in apoptosis-sensitive hiPSCs reduced the expression levels of BAX after irradiation and significantly decreased the number of apoptotic cells induced by radiation. CONCLUSION: Taken together, these results indicate that ΔNp63α represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells.

Long-chain saturated fatty acids in breast milk are associated with the pathogenesis of atopic dermatitis via induction of inflammatory ILC3s.

Breastfeeding influences the immune system development in infants and may even affect various immunological responses later in life. Breast milk provides a rich source of early nutrition for infant growth and development. However, the presence of certain compounds in breast milk, related to an unhealthy lifestyle or the diet of lactating mothers, may negatively impact infants. Based on a cohort study of atopic dermatitis (AD), we find the presence of damage-associated molecular patterns (DAMPs) activity in the mother's milk. By non-targeted metabolomic analysis, we identify the long-chain saturated fatty acids (LCSFA) as a biomarker DAMPs (+) breast milk samples. Similarly, a mouse model in which breastfed offspring are fed milk high in LCSFA show AD onset later in life. We prove that LCSFA are a type of damage-associated molecular patterns, which initiate a series of inflammatory events in the gut involving type 3 innate lymphoid cells (ILC3s). A remarkable increase in inflammatory ILC3s is observed in the gut, and the migration of these ILC3s to the skin may be potential triggers of AD. Gene expression analysis of ILC3s isolated from the gut reveal upregulation of genes that increase ILC3s and chemokines/chemokine receptors, which may play a role in ILC migration to the skin. Even in the absence of adaptive immunity, Rag1 knockout mice fed a high-LCSFA milk diet develop eczema, accompanied by increased gut ILC3s. We also present that gut microbiota of AD-prone PA milk-fed mice is different from non-AD OA/ND milk-fed mice. Here, we propose that early exposure to LCSFAs in infants may affect the balance of intestinal innate immunity, inducing a highly inflammatory environment with the proliferation of ILC3s and production of interleukin-17 and interleukin-22, these factors may be potential triggers or worsening factors of AD.

Chromosomal translocation t(11;14) and p53 deletion induced by the CRISPR/Cas9 system in normal B cell-derived iPS cells.

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain potential to transform by reprograming, and then chromosomal aberrations cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we have already established normal B cell-derived induced pluripotent stem cells (BiPSCs). Here we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eµ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5'-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14). These BiPSCs differentiated into hematopoietic progenitor cells (HPCs). However, unlike cord blood, those HPCs did not differentiated into B lymphocytes by co-culture with BM stromal cell. Therefore, further ingenuity is required to differentiate those BiPSCs-derived HPCs into B lymphocytes.

A novel signaling pathway associated with Lyn, PI 3-kinase and Akt supports the proliferation of myeloma cells.

Interleukin-6 (IL-6) is a growth factor for human myeloma cells. We have recently found that in myeloma cells the activation of both signal transducer and activator of transcription (STAT) 3 and extracellular signal-regulated kinase (ERK) 1/2 is not sufficient for the IL-6-induced proliferation, which further requires the activation of the src family kinases, such as Lyn. Here we showed that the Lyn-overexpressed myeloma cell lines had the higher proliferative rate with IL-6 and the enhanced activation of the phosphatidylinositol (PI) 3-kinase and Akt. The IL-6-induced phosphorylation of STAT3 and ERK1/2 was not up-regulated in the Lyn-overexpressed cells, indicating that the Lyn-PI 3-kinase-Akt pathway is independent of these pathways. The PI 3-kinase was co-precipitated with Lyn in the Lyn-overexpressed cells of which proliferation with IL-6 was abrogated by the specific inhibitors for PI 3-kinase or Akt, suggesting that the activation of the PI 3-kinase-Akt pathway associated with Lyn is indeed related to the concomitant augmentation of myeloma cell growth. Furthermore, the decreased expression of p53 and p21(Cip1) proteins was observed in the Lyn-overexpressed cells, implicating a possible downstream target of Akt. This study identifies a novel IL-6-mediated signaling pathway that certainly plays a role in the proliferation of myeloma cells and this novel mechanism of MM tumor cell growth associated with Lyn would eventually contribute to the development of MM treatment.

Induction of t(11;14) IgH enhancer/promoter-cyclin D1 gene translocation using CRISPR/Cas9.

Chromosomal translocation is a key process in the oncogenic transformation of somatic cells. Previously, artificial induction of chromosomal translocation was performed using homologous recombination-mediated loxP labeling of target regions followed by Cre-mediated recombination. Recent progress in genome editing techniques has facilitated the easier induction of artificial translocation by cutting two targeted genome sequences from different chromosomes. The present study established a system to induce t(11;14)(q13;q32), which is observed primarily in multiple myeloma (MM) and involves the repositioning of the cyclin D1 (CCND1) gene downstream of the immunoglobulin heavy chain (IgH) constant region enhancers by translocation. The placing of tandem gRNAs designed to cut both the IgH Eµ and CCND1 15-kb upstream regions in lentiCRISPRv2 enabled the induction of chromosomal translocation in 293T cells, with confirmation by translocation-specific PCR and fluorescence in situ hybridization probing with IgH and CCND1. At the translocation junctions, small deletions and the addition of DNA sequences (indels) were observed in several clones. Cloned cells with t(11;14) exhibited slower growth and lower CCND1 mRNA expression compared to the parent cells, presenting the opposite phenomena induced by t(11;14) in MM cells, indicating that the silent IgH gene juxtaposed to CCND1 may negatively affect CCND1 gene expression and cell proliferation in the non-B lymphocyte lineage. Therefore, the present study achieved the induction of silent promoter/enhancer translocation in t(11;14)(q13;q32) as a preparatory experiment to study the role of IgH constant region enhancer-driven CCND1 overexpression in oncogenic transformation processes in B lymphocytes.

Investigation of the cumulative number of chromosome aberrations induced by three consecutive CT examinations in eight patients.

In our previous study, we found that chromosomes were damaged by the radiation exposure from a single computed tomography (CT) examination, based on an increased number of dicentric chromosomes (Dics) formed in peripheral blood lymphocytes after a CT examination. We then investigated whether a cumulative increase in the frequency of Dics and chromosome translocations (Trs) formation could be observed during three consecutive CT examinations performed over the course of 3-4 years, using lymphocytes in peripheral bloods of eight patients (five males and three females; age range 27-77 years; mean age, 64 years). The effective radiation dose per CT examination estimated from the computational dosimetry system was 22.0-73.5 mSv, and the average dose per case was 40.5 mSv. The frequency of Dics formation significantly increased after a CT examination and tended to decrease before the next examination. Unlike Dics analysis, we found no significant increase in the frequency of Trs formation before and after the CT examination, and we observed no tendency for the frequency to decrease before the next CT examination. The frequency of Trs formation was higher than that of Dics formation regardless of CT examination. Furthermore, neither analysis of Dics nor Trs showed a cumulative increase in the frequency of formation following three consecutive CT examinations.

Ectopic cyclin D1 overexpression increases chemosensitivity but not cell proliferation in multiple myeloma.

We established a myeloma cell line (RPMI8226) with cyclin D1 overexpression in which the transfected cyclin D1 gene was stably expressed. D1 transfectants showed down-regulation of cyclin D2. Cell proliferation analysis did not show any differences among RPMI8226, mock control, and D1 transfectants. The number of S-phase cells increased while the number of G0/G1- and G2/M-phase cells decreased in D1 transfectants, which indicates a prolonged S-phase caused by cyclin D1 transfection. A decreased number of G2/M-phase cells was also detected in myeloma cells of patients with translocation t(11;14)(q13;q32). Western blot analysis revealed an increase in the hyperphosphorylated form of retinoblastoma (Rb) protein in D1 transfectants; however, the expression of p53, p16, Bax, Bad, Bcl-2, and Mcl-1 did not significantly change. Treatment with anti-myeloma drugs (melphalan, dexamethasone, bortezomib and immunomodulatory compounds) induced apoptosis earlier in D1 transfectants compared with RPMI8226 and mock control via the activation of both caspase-8 and -9. However, we could not detect a relationship between cyclin D1 expression and the response to treatment with VAD and bortezomib. Therefore, we assume that high sensitivity to anti-myeloma drugs depends on the duration of the S-phase, but a clinical response might depend on the number of myeloma cells with cyclin D1 overexpression.

Live single-cell video-mass spectrometry for cellular and subcellular molecular detection and cell classification.

The molecular content from the cytoplasm of a live, single mammalian cell and its organelle were trapped with a nano-electrospray ionization (ESI) tip acting as a micropipette under a video microscope, and hundreds of small molecular peaks were detected by direct nano-ESI mass spectrometry (MS). Granule- or cytoplasm-specific peaks in a mast cell (RBL 2H3) model were extracted by paired t-test to demonstrate their specific localization. Some of the typical and specific molecules were successfully identified by MS/MS analysis. This method was also applied to the cell classification of seven types of cell lines at the single-cellular level by principal component analysis (PCA), revealing seven clusters in the multivariate score plot.

Live single-cell molecular analysis by video-mass spectrometry.

The direct molecular analysis of a live single cell viewed under a video-microscope has been developed. The cell contents are sucked into a nano-electrospray tip, and hundreds of peaks of ionic compounds of low molecular weight are detected by nano-ESI Q-TOF mass spectrometry (MS). Cell-specific MS peaks in a single mouse-embryonic fibroblasts cell are extracted by a t-test, and one of the peaks is proceeded to MS/MS analysis for molecular identification. This method is direct and quick to identify the molecules of a cell with simultaneous observation by a video-microscope.

Dose-response curves for analyzing of dicentric chromosomes and chromosome translocations following doses of 1000 mGy or less, based on irradiated peripheral blood samples from five healthy individuals.

In terms of biological dosimetry at the time of radiation exposure, the dicentric chromosome (Dic) assay (DCA) is the gold standard for assessing for the acute phase and chromosome translocation (Tr) analysis is the gold standard for assessing the chronic phase. It is desirable to have individual dose-response curves (DRCs) for each laboratory because the analysis criteria differ between laboratories. We constructed the DRCs for radiation dose estimation (with three methods) using peripheral blood (PB) samples from five healthy individuals. Aliquots were irradiated with one of eight gamma-ray doses (0, 10, 20, 50, 100, 200, 500 or 1000 mGy), then cultured for 48 h. The number of chromosome aberrations (CAs) was analyzed by DCA, using Giemsa staining and centromere-fluorescence in situ hybridization (centromere-FISH) and by chromosome painting (chromosome pairs 1, 2 and 4) for Tr analysis. In DCA, there was large variation between individuals in the frequency of Dics formed, and the slopes of the DRCs were different. In Tr analysis, although variation was observed in the frequency of Tr, the slopes of the DRCs were similar after adjusting the background for age. Good correlation between the irradiation dose and the frequency of CAs formed was observed with these three DRCs. However, performing three different biological dosimetry assays simultaneously on PB from five donors nonetheless results in variation in the frequency of CAs formed, especially at doses of 50 mGy or less, highlighting the difficulty of biological dosimetry using these methods. We conclude that it might be difficult to construct universal DRCs.

Dehydroepiandrosterone can inhibit the proliferation of myeloma cells and the interleukin-6 production of bone marrow mononuclear cells from patients with myeloma.

The serum levels of an adrenal sex hormone, dehydroepiandrosterone sulfate (DHEA-S), are significantly more decreased in human myelomas compared with the reduction brought by physiologic decline with age. In order to clarify the effect of DHEA on myeloma cells, we investigated whether DHEA and DHEA-S could inhibit interleukin-6 (IL-6) production of bone marrow mononuclear cells and the proliferation of myeloma cells from patients with myeloma. DHEA-S and DHEA suppressed IL-6 production from a bone marrow stromal cell line, KM-102, as well as in bone marrow mononuclear cells from patients with myeloma. Furthermore, DHEA inhibited in vitro growth of the U-266 cell line and primary myeloma cells from the patients, as well as the in vivo growth of U-266 cells implanted i.p. in severe combined immunodeficiency-hIL6 transgenic mice. DHEA up-regulated the expression of peroxisome proliferator-activated receptor (PPAR), PPAR beta, but not PPARgamma or PPARalpha, and the expression of IkappaBalpha gene in myeloma cells and bone marrow stromal cells, which could explain the suppressive effect of DHEA on IL-6 production through the down-regulation of NF-kappaB activity. Therefore, these data revealed that DHEA-S, as well as DHEA, had a direct effect on myeloma and bone marrow stromal cells to inhibit their proliferation and IL-6 production, respectively.

Academic Responses to Fukushima Disaster.

Since radiation accidents, particularly nuclear disasters, are rarer than other types of disasters, a comprehensive radiation disaster medical curriculum for them is currently unavailable. The Fukushima compound disaster has urged the establishment of a new medical curriculum in preparation for any future complex disaster. The medical education will aim to aid decision making on various health risks for workers, vulnerable people, and residents addressing each phase in the disaster. Herein, we introduce 3 novel educational programs that have been initiated to provide students, professionals, and leaders with the knowledge of and skills to elude the social consequences of complex nuclear disasters. The first program concentrates on radiation disaster medicine for medical students at the Fukushima Medical University, together with a science, technology, and society module comprising various topics, such as public risk communication, psychosocial consequences of radiation anxiety, and decision making for radiation disaster. The second program is a Phoenix Leader PhD degree at the Hiroshima University, which aims to develop future leaders who can address the associated scientific, environmental, and social issues. The third program is a Joint Graduate School of Master's degree in the Division of Disaster and Radiation Medical Sciences at the Nagasaki University and Fukushima Medical University.

Establishment of induced pluripotent stem cells from normal B cells and inducing AID expression in their differentiation into hematopoietic progenitor cells.

B cell derived induced pluripotent stem cells (BiPSCs) were recently established from peripheral blood B cells by the simultaneous transfection of Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) and C/EBPα using a Sendai virus vector. Here, using a different method, we established BiPSCs with immunoglobulin heavy chain (IgH) gene rearrangement from normal B cells purified from lymph nodes. The critical points of our method are pre-stimulation of B cells with IL-21 and CD40-ligand (CD40L), followed by consecutive transfection of highly concentrated Yamanaka factors using a retroviral vector. Following each transfection the cells were centrifuged onto a retronectin coated plate and the activated by IL-4, IL-2, and CD40L. Furthermore, we established BiPSCs (BiPSC-A) in which activation-induced cytidine deaminase (AID) could be induced using the doxycycline-controlled. Both the parental BiPSC and BiPSC-A showed the capability of differentiating into hematopoietic progenitor cells (HPCs) based on confirmation of CD34 expression and colony-formation from CD34-positive cells. The findings that BiPSC-A can differentiate into HPCs suggest that there is a possibility that induction of AID expression would result in chromosomal translocations in the process of differentiation from BiPSCs, and therefore that these BiPSCs could be useful in elucidating the tumor origin of abnormal B cells in myelomagenesis.

Accelerated proliferation of myeloma cells by interleukin-6 cooperating with fibroblast growth factor receptor 3-mediated signals.

Interleukin-6 (IL-6) is a cytokine that regulates the proliferation of some tumor cells including multiple myeloma (MM). Ectopic expression of fibroblast growth factor receptor 3 (FGFR 3) associated with the chromosomal translocation, t(4;14)(p16.3;q32), is frequently found in MM, and therefore, has been implicated in the neoplastic transformation of this disease. Here, we show that IL-6 together with FGF enhanced proliferation of a myeloma cell line, KMS-11 carrying t(4;14)(p16.3;q32) and the FGFR 3-transfected U 266 myeloma cell line which ectopically expressed FGFR 3 but responded to neither IL-6 nor FGF alone. In KMS-11, IL-6 activated signal transducer and activator of transcription 3 (STAT 3) while FGF activated extracellular signal-regulated kinase 1/2 (ERK 1/2) and phosphatidylinositol (PI)-3 kinase. As both MEK inhibitors and a PI 3-kinase inhibitor abolished the effect of IL-6 and FGF, the activation of both the ERK 1/2 and PI 3-kinase signaling cascades is essential for the proliferation of KMS-11 enhanced by IL-6 and FGF. Furthermore, the FGF-induced activation of ERK 1/2 contributed to the serine phosphorylation of STAT 3, suggesting that the signaling crosstalk between the cytokine receptor, IL-6 receptor alpha/gp 130 and the growth factor receptor tyrosine kinase, FGFR 3. These results indicate that FGFR 3 plays a crucial role in the accelerated proliferation of MM carrying t(4;14)(p16.3;q32).

Analysis of chromosome translocation frequency after a single CT scan in adults.

We recently reported an increase in dicentric chromosome (DIC) formation after a single computed tomography (CT) scan (5.78-60.27 mSv: mean 24.24 mSv) and we recommended analysis of 2000 metaphase cells stained with Giemsa and centromere-FISH for dicentric chromosome assay (DCA) in cases of low-dose radiation exposure. In the present study, we analyzed the frequency of chromosome translocations using stored Carnoy's-fixed lymphocyte specimens from the previous study; these specimens were from 12 patients who were subject to chromosome painting of Chromosomes 1, 2 and 4. Chromosomes 1, 2 and 4 were analyzed in ∼5000 cells, which is equivalent to the whole-genome analysis of almost 2000 cells. The frequency of chromosome translocation was higher than the number of DICs formed, both before and after CT scanning. The frequency of chromosome translocations tended to be higher, but not significantly higher, in patients with a treatment history compared with patients without such a history. However, in contrast to the results for DIC formation, the frequency of translocations detected before and after the CT scan did not differ significantly. Therefore, analysis of chromosome translocation may not be a suitable assay for detecting chromosome aberrations in cases of low-dose radiation exposure from a CT scan. A significant increase in the frequency of chromosome translocations was not likely to be detected due to the high baseline before the CT scan; the high and variable frequency of translocations was probably due to multiple confounding factors in adults.