Language：English   Publishing type：Research paper (scientific journal)  

Background and Aim: Preoperative histological evaluation of pancreatic neoplasms is important for guiding the resection strategy and preventing postoperative adverse events. However, conventional endoscopic methods have technical limitations that reduce the accuracy of the histopathological examination. Probe electrospray ionization mass spectrometry (PESI-MS) may be a useful technique for rapidly evaluating small specimens. Methods: This single-center prospective study included patients with pancreatic neoplasms between October 2018 and December 2019. Pancreatic ductal adenocarcinoma (PDAC) specimens were obtained via endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) and non-neoplastic tissue was obtained via surgery. Specimens were subjected to PESI-MS and the mass spectra were analyzed using partial least squares regression-discriminant analysis. Results: The study included 40 patients with 20 nonneoplastic specimens and 19 PDAC specimens (1 case of neuroendocrine carcinoma was omitted). All nonneoplastic specimens were sufficient for PESI-MS analysis, although only 7 of 19 PDAC specimens were sufficient for PESI-MS analysis because of poor sample quality or insufficient quantity (<1 mg). Among the 27 analyzed cases, the mass spectra clearly differentiated between the PDAC and nonneoplastic specimens. Conclusions: This study revealed that PESI-MS could differentiate between PDAC and nonneoplastic specimens, even in cases where EUS-FNA produced very small specimens.

DOI： 10.1002/jgh3.12617

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Spandidos Publications  

Biomarkers may be of value for the early detection of gastric cancer (GC) and the preoperative identification of tumor characteristics to guide treatment strategies. The present study analyzed the expression levels of phospholipids in plasma from patients with GC using liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) to detect reliable biomarkers for GC. Furthermore, combining the results with a machine learning strategy, the present study attempted to establish a diagnostic system for GC. A total of 20 plasma samples from preoperative patients with GC and 16 plasma samples from tumor-free patients (controls) were selected from our biobank named 'SHINGEN (Yamanashi Biobank of Gastroenterological Cancers)', which includes a total of 1,592 plasma samples, and were analyzed by LC/ESI-MS. The obtained data were discriminated using a machine learning-based diagnostic algorithm, whose discriminant ability was confirmed through leave-one-out cross-validation. Using LC/ESI-MS, the levels of 236 lipid molecules were determined. Biomarker analysis revealed that a few lipids that were downregulated in the GC group could discriminate between the GC and control groups. Whole lipid composition analysis using partial least squares regression revealed good discrimination ability between the GC and control groups. Integrative analysis of all molecules using the aforementioned machine learning method exhibited a diagnostic accuracy of 94.4% (specificity, 93.8%; sensitivity, 95.0%). In conclusion, the outcomes of the present study suggested the potential future application of the aforementioned system in clinical settings. By accumulating more reliable data, the present system will be able to detect early-stage cancer and will be capable of predicting the efficacy of each therapeutic strategy.

DOI： 10.3892/ol.2021.12666

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Ovid Technologies (Wolters Kluwer Health)  

<title>ABSTRACT</title>
<sec>
<title>BACKGROUND</title>
We have previously developed a medical diagnostic pipeline that employs mass spectrometry and machine learning. It does not annotate molecular markers that are specific to cancer but uses entire mass spectra for predicting the properties of glioma.


</sec>
<sec>
<title>OBJECTIVE</title>
To validate the power of our diagnostic method in predicting the pathological and radiological properties of glioma with a simple sample preparation procedure.


</sec>
<sec>
<title>METHODS</title>
A total of 10 patients with glioma and 4 nonglioma patients who went through surgical resection were enrolled in our hospital. A total of 1020 mass spectra were acquired from 88 specimens. In order to examine the prediction power of the diagnostic pipeline that we have developed, we performed 10-fold cross-validation for pathological and radiological findings and calculated agreement rates with the conventional methods such as pathological diagnosis (World Health Organization [WHO] grading, MIB-1 labeling index [LI], mutations in the isocitrate dehydrogenase [IDH]-1 gene, and positive 5-aminolevulinic acid [5-ALA] fluorescence) and radiological information (gadolinium [Gd]-enhanced area and high-intensity area on fluid-attenuated inversion recovery [FLAIR] imaging).


</sec>
<sec>
<title>RESULTS</title>
Prediction accuracy for WHO malignant grade was 91.37%. Those for MIB-1 LI ≥ 10% and IDH-1 mutation-positive were 82.84% and 87.75%, respectively. Our method achieved an accurate prediction of 95.00% for the 5-ALA-positive lesion. The present method displayed an accuracy of 82.36% in predicting the area of FLAIR hyperintensity and 81.27% for the Gd-enhanced area.


</sec>
<sec>
<title>CONCLUSION</title>
Our methodology achieved a higher rate of prediction of glioma in terms of pathology and radiology. Research is ongoing to develop a validation cohort to verify the biological profiles of glioma specimens.


</sec>

DOI： 10.1093/neuopn/okaa026

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title><sec>
<title>Background</title>
Probe electrospray ionization-mass spectrometry (PESI-MS) can rapidly visualize mass spectra of small, surgically obtained tissue samples, and is a promising novel diagnostic tool when combined with machine learning which discriminates malignant spectrum patterns from others. The present study was performed to evaluate the utility of this device for rapid diagnosis of colorectal liver metastasis (CRLM).


</sec><sec>
<title>Methods</title>
A prospectively planned study using retrospectively obtained tissues was performed. In total, 103 CRLM samples and 80 non-cancer liver tissues cut from surgically extracted specimens were analyzed using PESI-MS. Mass spectra obtained by PESI-MS were classified into cancer or non-cancer groups by using logistic regression, a kind of machine learning. Next, to identify the exact molecules responsible for the difference between CRLM and non-cancerous tissues, we performed liquid chromatography-electrospray ionization-MS (LC-ESI-MS), which visualizes sample molecular composition in more detail.


</sec><sec>
<title>Results</title>
This diagnostic system distinguished CRLM from non-cancer liver parenchyma with an accuracy rate of 99.5%. The area under the receiver operating characteristic curve reached 0.9999. LC-ESI-MS analysis showed higher ion intensities of phosphatidylcholine and phosphatidylethanolamine in CRLM than in non-cancer liver parenchyma (<italic>P</italic> &lt; 0.01, respectively). The proportion of phospholipids categorized as monounsaturated fatty acids was higher in CRLM (37.2%) than in non-cancer liver parenchyma (10.7%; <italic>P</italic> &lt; 0.01).


</sec><sec>
<title>Conclusion</title>
The combination of PESI-MS and machine learning distinguished CRLM from non-cancer tissue with high accuracy. Phospholipids categorized as monounsaturated fatty acids contributed to the difference between CRLM and normal parenchyma and might also be a useful diagnostic biomarker and therapeutic target for CRLM.


</sec>

DOI： 10.1186/s12885-021-08001-5

PubMed

Other Link： http://link.springer.com/article/10.1186/s12885-021-08001-5/fulltext.html

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

A moving string sampling probe and a new ESI based ionization source that can be readily incorporated into the existing endoscopes are developed for performing in vivo mass spectrometry during the endoscopic procedure. The medical-grade silk suture driven by a stepping motor is used to perform the sampling on the region of interest when the probe head is brought gently into contact with the surface of the gastrointestinal tissue. The tissues and the compounds adhered to the sampling string are transported to an ionization region inside the ion inlet tube in which they are extracted and ionized by the charging droplets generated from an electrospray outside the ion inlet. Since the extraction/ionization and sampling processes are isolated, organic solvents, high voltage (HV), and heating can be used for the optimization of ionization without compromising the biocompatibility of the sampling probe. The demonstration of the in vivo analysis of the gastric mucosa of a mouse is performed using a 2 m long gastrointestinal endoscope.

DOI： 10.1021/jasms.0c00366

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Mass Spectrometry Society of Japan  

In 2007, probe electrospray ionization/mass spectrometry (PESI/MS) was developed. In this technique, the needle is moved down along a vertical axis and the tip of the needle touched to the sample. After capturing the sample at the needle tip, the needle is then moved up and a high voltage is applied to the needle at the highest position to generate electrospray. Due to the discontinuous sampling followed by the generation of spontaneous electrospray, sequential and exhaustive electrospray takes place depending on the surface activity of the analytes. As modified versions of PESI, dipping PESI (dPESI), sheath-flow PESI (sfPESI) and adjustable sfPESI (ad-sfPESI) have been developed. These methods are complementary to each other and they can be applicable to surface and bulk analysis of various biological samples. In this article, the characteristics of these methods and their applications to real samples will be reviewed.

DOI： 10.5702/massspectrometry.A0092

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

BACKGROUND AND AIMS: Complete surgical resection with negative margin is one of the pillars in treatment of liver tumours. However, current techniques for intra-operative assessment of tumour resection margins are time-consuming and empirical. Mass spectrometry (MS) combined with artificial intelligence (AI) is useful for classifying tissues and provides valuable prognostic information. The aim of this study was to develop a MS-based system for rapid and objective liver cancer identification and classification. METHODS: A large dataset derived from 222 patients with hepatocellular carcinoma (HCC, 117 tumours and 105 non-tumours) and 96 patients with mass-forming cholangiocarcinoma (MFCCC, 50 tumours and 46 non-tumours) were analysed by Probe Electrospray Ionization (PESI) MS. AI by means of support vector machine (SVM) and random forest (RF) algorithms was employed. For each classifier, sensitivity, specificity and accuracy were calculated. RESULTS: The overall diagnostic accuracy exceeded 94% in both the AI algorithms. For identification of HCC vs non-tumour tissue, RF was the best, with 98.2% accuracy, 97.4% sensitivity and 99% specificity. For MFCCC vs non-tumour tissue, both algorithms gave 99.0% accuracy, 98% sensitivity and 100% specificity. CONCLUSIONS: The herein reported MS-based system, combined with AI, permits liver cancer identification with high accuracy. Its bench-top size, minimal sample preparation and short working time are the main advantages. From diagnostics to therapeutics, it has the potential to influence the decision-making process in real-time with the ultimate aim of improving cancer patient cure.

DOI： 10.1111/liv.14604

PubMed

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/liv.14604

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Probe electrospray ionization mass spectrometry (PESI-MS) is an ambient ionization-based mass spectrometry method that surpasses the original electrospray ionization technique in features such as the rapidity of analysis, simplicity of the equipment and procedure, and lower cost. This study found that the PESI-MS system with machine learning has the potential to establish a lipid-based diagnosis of breast cancer with higher accuracy, using a simpler approach. Rapid MS for breast cancer.

DOI： 10.1002/bjs.11613

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Real-time and in-situ mass-spectrometry analyses of living animal and biological sample were performed using a novel remote sampling electrospray ionization (RS-ESI) probe. Unlike conventional ESI, in which injection or syringe loading is required for sample introduction, the RS-ESI probe ionizes the samples when the sampling capillary is in contact with the sample. As the sampling capillary is electrically held at ground potential, the safety of the animal and operator is assured. The liquid sample is aspirated to the ESI emitter at the other end of the capillary by the Venturi effect. Subsequently, the electrospray is generated when a high voltage is applied to the counter electrode placed inside the ion source chamber. The probe unit is attached to the mass spectrometer with a long flexible tube and its position can be freely manipulated during the analysis. In this report, we demonstrate a real-time analysis of a living mouse liver and an automatic analysis of 138 serum samples using this new technique.

DOI： 10.1016/j.jpba.2019.04.050

PubMed

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer US  

Introduction: Atherosclerotic diseases are the leading cause of death worldwide. Biomarkers of atherosclerosis are required to monitor and prevent disease progression. While mass spectrometry is a promising technique to search for such biomarkers, its clinical application is hampered by the laborious processes for sample preparation and analysis. Methods: We developed a rapid method to detect plasma metabolites by probe electrospray ionization mass spectrometry (PESI-MS), which employs an ambient ionization technique enabling atmospheric pressure rapid mass spectrometry. To create an automatic diagnosis system of atherosclerotic disorders, we applied machine learning techniques to the obtained spectra. Results: Using our system, we successfully discriminated between rabbits with and without dyslipidemia. The causes of dyslipidemia (genetic lipoprotein receptor deficiency or dietary cholesterol overload) were also distinguishable by this method. Furthermore, after induction of atherosclerosis in rabbits with a cholesterol-rich diet, we were able to detect dynamic changes in plasma metabolites. The major metabolites detected by PESI-MS included cholesterol sulfate and a phospholipid (PE18:0/20:4), which are promising new biomarkers of atherosclerosis. Conclusion: We developed a remarkably fast and easy method to detect potential new biomarkers of atherosclerosis in plasma using PESI-MS.

DOI： 10.1007/s11306-018-1334-z

Scopus

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

Objectives: Intraoperative identification of tumor margins is essential to achieving complete tumor resection. However, the process of intraoperative pathological diagnosis involves cumbersome procedures, such as preparation of cryosections and microscopic examination, thus requiring more than 30 min. Moreover, intraoperative diagnoses made by examining cryosections are occasionally inconsistent with postoperative diagnoses made by examining paraffin-embedded sections because the former are of poorer quality. We sought to establish a more rapid accurate method of intraoperative assessment.
Materials and methods: A diagnostic algorithm of head and neck squamous cell carcinoma (HNSCC) using machine learning was constructed by mass spectra obtained from 15 non-cancerous and 19 HNSCC specimens by probe electrospray ionization mass spectrometry (PESI-MS). The clinical validity of this system was evaluated using intraoperative specimens of HNSCC and normal mucosa.
Results: A total of 114 and 141 mass spectra were acquired from non-cancerous and cancerous specimens, respectively, using both positive- and negative-ion modes of PESI-MS. These data were fed into partial least squares-logistic regression (PLS-LR) to discriminate tumor-specific spectral patterns. Leave-one-patient-out cross validation of this algorithm in positive- and negative-ion modes showed accuracies in HNSCC diagnosis of 90.48% and 95.35%, respectively. In intraoperative specimens of HNSCC, this algorithm precisely defined the borders of the cancerous regions; these corresponded with those determined by examining histologic sections. The procedure took approximately 5 min.
Conclusion: This diagnostic system, based on machine learning, enables accurate discrimination of cancerous regions and has the potential to provide rapid intraoperative assessment of HNSCC margins.

DOI： 10.1016/j.oraloncology.2017.11.008

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

In this paper, we briefly review the remote mass spectrometric techniques that are viable to perform "endoscopic mass spectrometry," i.e., in-situ and in-vivo MS analysis inside the cavity of human or animal body. We also report our experience with a moving string sampling probe for the remote sample collection and the transportation of adhered sample to an ion source near the mass spectrometer. With a miniaturization of the probe, the method described here has the potential to be fit directly into a medical endoscope.

DOI： 10.5702/massspectrometry.S0070

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry  

At present, endoscopy relies almost exclusively on optical microscopy and the accurate analysis such as MS interrogation is performed ex situ using biopsy. In this work, a novel probing system is developed to perform in situ and in vivo endoscopic mass spectrometry using a moving string for the sampling and transportation of material. A prototype of a mass spectrometric endoscope is constructed using an industrial endoscope and a commercial mass spectrometer. The sampling system consists of a moving cotton thread driven by motorized pulleys. When the target surface is touched by the sampling probe, the cotton thread "wipes" and transports the adhered sample to the ion source. Depending on the target analytes, desorption electrospray and atmospheric pressure chemical ionization sources are employed interchangeably for the desorption and ionization. The surface under analysis is not subjected to heat, organic solvents, high voltage or charged droplets. In situ endoscopic MS of a living mouse and surface analysis inside a volunteer subject's mouth are demonstrated.

DOI： 10.1039/c7an00650k

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

The relationship between hepatitis B virus (HBV) infection and lipid accumulation remains largely unknown. In this study, we investigated the effect of HBV propagation on lipid droplet growth in HBV-infected cells and HBV-producing cell lines, HepG2.2.15 and HBV-inducible Hep38.7-Tet. The amount of intracellular triglycerides was significantly reduced in HBV-infected and HBV-producing cells compared with HBV-lacking control cells. Electron and immunofluorescent microscopic analyses showed that the average size of a single lipid droplet (LD) was significantly less in the HBV-infected and HBV-producing cells than in the HBV-lacking control cells. Cell death-inducing DFF45-like effectors (CIDEs) B and C (CIDEB and CIDEC), which are involved in LD expansion for the improvement of lipid storage, were expressed at a significantly tower level in HBV-infected or HBV-producing cells than in HBV-lacking control cells, while CIDEA was not detected in those cells regardless of HBV production. The activity of the CIDEB and CIDEC gene promoters was impaired in HBV-infected or HBV-producing cells compared to HBV-lacking control cells, while CIDEs potentiated HBV core promoter activity. The amount of HNF4 alpha, that can promote the transcription of CIDEB was significantly tower in HBV-producing cells than in HBV-Lacking control cells. Knockout of CIDEB or CIDEC significantly reduced the amount of supernatant HBV DNA, intracellular viral RNA and nucleocapsid-associated viral DNA, while the expression of CIDEB or CIDEC recovered HBV production in CIDEB- or CIDEC-knockout cells. These results suggest that HBV regulates its own viral replication via CIDEB and CIDEC.

DOI： 10.1099/jgv.0.000813

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Mass Spectrometry Society of Japan  

In mass spectrometry, analytes must be released in the gas phase. There are two representative methods for the gasification of the condensed samples, i.e., ablation and desorption. While ablation is based on the explosion induced by the energy accumulated in the condensed matrix, desorption is a single molecular process taking place on the surface. In this paper, desorption methods for mass spectrometry developed in our laboratory: flash heating/rapid cooling, Leidenfrost phenomenon-assisted thermal desorption (LPTD), solid/solid friction, liquid/solid friction, electrospray droplet impact (EDI) ionization/desorption, and probe electrospray ionization (PESI), will be described. All the methods are concerned with the surface and interface phenomena. The concept of how to desorb less-volatility compounds from the surface will be discussed.

DOI： 10.5702/massspectrometry.S0059

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Mass Spectrometry Society of Japan  

Tissue samples from renal cell carcinoma patients were analyzed by electrospray droplet ion beam-induced secondary ion mass spectrometry (EDI/SIMS). Positively- and negatively-charged secondary ions were measured for the cancerous and noncancerous regions of the tissue samples. Although specific cancerous species could not be found in both the positive and negative secondary ion spectra, the spectra of the cancerous and noncancerous tissues presented different trends. For instance, in the m/z range of 500-800 of the positive secondary ion spectra for the cancerous tissues, the intensities for several m/z values were lower than those of the m/z+2 peaks (indicating one double bond loss for the species), whereas, for the noncancerous tissues, the inverse trend was obtained. The tandem mass spectrometry (MS/MS) was also performed on the tissue samples using probe electrospray ionization (PESI), and some molecular ions produced by PESI were found to be fragmented into the ions observed in EDI/SIMS analysis. When the positive secondary ion spectra produced by EDI/SIMS were analyzed by principal component analysis, the results for cancerous and noncancerous tissues were separated. The EDI/SIMS method can be applied to distinguish between a cancerous and a noncancerous area with high probability.

DOI： 10.5702/massspectrometry.A0053

PubMed

Language：Japanese   Publishing type：(MISC) Other article   Publisher：医学書院  

DOI： 10.11477/mf.1430200141

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

Melanin-concentrating hormone (MCH) receptor 1 (MCHR1) is a class A G-protein-coupled receptor (GPCR). The MCH-MCHR1 system has been implicated in the regulation of feeding, emotional processing, and sleep in rodents. Recent work revealed that MCHR1 is selectively expressed in neuronal primary cilia of the central nervous system. Cilia have various chemosensory functions in many types of cell, and ciliary dysfunction is associated with ciliopathies such as polycystic kidney disease and obesity. Although dynamic modulation of neuronal cilia length is observed in obese mice, the functional interaction of neuronal ciliary GPCR and its endogenous ligand has not yet been elucidated. We report here that MCH treatment significantly reduced cilia length in hTERT-RPE1 cells (hRPE1 cells) transfected with MCHR1. Quantitative analyses indicated that MCH-induced cilia shortening progressed in a dose-dependent manner with an EC50 lower than 1 nM when cells were treated for 6 h. Although the assembly and disassembly of primary cilia are tightly coupled to the cell cycle, cell cycle reentry was not a determinant of MCH-induced cilia shortening. We confirmed that MCH elicited receptor internalization, Ca2+ mobilization, ERK and Akt phosphorylation, and inhibition of cyclic AMP accumulation in MCHR1-expressing hRPE1 cells. Among these diverse pathways, we revealed that Gi/o-dependent Akt phosphorylation was an important component in the initial stage of MCH-induced cilia length shortening. Furthermore, induction of fewer cilia by Kif3A siRNA treatment significantly decreased the MCH-mediated phosphorylation of Akt, indicating the functional importance of the MCHR1-Akt pathway in primary cilia. Taken together, the present data suggest that the MCH-MCHR1 axis may modulate the sensitivity of cells to external environments by controlling the cilia length. Therefore, further characterization of MCHR1 as a ciliary GPCR will provide a potential molecular mechanism to link cilia length control with obesity. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cellsig.2016.02.018

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl) cycloheximide impaired the formation of a homo-or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

DOI： 10.1038/srep16699

Web of Science

PubMed

Language：Japanese   Publishing type：(MISC) Other article  

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry  

The present paper describes the application of solid probe assisted nanoelectrospray ionization mass spectrometry (SPA-nanoESI-MS) for the direct analysis of samples in solid or dried form. The experimental procedure is simple and requires a metallic needle to touch the sample surface followed by inserting the needle into a solvent preloaded nano-capillary. A number of real-world samples in solid or dried form comprising proteins, drugs in tablets, illicit drugs in dried urine, and lipids in tissues were analyzed to evaluate the applicability of the technique in diverse fields. This technique can produce high quality mass spectra without clogging the capillary tip. The quantitative aspect of this technique was evaluated using morphine from dried urine. In addition, biofluids/biomolecules captured on the needle tip and stored for several days at room temperature produced almost similar spectra to those obtained for fresh samples of cancerous and noncancerous tissues. The high sensitivity, versatile applicability and capability of dried sample analysis extend the scope of SPA-nanoESI to new ventures like bioanalytical and forensic analysis as well as clinical diagnosis.

DOI： 10.1039/C4AY02607A

Web of Science

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Mass Spectrometry Society of Japan  

Ambient ionization mass spectrometry is one of the most challenging analytical tools in the field of biomedical research. We previously demonstrated that probe electrospray ionization mass spectrometry (PESI-MS) could potentially be used in the rapid diagnosis of cancer. Although this technique does not require a tedious sample pretreatment process, it was not possible for our previously reported setup to be applied to cases involving the direct sampling of tissues from living animal and large animal subjects, because there would not be enough room to accommodate the larger bodies juxtaposed to the ion inlet. To make PESI-MS more applicable for the real-time analysis of living animals, a long auxiliary ion sampling tube has been connected to the ion inlet of the mass spectrometer to allow for the collection of ions and charged droplets from the PESI source (hereafter, referred to as non-proximate PESI). Furthermore, an additional ion sampling tube was connected to a small diaphragm pump to increase the uptake rate of air carrying the ions and charged droplets to the ion inlet. This modification allows for the extended ion sampling orifice to be positioned closer to the specimens, even when they are too large to be placed inside the ionization chamber. In this study, we have demonstrated the use of non-proximate PESI-MS for the real-time analysis for biological molecules and pharmacokinetic parameters from living animals.

DOI： 10.5702/massspectrometry.S0048

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

A metal wire-inserted disposable gel-loading tip was examined as an electrospray emitter. Its performance was similar to that of conventional electrospray ionization (ESI) with a relatively low flow rate (similar to 100 nL/min) and without the need for solvent pumps. It was also used as an emitter for solid probe-assisted ESI (SPA-ESI) (e.g., biofluid Was sampled from the biological tissue by a needle and was inserted into the solvent-preloaded gel-loading tip). Selective detection of lipids and proteins, such alpha and beta chains of hemoglobin could be accomplished by choosing appropriate solvents. A suitable protocol for cancer diagnosis was established by this method. A good figure of merit of this method is its applicability to biological tissue diagnostics with high cost efficiency and on a disposable basis.

DOI： 10.1021/ac403261s

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

This study presents a novel direct analysis strategy for rapid mass spectrometric profiling of biochemicals in real-world samples via a direct sampling probe (DSP) without sample pretreatments. Chemical modification is applied to a disposable stainless steel acupuncture needle to enhance its surface area and hydrophilicity. After insertion into real-world samples, biofluid can be attached on the DSP surface. With the presence of a high DC voltage and solvent vapor condensing on the tip of the DSP, analyte can be dissolved and electrosprayed. The simplicity in design, versatility in application aspects, and other advantages such as low cost and disposability make this new method a competitive tool for direct analysis of real-world samples.

DOI： 10.1007/s13361-013-0697-7

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Real-time analyses of hepatocellular carcinoma were performed in living mice to assess the applicability of probe electrospray ionization-mass spectrometry (PESI-MS) in medical diagnosis. The number of peaks and the abundance of ions corresponding to triacylglycerols (TAGs) were higher in cancerous tissues than in noncancerous tissues. Multiple sequential scans of the specimens were performed along a predetermined line extending over the noncancerous region to detect the boundary of the cancerous region. Our system successfully discriminated the noncancerous and cancerous tissues based on the intensities of the TAG ions. These results highlight the potential application of PESI-MS for clinical diagnosis in cancer. (c) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2013.06.017

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Melanin-concentrating hormone (MCH) is the natural peptide ligand for MCHR1 and MCHR2, which belong to the G protein-coupled receptor (GPCR) superfamily. The MCH-MCHR1 system is involved in the regulation of feeding, energy homeostasis and emotional processing in rodents. Recently, MCHR1 expression was discovered in neuronal immotile primary cilia of the central nervous system in mice. The cilium has an important chemosensory function in many types of cell and ciliary dysfunction is associated with cliopathies such as polycystic kidney disease, retinal dystrophy, and obesity. The targeting sequence of ciliary membrane proteins is thought to be unique. Although these sequences have been predicted in the cytoplasmic third loop and/or C-terminus of GPCRs, little is known about the characteristics of MCHR1. We thus explored the molecular mechanisms of MCHR1 targeting by transiently expressing a series of MCHR1 mutants into ciliated hRPE1 cells and evaluated the effects of these mutations on the ciliary localization of the heterologous receptor. This approach demonstrated that an Ala-to-Gly mutation (A242G) within the third intracellular loop induced a significant reduction in ciliary localization of the receptor without affecting the ciliogenesis. In contrast, no C-terminal truncation mutant had any effect on ciliary localization or cilia length. This study provides a potential molecular link between defective cilia and clinical manifestations such as obesity. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen

Web of Science

PubMed

Language：Japanese   Publishing type：(MISC) Other article   Publisher：オーム社/島津評論編集部  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry  

We have examined several combinations of solvents and probes with the aim of optimizing the ionization conditions for biomolecules e. g., proteins, peptides and lipids by negative mode probe electrospray ionization mass spectrometry (PESI-MS). With the data presented in this study, negative-mode PESI-MS can be considered as a potential tool for biomolecular analysis and cancer diagnostics because of its simplicity in instrumental configuration. A sharper sampling probe was found to be better for obtaining high quality mass spectra because it can generate stable electrospray without the occurrence of gas breakdown. Although the best conditions may depend on each sample, aqueous organic solvent solutions, especially isopropanol-H2O (1/1) with a pH of &gt;= 7, are shown to be preferable for negative-mode PESI-MS, which was successfully applied to colon cancer diagnosis.

DOI： 10.1039/c3an365

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

We have examined several combinations of solvents with the aim of optimizing the ionization conditions for molecular diagnosis of malignant tumours by PESI-MS. Although the best conditions may depend on the actual species in the sample, the optimal conditions for renal cell carcinoma (RCC) were achieved by using alcohols. PESI-MS successfully delineated the differential expression of phospholipids (PCs) and triacylglycerols (TAGs) in noncancerous and RCC tissues by using these solvent systems. This study paves the way for the application of PESI-MS in medical samples.

DOI： 10.1007/s13361-012-0462-3

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry  

To perform remote and direct sampling for mass spectrometry, solid probe assisted nanoelectrospray ionization (SPA-nanoESI) has been newly developed. After capturing the sample on the tip of the needle by sticking it to the biological tissue, the needle was inserted into the solvent-preloaded nanoESI capillary from the backside. NanoESI gave abundant ion signals for human kidney tissues and the liver of a living mouse. The method is easy to operate and versatile because any biological specimen could be sampled away from the mass spectrometer. Minimal invasiveness is another merit of this method.

DOI： 10.1039/c2an36006c

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

Immediate diagnosis of human specimen is an essential prerequisites in medical routines. This study aimed to establish a novel cancer diagnostics system based on probe electrospray ionization-mass spectrometry (PESI-MS) combined with statistical data processing. PESI-MS uses a very fine acupuncture needle as a probe for sampling as well as for ionization. To demonstrate the applicability of PESI-MS for cancer diagnosis, we analyzed nine cases of clear cell renal cell carcinoma (ccRCC) by PESI-MS and processed the data by principal components analysis (PCA). Our system successfully delineated the differences in lipid composition between non-cancerous and cancerous regions. In this case, triacylglycerol (TAG) was reproducibly detected in the cancerous tissue of nine different individuals, the result being consistent with well-known profiles of ccRCC. Moreover, this system enabled us to detect the boundaries of cancerous regions based on the expression of TAG. These results strongly suggest that PESI-MS will be applicable to cancer diagnosis, especially when the number of data is augmented.

DOI： 10.1007/s13361-012-0447-2

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Lifescience Global  

Recent innovations in mass spectrometry make it possible to diagnose malignant tumors through a rapid, nondestructive and less-expensive way. One of the important facets in this achievement lies in the development of several superior ionization techniques that are essentially derivatives of two authentic methods
matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). In this review article, we introduce a novel cancer diagnostic system based on probe electrospray ionization (PESI) and logistic regression algorithm. This method uses a very fine needle with a tip diameter of several hundreds nm, which serves as a sampling as well as ionization device. Only a few picolitre (pL) of sample are sufficient to acquire mass spectra for making a diagnosis. Furthermore, as this method does not require any sample pre-treatments that often disorganize the original molecular composition of samples, it has a potential in delineating substances that have been missed by conventional analytical methods. By implementing this technology, we have successfully made in situ diagnosis of malignant tumors in human tissues and in living animals. On the other hand, there are two promising and competitive diagnostic methods
one is desorption ionization mass spectrometry (DESI-MS), and the other is rapid evaporation ionization mass spectrometry (REI-MS) coupled with electrical surgical knife. They are also promising technologies in the new era of analytical oncology. We compare these three methods briefly and attempt to give a new perspective in cancer diagnostics. © 2012 Takeda et al.

DOI： 10.6000/1927-7229.2012.01.01.11

Scopus

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Myelination is an essential prerequisite for the nervous system to transmit an impulse efficiently by a saltatory conduction. In the peripheral nervous system (MIS), Schwann cells (SCs) engage in myelination. However, a detailed molecular mechanism underlying myelination still remains unclear. In this study, we hypothesized that the primary cilia of SCs are the regulators of Hedgehog (Hh) signaling-mediated myelination. To confirm our hypothesis, we used mouse dorsal root ganglion (DRG)/SC co-cultures, wherein the behavior of SCs could be analyzed by maintaining the interaction of SCs with DRG neurons. Under these conditions, SCs had primary cilia, and Hh signaling molecules accumulated on the primary cilia. When the SCs were stimulated by the addition of desert hedgehog or smoothened agonist, formation of myelin segments on the DRG axons was facilitated. On the contrary, upon administration of cyclopamine, an inhibitor of Hh signaling, myelin segments became comparable to those of controls. Of note, the ratio of SCs harboring primary cilium reached the highest point during the early phase of myelination. Furthermore, the strongest effects of Hh on myelination were encountered during the same stage. These results collectively indicate that Hh signaling regulates myelin formation through primary cilia in the MIS. (C) 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.diff.2011.10.006

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Probe electrospray ionization (PESI) is one of the most promising methods in biochemical analysis because it enables us to analyze biological samples very quickly without any special pretreatment. Moreover, due to the small size of the needle tip, this method has advantages such as low invasiveness to the samples, making it possible to analyze the biological profiles of organs or tissues in living animal in situ. In this study, we performed a real-time analysis of living mice that delineates the differences in lipid composition of hepatocytes between normal and steatotic mice. In steatotic mice, the number of peaks and the ion abundance for triacylglycerols were much higher compared with those of control mice. All mice used in this study tolerated the procedure well and survived for more than a month until sacrificed for further analysis. To test a potential for medical diagnosis, human tumor tissues were also measured and we obtained discriminative results judged as useful for diagnostics. These results pave the way into the application of PESI to the in vivo analysis of biological molecules. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2011.06.020

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LIMITED  

Sensing extracellular milieu is a fundamental requirement of cells. To facilitate and specify sensory reception, mammalian cells develop an antenna-like structure denoted as the primary cilia. Nearly all interphase and nondividing cells in vertebrates have a single, nonmotile seemingly unspecialized cilium (called a primary cilium). In the central nervous system, astrocytes express primary cilia, but their function in astrocytes has not been examined. Recent studies have shown that primary cilia unite receptors and the machinery of signal-transduction components, such as Wnt and Hedgehog (Rh) signaling cascades. Although, Hh signaling cascades are known to be activated in various cells during development, their physiological functions in the adult nervous system, especially in glial cells, are still unknown. In this study, we reveal that glial primary cilia receive the Hh signal and regulate the survival of astrocytes under stressed conditions such as starvation. Interestingly, increased astrocyte survival was reversed by knockdown of Ift20, which is one of the main components for building primary cilia. These results collectively indicate that the activation of Rh signaling in the primary cilia plays an important role in the survival of astrocytes under stressed conditions. (C) 2010 Wiley-Liss, Inc.

DOI： 10.1002/glia.21105

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LIMITED  

Electrospray droplet impact (EDI)/secondary ion mass spectrometry (SIMS) is a new desorption/ionization technique for mass spectrometry in which highly charged water clusters produced from the atmospheric-pressure electrospray are accelerated in vacuum by 10 kV and impact the sample deposited on the metal substrate. EDI/SIMS was shown to enhance intact molecular ion formation dramatically compared to conventional SIMS. EDI/SIMS has been successfully applied to the analysis of mouse brain without any sample preparation. Five types of lipids, i.e. phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), galactocerebroside (GC) and sulfatide (ST), were readily detected from mouse brain section. In addition, by EDI/SIMS, six different regions of the mouse brain (cerebral cortex, corpus callosum, striatum, medulla oblongata, cerebellar cortex and cerebellar medulla) were examined. While GCs and STs were found to be rich in white matter, PIs were rich in gray matter. Copyright (C) 2010 John Wiley & Sons, Ltd.

DOI： 10.1002/jms.1729

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2010.01.121

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LIMITED  

Although being an atmospheric pressure ion source, electrospray ionization (ESI) has rarely been used directly for ambient imaging mass spectrometry because the sample has to be introduced as liquid solution through the capillary. Instead of capillary, probe electrospray ionization (PESI), which has been developed recently, uses a solid needle as the sampling probe, as well as the electrospray emitter, and has been applied not only for liquid solutions but also for the direct sampling on wet samples. Biological tissues are composed of cells that contain 70-90% water, and when the surface is probed by the needle tip, the biological fluid adhering to the needle can be electrosprayed directly or assisted by additional solvent added onto the needle surface. Here, we demonstrate ambient imaging mass spectrometry of mouse brain section using PESI, incorporated with an auxiliary heated capillary sprayer. The solvent vapor generated from the sprayer condensed on the needle tip, re-dissolving the adhered sample, and at the same time, providing an indirect means for needle cleaning. The histological sections were prepared by fixation using paraformaldehyde, and the spatial analysis was automated by maintaining an equal sampling depth into the sample in addition to raster scan. Phospholipids and galactosylceramides were readily detected from the mouse brain section in the positive ion mode, and were mapped with 60 gm lateral resolution to form mass spectrometric images. Copyright (C) 2009 John Wiley & Sons, Ltd.

DOI： 10.1002/jms.1632

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LIMITED  

Probe electrospray ionization (PESI) is a recently developed technique that uses a fine solid needle as a probe for sampling biological materials. In this study, we quantified the volume of liquid sample picked up by the solid needle with the tip diameter of similar to 700 nm and the apex angle of similar to 60 degrees. The amounts of low-viscosity samples (rat urine) loaded on the tip of the needle by a single stroke were 0.35 +/- 0.09 pl. Interestingly, the amount of liquid adhered to the tip did not significantly depend on the protein concentration, but viscosity and surface tension of the sample. Under these conditions, we successfully obtained mass spectra for each biological sample. Copyright (C) 2009 John Wiley & Sons, Ltd.

DOI： 10.1002/jms.1576.

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2009.03.039.

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER DISTRIBUTION CENTER  

Although the gap junction or connexin (Cx) is considered to be a tumor-suppressor, it is also required for tumor promotion. Therefore, we examined hepatic gap junctions in hepatocarcinogen-resistant (DRH) rats. Specifically, we investigated gap junction structure and Cx32 expression during normal conditions and in response to a hepatocarcinogen, 3&apos;-methyl-4-dimethylaminoazobenzene (3&apos;-MeDAB). On a basal diet without 3&apos;-MeDAB, hepatic gap junctions and Cx32 protein expression were greater in DRH rats than in control Donryu rats, as evidenced by morphometry, immunohistochemistry and immunoblotting. On a diet containing 3&apos;-MeDAB, gap junctions and expressed Cx32 were increased significantly in Donryu rats, but not in DRH rats. In this condition, Donryu rats lost weight but DRH rats increased relative liver weight. After 3&apos;-MeDAB treatment, cathepsin D expression in hepatocytes was significantly increased only in Donryu rats, indicating that DRH rats were less susceptible to 3&apos;-MeDAB. The abundance of mitogen-activated protein kinase, some constituent of which might be associated with the degree of Cx protein phosphorylation, was reduced to a greater extent in Donryu than in DRH rats after 3&apos;-MeDAB treatment. The resistance of DRH rats to carcinogenesis may be due partially to their stabilized gap junctions, which could coordinate metabolic coupling to evade 3&apos;-MeDAB toxicity.

DOI： 10.1007/s00418-008-0473-0

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Klotho mutant mice, defective in the klotho gene, develop multiple age-related disorders with very short lifespans. Introduction of the exogenous klotho gene into these mutant mice leads to an improvement in their phenotypes, while overexpression of this gene in wild-type mice significantly extends their lifespan. These observations suggest that the klotho gene/protein has an anti-aging function. Since there have been only a few reports with some disagreement about results on the CNS of the mutant mice, we tried to clarify whether the CNS neurons generate aging-like features, even in premature stages, using biochemical and morphological approaches. Results obtained from the mutant mice, when compared with wild-type mice, were as follows. Neurofilaments (NFs) were increased significantly in axons, with the subunit proteins showing a significant enhancement in phosphorylation or expression of NF-H or NF-L, respectively. Microtubules in Purkinje cell dendrites were closer to each other, and in the CNS tissue tubulin was unaltered, but microtubule-associated protein (MAP) 2 was significantly reduced in expression. Neuronal cellular organelles were morphologically disordered. Lysosomes, cathepsin D and light chain 3 of MAP1A/B (LC3) were augmented with the appearance of putative autophagy-related structures. Antiapoptotic Bcl-xL and proapoptotic Bax were reduced and enhanced, respectively, and mitogen-activated protein kinase was reduced. Synapse-related proteins and structures were decreased. Neuronal degeneration was evident in hippocampal pyramidal cells, and possibly in Purkinje cells. Astrocytic glial filaments and glial fibrillary acidic protein were increased in density and expression, respectively. Together, the CNS neuronal alterations in klotho mutant mice were quite similar to those found in aged animals, including even premature death, so this mouse should be a more appropriate animal model for CNS aging than those previously reported. (c) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2008.01.032.

Web of Science

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER DISTRIBUTION CENTER  

"Autophagy" is a highly conserved pathway for degradation, by which wasted intracellular macromolecules are delivered to lysosomes, where they are degraded into biologically active monomers such as amino acids that are subsequently re-used to maintain cellular metabolic turnover and homeostasis. Recent genetic studies have shown that mice lacking an autophagy-related gene (Atg5 or Atg7) cannot survive longer than 12 h after birth because of nutrient shortage. Moreover, tissue-specific impairment of autophagy in central nervous system tissue causes massive loss of neurons, resulting in neurodegeneration, while impaired autophagy in liver tissue causes accumulation of wasted organelles, leading to hepatomegaly. Although autophagy generally prevents cell death, our recent study using conditional Atg7-deficient mice in CNS tissue has demonstrated the presence of autophagic neuron death in the hippocampus after neonatal hypoxic/ischemic brain injury. Thus, recent genetic studies have shown that autophagy is involved in various cellular functions. In this review, we introduce physiological and pathophysiological roles of autophagy.

DOI： 10.1007/s00418-008-0406-y

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Landes Bioscience10.4161/auto.2744  

Atg4B, a mammalian homologue of yeast Atg4, has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To better understand the role of Atg4B in rat tissue cells, we prepared antibodies against Atg4B, and PC 12 cells in which the expression of Atg4B was knocked down by RNA interference. In the RNA interference-treated PC 12 cells, for which the expression of Atg4B was 10% of wild-type PC 12 cells, the expression of cytosolic LC3-1 was similar to that in wild-type cells. Knockdown cell lysates, however, suppressed the cleavage of recombinant proLC3 to LC3-1. Moreover, the expression of Atg4B protein and mRNA was ubiquitous in rat tissues; however, the expression levels were not identical, but were dependent on the tissue, with the expression high in brain and testicular tissue, and low in muscular and heart tissue. In brain tissue, the expression of Atg4B protein and mRNA was higher in neurons, especially in the cerebellum and olfactory bulb, as evidenced by immunohistochemistry and in situ hybridization. These lines of evidence suggest that Atg4B plays a major role in the processing of LC3 and is widely distributed in rat tissues. In particular, in brain tissues, autophagy may be deeply associated with the metabolism of neurons, especially in the cerebellum.

DOI： 10.4161/auto.2744

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Investigative Pathology  

In cathepsin D-deficient (CD-/-) and cathepsins B and L double-deficient (CB-/-CL-/-) mice, abnormal vacuolar structures accumulate in neurons of the brains. Many of these structures resemble autophagosomes in which part of the cytoplasm is retained but their precise nature and biogenesis remain unknown. We show here how autophagy contributes to the accumulation of these vacuolar structures in neurons deficient in cathepsin D or both cathepsins B and L by demonstrating an increased conversion of the molecular form of MAP1-LC3 for autophagosome formation from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). in both CD-/- and CB-/-CL-/- mouse brains, the membrane-bound LC3-II form predominated whereas MAP1-LC3 signals accumulated in granular structures located in neuronal perikarya and axons of these mutant brains and were localized to the membranes of autophagosomes, evidenced by immunofluorescence microscopy and freeze-fracture-replica immunoelectron microscopy. Moreover, as in CD-/- neurons, autofluorescence and subunit c of mitochondrial ATP synthase accumulated in CB-/-CL-/- neurons. This suggests that not only CD-/- but also CB-/-CL-/- mice could be useful animal models for neuronal ceroid-lipofuscinosis/Batten disease. These data strongly argue for a major involvement of autophagy in the pathogenesis of Batten disease/lysosomal storage disorders.

DOI： 10.1016/S0002-9440(10)61253-9

Web of Science

PubMed

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：東京ビッグサイト  

Language：English   Presentation type：Oral presentation(invited, special)  

Venue：株式会社島津製作所  

Event date： 2019.7

Language：English   Presentation type：Oral presentation(invited, special)  

Event date： 2019.7

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：株式会社島津製作所  

Event date： 2019.5

Language：Japanese   Presentation type：Poster presentation  

Event date： 2019.5

Language：Japanese   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：つくば国際会議場エポカルつくば、茨城県、つくば市  

Language：English   Presentation type：Poster presentation  

Venue：つくば国際会議場エポカルつくば、茨城県、つくば市  

Language：Japanese   Presentation type：Poster presentation  

Venue：朱鷺メッセ 新潟コンベンションセンター  

Event date： 2019.1

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：JST東京本部別館1Fホール、東京都、市ヶ谷  

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：JST東京本部別館1Fホール、東京都、市ヶ谷  

Language：English   Presentation type：Poster presentation  

Venue：タワーホール船堀、東京都、江戸川区  

Event date： 2018.6

Language：English   Presentation type：Poster presentation  

Event date： 2018.3

Language：Japanese   Presentation type：Poster presentation  

Venue：日本医科大学武蔵境校舎・日本獣医生命科学大学  

Language：Japanese   Presentation type：Poster presentation  

Venue：日本医科大学武蔵境校舎・日本獣医生命科学大学  

Event date： 2017.10

Language：Japanese   Presentation type：Poster presentation  

Venue：金沢東急ホテル  

Language：Japanese   Presentation type：Poster presentation  

Venue：金沢東急ホテル  

Event date： 2017.9

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：一橋講堂  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：一橋講堂（学術総合センター2F）  

Event date： 2017.5

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：つくば国際会議場エポカルつくば、茨城県、つくば市  

Event date： 2017.3

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：長崎大学坂本キャンパス  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：長崎大学坂本キャンパス  

Event date： 2017.2

Language：Japanese   Presentation type：Poster presentation  

Venue：金沢東急ホテル  

Language：Japanese   Presentation type：Poster presentation  

Venue：金沢東急ホテル  

Event date： 2016.5

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：ホテル阪急エキスポパーク、大阪府、吹田市  

Event date： 2016.3

Language：Japanese   Presentation type：Poster presentation  

Venue：ビッグバレットふくしま  

Language：Japanese   Presentation type：Poster presentation  

Venue：ビッグパレットふくしま、福島県、郡山市  

Event date： 2015.7

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：神戸国際会議場、兵庫県、神戸市  

Event date： 2015.6

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：つくば国際会議場エポカルつくば、茨城県、つくば市  

Event date： 2015.3

Language：Japanese   Presentation type：Poster presentation  

Language：Japanese   Presentation type：Poster presentation  

Venue：神戸国際会議場  

Event date： 2014.9

Language：English   Presentation type：Poster presentation  

Language：Japanese   Presentation type：Poster presentation  

Venue：パシフィコ横浜、神奈川県、横浜市  

Event date： 2014.8

Language：Japanese   Presentation type：Poster presentation  

Event date： 2014.5

Language：Japanese   Presentation type：Oral presentation(general)  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：アクトシティ浜松研修交流センター、静岡県、浜松市  

Event date： 2014.5

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：ホテル阪急エキスポパーク、大阪  

Event date： 2014.3

Language：Japanese   Presentation type：Poster presentation  

Venue：自治医科大学キャンパス、栃木県、下野市  

Language：Japanese   Presentation type：Poster presentation  

Venue：自治医科大学キャンパス、栃木県、下野市  

Event date： 2014.3

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：大阪 (大阪大学理学研究科H棟)  

Language：Japanese   Presentation type：Oral presentation(invited, special)  

Venue：大阪 (大阪大学理学研究科H棟)、大阪府、豊中市  

Event date： 2013.9

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：つくば国際会議場エポカルつくば、茨城県、つくば市  

Event date： 2013.7

Language：English   Presentation type：Poster presentation  

Venue：Taipei International Convention Center, Taipei, Taiwan  

Event date： 2013.3

Language：Japanese   Presentation type：Poster presentation  

Venue：サンポートホール高松・かがわ国際会議場、香川県  

Language：Japanese   Presentation type：Poster presentation  

Venue：サンポートホール高松・かがわ国際会議場、香川県  

Event date： 2012.10

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：じゅうろくプラザ、岐阜  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：じゅうろくプラザ、岐阜  

Event date： 2012.9

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：名古屋国際会議場  

Event date： 2012.9

Language：English   Presentation type：Poster presentation  

Event date： 2012.9

Language：Japanese   Presentation type：Poster presentation  

Venue：ベルクラシック甲府、山梨  

Language：Japanese   Presentation type：Poster presentation  

Venue：ベルクラシック甲府、山梨  

Event date： 2012.6

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：東京大学理学部  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：東京大学理学部  

Event date： 2012.3

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨大学、山梨  

Language：Japanese   Presentation type：Oral presentation(general)  

Venue：山梨大学、山梨  

Event date： 2011.9

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：横浜  

Event date： 2011.9

Language：English   Presentation type：Poster presentation  

Event date： 2011.9

Language：English   Presentation type：Oral presentation(general)  

Language：English   Presentation type：Oral presentation(general)  

Venue：ホテル阪急エキスポパーク、大阪  

Event date： 2011.6

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：北海道大学、札幌  

Event date： 2011.6

Language：English   Presentation type：Poster presentation  

Event date： 2011.3

Language：Japanese   Presentation type：Poster presentation  

Venue：パシフィコ横浜、横浜  

Language：Japanese   Presentation type：Poster presentation  

Venue：パシフィコ横浜, 神奈川  

Event date： 2010.12

Language：English   Presentation type：Poster presentation  

Venue：Philadelphia, PA  

Event date： 2010.9

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：神戸コンベンションセンター  

Event date： 2010.6

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：つくば国際会議場 エポカルつくば  

Event date： 2010.5

Language：English   Presentation type：Poster presentation  

Venue：Salt Lake City, Utah  

Event date： 2010.3

Language：Japanese   Presentation type：Poster presentation  

Venue：岩手県民会館  

Language：Japanese   Presentation type：Poster presentation  

Venue：岩手県民会館  

Event date： 2009.9

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：名古屋国際会議場  

Event date： 2009.5

Language：Japanese   Presentation type：Poster presentation  

Venue：大阪国際交流センター  

Language：Japanese   Presentation type：Poster presentation  

Venue：大阪国際交流センター  

Event date： 2009.3

Language：Japanese   Presentation type：Poster presentation  

Venue：岡山理科大学  

Language：Japanese   Presentation type：Poster presentation  

Venue：岡山理科大学  

Event date： 2007.3

Language：Japanese   Presentation type：Poster presentation  

Venue：大阪国際会議場  

Language：Japanese   Presentation type：Poster presentation  

Venue：大阪国際会議場  

Event date： 2006.10

Language：English   Presentation type：Poster presentation  

Event date： 2006.7

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：国立京都国際会館  

Event date： 2006.3

Language：Japanese   Presentation type：Poster presentation  

Venue：北里大学  

Language：Japanese   Presentation type：Poster presentation  

Venue：北里大学  

Event date： 2005.11

Language：English   Presentation type：Poster presentation  

Event date： 2005.4

Language：English   Presentation type：Poster presentation  

Venue：Il ciocco, Barga, Italy  

Event date： 2005.3

Language：Japanese   Presentation type：Poster presentation  

Venue：富山医科薬科大学  

Language：Japanese   Presentation type：Poster presentation  

Venue：富山医科薬科大学  

Event date： 2004.8

Language：English   Presentation type：Poster presentation  

Language：English   Presentation type：Poster presentation  

Venue：国立京都国際会館、京都府、京都市  

Event date： 2003.4

Language：Japanese   Presentation type：Poster presentation  

Venue：アクロス福岡  

Language：Japanese   Presentation type：Poster presentation  

Venue：アクロス福岡  

Applicant：国立大学法人山梨大学

Application no：特願2017-079391  Date applied：2017.4

Announcement no：特開2018-181600  Date announced：2018.11

Patent/Registration no：特許7025621号  Date registered：2022.2 

Country of applicant：Domestic  

Applicant：国立大学法人山梨大学

Application no：特願2015-078992  Date applied：2015.4

Announcement no：特開2016-200435  Date announced：2016.12

Patent/Registration no：特許6715451号  Date registered：2020.6 

Country of applicant：Domestic  

Applicant：株式会社島津製作所、田邉國士、国立大学法人山梨大学

Application no：特願2012-186490  Date applied：2012.8

Announcement no：特開2014-44110  Date announced：2014.3

Patent/Registration no：特許6189587号  Date registered：2017.8 

Country of applicant：Domestic  

Award type：Award from Japanese society, conference, symposium, etc. 

画期的イオン化法を用いた質量分析型迅速がん診断装置の開発

Award type：Award from Japanese society, conference, symposium, etc. 

探針エレクトロスプレーイオン化法を用いた生体マススペクトル解析

シュワン細胞の髄鞘形成におけるHhシグナルとレチノイドの相互関係の解明