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By developing the equipment and constructing sample-specific pretreatment methods, we have developed a two-dimensional electrophoresis (2DE) method for the social implementation with (1) high-throughput, (2) high sensitivity, and (3) high reproducibility performance. Furthermore, by taking advantage of the fact that the data obtained by 2DE are images that are compatible with machine learning and the high-throughput performance of the previously developed method, we have succeeded in AI-aided proteomic disease diagnosis using 2DE images of sepsis patient sera. In machine learning, the accuracy and quality (the types of things that can be discriminated) improve as the amount of data increases, and since the 2DE system can in principle be miniaturized, and the practical application of the system can foster big data, it is expected that proteomics will be implemented in society using general-purpose 2DE in the near future. This paper introduces the results of the authors' research and development to date and discusses new developments in 2DE driven by open innovation accelerated by AI. [ABSTRACT FROM AUTHOR]

Phosphorylation is an important post-translational modification of proteins involved in a wide variety of biological processes. The phosphorylation status reflects the relative activities of protein kinases and phosphatases, resulting in the diversity of proteins in the cell. Changes in phosphorylation status may also affect protein function by changing its conformational state, stability, or interactions with other molecules. Various analytical strategies have been proposed to analyze the phosphorylation status of proteins. Here, we will introduce powerful analytical tools for phosphoproteomics based on electrophoresis and mass spectrometry to obtain information on the protein phos-phorylation involved in biological processes. Together, we believe that these analytical methods have the potential to accelerate the progress of proteomics research. [ABSTRACT FROM AUTHOR]

Copyright of Journal of the Society of Japanese Women Scientists / Nihon Josei Kagakusha no Kai Gakujutsushi is the property of Society of Japanese Women Scientists and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Food Hygienic & Safety Science / Shokuhin Eiseigaku Zasshi is the property of Japanese Society for Food Hygiene & Safety and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Genusmalassezia are now divided to fourteen species. Different species will start or aggravate different skin diseases. In the seborrheic dermatitis, M. restricta will play an important role, in the atopic dermatitis, M. globosa and/or M. restricta are maior cutaneous microflora. The availability of new tools such as genomic and proteomic analyses has begun to provide a new insight into the pathogenetic mechanisms involved. [ABSTRACT FROM AUTHOR]

Although 82% of the cerebrospinal fluid (CSF) is localized in the subarachnoid space, the function of the blood-arachnoid barrier (BAB) has not been elucidated. We found an abundance of drug efflux transporter proteins in the BAB. Moreover, several transporter proteins, including organic anion transporters and organic cation transporters have been found in the BAB. Significant in vivo transport activity of the BAB has been observed, suggesting that the BAB plays a pivotal role in the CNS barrier. Using quantitative targeted absolute proteomic analysis, we determined the similarities and differences in transport function and tight junction formation among the BBB, BAB, blood-CSF barrier (BCSFB), and blood-spinal cord barrier (BSCB). Furthermore, we proposed several hypotheses regarding the molecular mechanism underlying CNS barrier disintegration in the presence of oxidative stress, TNF-α release, and multiple sclerosis.

Human epidermal growth factor receptor 2 (Her2)-targeting antibodies and anti-hormone therapy are effective for most breast cancer patients. However, such approaches are not viable with resistant cases or in triple-negative breast cancer (TNBC) patients, given the lack of Her2 and estrogen and progesterone receptors in these patients. Thus, new drug targets are urgently required. From this perspective, we searched for novel drug targets using proteomic analysis, and identified Eph receptor A10 (EphA10), which is elevated in breast cancer cells as compared to normal breast tissue. Here, we evaluated the potential of EphA10 as a drug target by analyzing its protein expression profile/function in cancer cells, and then by using an anti-EphA10 antibody to treat EphA10-expressing tumor-bearing mice. Protein expression profile analysis showed that EphA10 was expressed in various breast cancer subtypes, including TNBCs, with no expression observed in normal tissues, apart from the testes. Moreover, functional analysis of the cancer cells revealed that ligand-dependent proliferation was observed in EphA10-expressed cancer cells. Thus, we developed our novel anti-EphA10 antibody, which binds to EphA10 with high specificity and affinity at the nanomolar level. Finally, therapeutic analysis indicated that tumor growth was significantly suppressed in the mAb-treated mice in a dose-dependent manner. These results suggest that the EphA10-targeting therapy may be a novel therapeutic option for the management of breast cancer, including in TNBCs which aren't currently treated with molecular-targeted agents. Consequently, we hope that these findings will contribute to the development of a new targeting therapy for refractory breast cancer patients.

Down syndrome, caused by the triplication of human chromosome 21, is the most frequent genetic cause of mental retardation. Mice with a segmental trisomy for mouse chromosome 16, which is orthologous to human chromosome 21, exhibit abnormalities similar to those in individuals with Down syndrome and therefore offer the opportunity for a genotype-phenotype correlation. In the current review, I present several mouse lines with trisomic regions of various lengths and discuss their usefulness for elucidating the mechanisms underlying Down syndrome-associated developmental cognitive disabilities. In addition, our recent comprehensive study attempting to identify molecules with disturbed expression in the brain of a mouse model of Down syndrome in order to develop a pharmacologic therapy for Down syndrome is described.

Nicotinamide adenine dinucleotide(NAD') is a classic coenzyme playing a critical role in cellular redox reactions. Emerging evidence demonstrates that NAD' and its key mediators, NAD+-dependent protein deacetylases (sirtuins), together regulate many important meta- bolic pathways including mitochondrial function. Thus, impaired NAD' biosynthesis is critically involved in the pathophysiology of aging and age-associated diseases. Importantly, administration of key NAD+ intermediates, such as nicotinamide mononucleotide(NMN) or nicotinamide riboside (NR), improves mitochondrial function and exerts remarkable therapeutic effects for various age-associated diseases, such as diabetes and cancer, in mice. In this review, we will summarize and discuss pathophysiological relevance and translational potential of NAD' biology and mitochondrial sirtuin(SIRT3) in age-associated diseases.

Amyloidosis is one of the most well-known protein-misfolding diseases caused by the deposition of insolu- ble amyloid fibrils in extracellular spaces. At least 31 amyloid fibril proteins have been identified. To elu- cidate the pathogenesis and diagnose the type of amyloidosis, mass spectrometric techniques, including liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization time of flight mass spectrometry, have been widely used for the analysis of biological samples, such as serum, urine, and tissues. We review new insights into the diagnosis and pathogenesis of amyloidosis using several mass spectrometric methods. [Review].

Our immune system resembles an intelligent security system, which continually monitors for foreign invaders (infectious diseases); however, in some cases, this system recognizes healthy parts as something harmful or foreign and then attacks them (autoimmune diseases). The defining characteristics of an autoimmune disease are the existence of T- and B-cell autoreactivity against self proteins (autoantigens). In addition to autoimmune diseases, aberrant host proteins that occur during a certain state of diseases (e.g., cancer) can be recognized as an autoantigen. Immune complexes (ICs) are produced during an immune response and may reflect some aspects of an ongoing immune response. Therefore, the identity of antigens incorporated into ICs provides the information that in the future may aid in the development of diagnosis and treatment strategies for autoimmune diseases, infection, cancer, and transplantation therapy, and this information might be more relevant than information on free antigens. We developed a novel proteomic strategy (immune complexome analysis) in which ICs are separated from serum, followed by direct tryptic digestion and nano-liquid chromatography-tandem mass spectrometry for the identification and profiling of antigens in circulating ICs. We applied this strategy to the analysis of circulating ICs in autoimmune diseases (rheumatoid arthritis, anti-neutrophil cytoplasmic antibody-associated vasculitis, Takayasu's arteritis, mixed connective tissue disease, dermatomyositis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus), infectious diseases and cancers. In this review, we mainly discuss the results for autoimmune diseases.

The incidence of prostate cancer is rapidly increasing in Japan. Currently, the biomarker of prostate cancer is widely used in clinical is serum PSA only. We need to develop novel molecular markers (biomarkers) that diagnose early prostate cancer so that we can treat appropriately. Recently, the whole genome sequencing analysis has advanced that has made finding of novel molecular markers easier. The best molecular markers identified using personalized genome information will be useful to select appropriate treatment. These therapeutic options for each patient will improve the survival rate. We give an outline about the latest molecular marker basic research with reference to the articles.

Various stresses on the heart, such as myocardial infarction and hemodynamic overload, activate the sympathetic nervous system and the renin-angiotensin system, ultimately reach the nuclei of cardiomyocytes, and change the pattern of gene expression associated with cardiac hypertrophy. Although present pharmacological therapy for heart failure targets such extracellular molecules, mortality due to heart failure is still high. A zinc finger protein, GATA4, is one of the hypertrophy-responsive transcription factors, forms a functional protein complex with an intrinsic histone acetyltransferase, p300, and regulates pathological cardiac hypertrophy. Disruption of this complex results in the inhibition of cardiac hypertrophy and heart failure in vivo. To establish a more effective therapy for heart failure, we have been analyzing a common nuclear pathway within cardiomyocytes. We identified 73 GATA4 binding proteins by tandem-affinity purification and mass spectrometric analysis. Noble GATA4 binding partners, such as cyclin-dependent kinase-9 (Cdk9: the core factor of positive transcription elongation factor b) and retinoblastoma-association protein 48/46 (RbAp48/46: the co-repressor complexes containing HDAC1/2), regulate the p300/GATA4-mediated signaling pathway and hypertrophic responses. Further analysis of p300/GATA4 complex is expected to identify target molecules for heart failure therapy.

Medical strategy for rheumatoid arthritis (RA) has markedly advanced in recent years. The introductions of biologics and methotrexate as an anchor drug have made it possible to not only suppress pain and inflammation (clinical remission), but also to inhibit joint destruction (structural remission), leading to cure of the disease. In order to achieve this target, it is the most important to diagnose RA early and promote disease remission. However, since the condition and pathology are diverse among patients, optimal treatment for each patient is desired (personalized medicine). Treatment should be performed under consideration of the disease state such as activity, prognosis regarding joint destruction, and complications. It is also important to clarify the patient characteristics, such as responsiveness to the drugs and risk of adverse effects. Biomarkers, such as proteomics and pharmacogenomics (genetic polymorphism, etc.), are indispensable for personalized medicine. We have established a predictive model for methotrexate hepatotoxicity, consisting of 13 SNPs with a sensitivity of 100% and specificity of 89%, although the model should be validated with a larger-scale prospective study. RA is a multifactorial disorder with clinically heterogeneous features. Gene-environment interaction is closely involved in the production of anti-CCP antibodies (ACPA); thereafter, secondary stimuli of joints may lead to symptoms of RA. Joint injury, emotional stress, and infections often trigger the onset of RA. Cure can be achieved through complete remission by early aggressive treatment and returning to the pre-clinical state of RA with environmental improvement.

We previously disclosed a novel extracellular matrix tenascin-X (TNX) , the largest member of the tenascin family. So far, we have made efforts to elucidate the roles of TNX. TNX is involved in collagen deposition, collagen fibrillogenesis, and modulation of collagen stiffness. Homozygous mutations in TNXB, the gene encoding TNX, cause a classic-type Ehlers-Danlos syndrome (EDS) , a heritable connective tissue disorder, whereas haploinsufficiency of TNXB and heterozygous mutations in TNXB are associated with hypermobility-type EDS. Recently, we performed proteomic analyses of calcific aortic valves (CAVs) compared with relatively adjacent normal tissues to understand the underlying molecular mechanisms of dystrophic valvular calcification. Interestingly, we found that TNX was the protein with the greatest decrease in expression among the differentially expressed proteins and that expression levels of proteins modulating collagen structure and function, such as type I collagen and decorin, were also decreased in CAVs. In this review, I will discuss about the decreased level of collagen due to the reduction of expression levels of proteins that play regulatory roles in collagen functions such as fibril organization and fibrillogenesis in CAVs.

While, in general, a certain number of clinical psychiatrists might not be familiar with molecular biology, the mechanisms of mental illnesses have been uncovered by molecular biology for decades. Among mental illnesses, even biological psychiatrists and neuroscientists have paid less attention to the biological treatment of autism spectrum disorder (ASD) than Alzheimer's disease and schizophrenia since ASD has been regarded as a developmental disorder that was seemingly untreatable. However, multifaceted methods of molecular biology have revealed the mechanisms that would lead to the medication of ASD. In this article, how molecular biology dissects the pathobiology of ASD is described in order to announce the possibilities of biological treatment for clinical psychiatrists.

The theme of this symposium was "Investigation by young members--Introduction of their Projects and Progress--". This theme was timely because the president of this meeting, Dr. Minoru Tozuka (TMDU), and colleagues have recently been devoted to educate students who are expected to be laboratory technologists with research-oriented minds. Three members presented their papers. The first speaker was Dr. Kurano from The University of Tokyo, and he discussed the crucial role of apolipoprotein M in "sphingosine 1-phosphatology". The second speaker was Dr. Nakayama from Bunkyo Gakuen University, and her talk was focused on the comprehensive analysis of urinary proteins aiming to identify novel markers of the renal function. The final presentation, by Dr. Matsumoto from Shinshu University, was on studies on bacterial small-colony variants obtained from clinical samples. I chaired this symposium and was greatly encouraged as one of the teachers by the power of young investigators in this field.

Biomarkers, reflecting disease states or predicting/assessing drug efficacy or adverse reactions, are expected to play pivotal roles in effective drug development and promoting proper usage of drugs. To accelerate biomarker identification and usage, administrative guidance can direct to design appropriate exploration, validation and utilization studies and show examination procedures. However, very limited number of guidance or its draft were released from Japanese, US and European regulatory authorities so far. From 2012, we have been conducting proteomic and metabolomic studies using blood and urine samples from human and rat, in order to establish draft guidance for sampling/storage of these biofluid and for extrapolation of biomarker candidates from animals in the non-clinical to humans in the clinical studies. The results are still partial and the rest of the analysis is ongoing. However, we developed sensitive proteomic system for urine and found large inter-sex differences in the proteomic profiles of rat. In addition, matrix-, sex- and generation-differences were also observed in the metabolite levels in human blood, some of which showed over 2-fold differences. We continue this regulatory science studies for contribution to accelerated novel biomarker findings and its usage by generation of the draft guidance.

Biopharmaceuticals represent new generation drugs that are more effective than conventional low molecular weight medicines for the treatment of cancer and other refractory diseases. In order to develop biopharmaceuticals it is critically important to explore suitable target molecules both for the diagnosis and treatment of disease. However, the choice of effective biomarker or drug target is often the most difficult part of the drug development process. Target discovery typically involves 'omics' techniques such as genomics, gene chip analysis and proteomics. Of these, proteomics is particularly important because proteins often comprise the final biomarker or drug target in the clinical sample (e.g., tissue or blood). Comprehensive proteomic analysis involves the identification of target proteins with different levels of expression between two states and is extremely useful at selecting promising candidates. Technology is then applied to further narrow down the list of relevant proteins. Here, I would like to introduce our novel procedure, termed "antibody proteomics technology", which can generate monoclonal antibody from a minute amount (i.e., nanogram level) of protein. Antibody proteomics technology can be used to easily identify suitable target molecules in order to develop effective biomarkers and drug targets.

Protein phosphorylation, one of the most ubiquitous post-translational modifications of proteins, is reversibly controlled by protein kinases and phosphatases, and regulates a variety of cell functions through cellular signal transduction pathways. These signals are involved in various diseases such as cancer, and often cause the disease itself or its progression. Phosphoproteomics has been explored to analyze protein phosphorylation comprehensively and has been applied to monitoring the entire network of cellular signal transduction. In this review, the frontlines of phosphoproteomics as well as its application to cancer molecular targeting drug discovery, therapy and diagnostics are described.

Cancer is a genetically and clinically diverse disease, and biomarkers are required to optimize the therapeutic strategies. Proteome is a functional translation of genome, directly regulating cancer phenotypes. Biomarker studies have been conducted by the proteomic approach. By the proteomic approach, we developed a novel prognostic biomarker in gastrointestinal stromal tumor(GIST). In GIST, adjuvant imatinib mesylate after resection improved recurrence-free survival. A prognostic biomarker is required to select the patients for the treatment with imatinib mesylate. Proteomic approach using two-dimensional difference gel electrophoresis(2D-DIGE) identified pfetin as a biomarker for favorable prognosis. Multi-institutional validation study by immunohistochemistry established the prognostic values of pfetin in GIST. Pfetin will be applicable for a novel prognostic modality.

The prevalence of diabetes is rapidly increasing worldwide, leading to an increasing risk of microangiopathic complications. To identify biomarkers of diabetic microangiopathy that are useful for prevention and early treatment of diabetic complications, proteomic analyses of clinical samples from diabetic patients have been performed. However, as diabetes is a systemic disease, it is difficult to define the pathologic conditions and foci causing changes in proteomic profile of clinical samples from diabetics. An approach using multiple reaction monitoring may be useful to systemically identify diabetic microangiopathy biomarkers from blood and urine samples from diabetic patients.

Prostate-specific antigen (PSA) has been widely used as a biomarker for prostate cancer detection and progression. However, PSA has limitations in terms of predicting the prognosis in specific cases of prostate cancer because it does not reflect cancer biology. Due to the development of new techniques in the fields of genomics and proteomics, many new biomarkers have been discovered in the last several years. In this review, we described some of these new and promising biomarkers including gene and protein markers, cytokines and circulating tumor cells. Moreover, we introduced our proteomic-based findings in our search for new prostate cancer biomarkers.

A majority of proteins encoded in genomes of limited size are post-translationally diversified by covalent modifications such as glycosylation and ubiquitination. Although recent advances in structural proteomics have enabled high-throughput structure determination of proteins, structural analyses of post-translationally modified proteins remain challenging because of the lack of appropriate determination methods. Therefore, we developed methodologies for characterizing the post-translational modifications of proteins from the structural viewpoint, focusing especially on glycosylation and ubiquitination. For instance, we established a systematic method for structural glycomics to address broader issues, including glycosylation profiling and 3D structure analyses of glycoproteins. Our stable-isotope-assisted NMR techniques in conjunction with X-ray crystallographic approach provide valuable information at the atomic level on conformations, dynamics, and interactions of glycoproteins such as antibody and proteins involved in the ubiquitin-proteasome system. These studies provide the structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans and mechanistic insights into ubiquitination reactions in glycoprotein-fate determination in cells. These approaches will allow new possibilities for structural studies on post-translationally modified proteins of clinical, pathological, and pharmaceutical interests.

Mitochondrial permeability transition (PT) is the phenomenon in which the mitochondrial inner membrane becomes permeable to various solutes and ions. When PT is induced by Ca(2+), cytochrome c is released from mitochondria into the cytosol where it then triggers subsequent steps of programmed cell death, apoptosis. Thus, the proteins that regulate PT and cytochrome c release could become druggable targets for various diseases. However, the mechanisms of PT and the release of cytochrome c have not yet been revealed. We previously showed that valinomycin, a potassium selective ionophore, also caused release of cytochrome c from mitochondria without inducing PT. This result indicates that cytochrome c could be released from mitochondria with or without induction of PT. In this study, to understand the difference of effects of valinomycin and Ca(2+) on mitochondria, we examined what protein species are released from valinomycin- and Ca(2+)-treated mitochondria by LC-MS/MS. As a result, only the proteins located in the intermembrane space were found to be released from valinomycin-treated mitochondria, while those in both the intermembrane space and in the matrix were released from Ca(2+)-treated mitochondria. Furthermore, the protein releases by each reagent occurred not selectively but in a concentration-dependent manner. Based on these results, the permeabilization effects of Ca(2+) and valinomycin on the inner and outer mitochondrial membranes are discussed.

Omics analysis is defined as a comprehensive analysis of various biological aspects from gene sequence to metabolite pattern via protein expression, and it might revolutionarily alter the design of scientific experiments as well as the process of biological investigation. Furthermore, omics technologies allow the generation of huge amounts of data at high-throughput. Particularly, proteomics is one of the omics technologies, which is a comprehensive analysis of protein, and it is hoped that proteomics accelerates translational research in the field of malignant neoplasm. Given the present status of proteomics technologies, we performed differential analysis between two categories such as a metastasis-positive group and a negative group using the clinical materials of a single kind of malignant neoplasm, and detected various kinds of protein molecules associated with metastasis. However, we could not obtain any evidence that these molecules were directly related with metastasis. The close relationship between clinical doctors, molecular pathologists and technical experts concerning proteomics is essential, because an experimental design, which they would generate, is extremely important for the detection of the targeted molecules. Additionally, in this paper some recent reports concerning the exploration of metastasis-related molecules using proteomic technology proteomics are introduced.

The Targeted Protein Research Program (TPRP) started in 2007 as a sequel of the Protein 3000 Project which lasted from 2002 to 2007. In the new project, four cores, Protein Production, Structure Analysis, Control of Protein Functions with Compounds, and Informatics, have been established as focus of methodology developments critical for functional and structural studies by the target protein research teams. Within the "Analysis Core" synchrotron radiation plays a pivotal role providing X-ray beams for structural analyses of the target proteins. The two large Japanese synchrotron radiation facilities, SPring-8 and Photon Factory (PF), along with three protein crystallography groups from Hokkaido, Kyoto and Osaka Universities have teamed up to develop two complementary micro-beam beamlines, one on each synchrotron site, and associated technologies for cutting edge structural biology research. At the PF, there are 5 operational beamlines which are equipped with state-of-the-art instrumentation for high-throughput protein crystallography experiments. Within the TPRP framework, the PF is developing a micro-focus beamline optimized for a lower energy single anomalous diffraction (SAD) experiment. This will be particularly useful for structure determination of difficult protein targets for which heavy atom derivatives or selenomethionine substitution does not work and other standard phasing methods fail to give structure solutions. This will augment the capabilities of the PF structural biology beamlines with similar look-and-feel experimental environments.

With recent development of the nanotechnology, nanomaterials have been successfully employed in various industrial applications such as medicine and cosmetics. Nanomaterials show the useful properties such as electronic reactivity and the tissue permeability that were not provided by micromaterials. Thus, nanomaterials are expected as innovative materials for the development of medicine and cosmetics. However, these innovative properties may show unknown biological responses that could not been detected by the conventional toxicity assay. For industrial development and affluent society establishment that enjoyed only a benefit of nanomaterials, it is urgent to gather information of the properties and the biological effects, and to establish the standard safety evaluation method of nanomaterials. So, we are analyzing association among property, biodistribution and biological effects of nanomaterials to search for the safety biomarker (functional-, molecular- and biochemical-biomarker) using nanosilicas (nSP) as a standard nanomaterial. Because nSP shows high uniform dispersibility and is already used in medicine, cosmetics and food additive, the results of this study are useful to extrapolate it to other nanomaterials and to make practicable as safety biomarker. In this report, we show the latest knowledge about the linkage information among property, biodistribution and biological effects of nSP by toxicokinetics and toxicoproteomics, and the search study of safety biomarker based on these basic information.