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Voltage-gated sodium channels, including Na V 1.7, Na V 1.8, and Na V 1.9, play important roles in pain transmission and chronic pain development. However, the specific mechanisms of their action remain unclear, highlighting the need for in vivo stimulation studies of these channels. Optogenetics, a novel technique for targeting the activation or inhibition of specific neural circuits using light, offers a promising solution. In our previous study, we used optogenetics to selectively excite Na V 1.7-expressing neurons in the dorsal root ganglion of mice to induce nocifensive behavior. Here, we further characterize the impact of nocifensive behavior by activation of Na V 1.7, Na V 1.8, or Na V 1.9-expressing neurons. Using CRISPR/Cas9-mediated homologous recombination, Na V 1.7-iCre, Na V 1.8-iCre, or Na V 1.9-iCre mice expressing iCre recombinase under the control of the endogenous Na V 1.7, Na V 1.8, or Na V 1.9 gene promoter were produced. These mice were then bred with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain Na V 1.7-ChR2, Na V 1.8-ChR2, or Na V 1.9-ChR2 mice. Blue light exposure triggered paw withdrawal in all mice, with the strongest response in Na V 1.8-ChR2 mice. These light sensitivity differences observed across Na V 1.x-ChR2 mice may be dependent on ChR2 expression or reflect the inherent disparities in their pain transmission roles. In conclusion, we have generated noninvasive pain models, with optically activated peripheral nociceptors. We believe that studies using optogenetics will further elucidate the role of sodium channel subtypes in pain transmission., (© 2024 The Author(s). Journal of Neuroscience Research published by Wiley Periodicals LLC.)

Chronic kidney disease (CKD) induces cardiac inflammation and fibrosis and reduces survival. We previously demonstrated that G protein-coupled receptor 68 (GPR68) promotes cardiac inflammation and fibrosis in mice with 5/6 nephrectomy (5/6Nx) and patients with CKD. However, no method of GPR68 inhibition has been found that has potential for therapeutic application. Here, we report that Cephalotaxus harringtonia var. nana extract and homoharringtonine ameliorate cardiac inflammation and fibrosis under CKD by suppressing GPR68 function. Reagents that inhibit the function of GPR68 were explored by high-throughput screening using a medicinal plant extract library (8,008 species), and we identified an extract from Cephalotaxus harringtonia var. nana as a GPR68 inhibitor that suppresses inflammatory cytokine production in a GPR68 expression-dependent manner. Consumption of the extract inhibited inflammatory cytokine expression and cardiac fibrosis and improved the decreased survival attributable to 5/6Nx. Additionally, homoharringtonine, a cephalotaxane compound characteristic of C. harringtonia, inhibited inflammatory cytokine production. Homoharringtonine administration in drinking water alleviated cardiac fibrosis and improved heart failure and survival in 5/6Nx mice. A previously unknown effect of C. harringtonia extract and homoharringtonine was revealed in which GPR68-dependent inflammation and cardiac dysfunction were suppressed. Utilizing these compounds could represent a new strategy for treating GPR68-associated diseases, including CKD., Competing Interests: Declaration of competing interest All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. All the authors have read and approved the submission for publication., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pulsed radiofrequency (PRF) is a safe method of treating neuropathic pain by generating intermittent electric fields at the needle tip. Resiniferatoxin (RTX) is an ultrapotent agonist of transient receptor potential vanilloid subtype-1 (TRPV1) receptors. We investigated the mechanism of PRF using a rat model of RTX-induced neuropathic pain. After administering RTX intraperitoneally, PRF was applied to the right sciatic nerve. We observed the changes in TRPV1, calcitonin gene-related peptide (CGRP), and brain-derived neurotrophic factor (BDNF) in the dorsal root ganglia by western blotting. Expressions of TRPV1 and CGRP were significantly lower in the contralateral (RTX-treated, PRF-untreated) tissue than in control rats (p<0.0001 and p<0.0001, respectively) and the ipsilateral tissues (p<0.0001 and p<0.0001, respectively). BDNF levels were significantly higher in the contralateral tissues than in the control rats (p<0.0001) and the ipsilateral tissues (p<0.0001). These results suggest that, while TRPV1 and CGRP are decreased by RTX-induced neuronal damage, increased BDNF levels result in pain development. PRF may promote recovery from neuronal damage with concomitant restoration of TRPV1 and CGRP, and exert its analgesic effect by reversing BDNF increase. Further research is required to understand the role of TRPV1 and CGRP restoration in improving mechanical allodynia., Competing Interests: No potential conflict of interest relevant to this article was reported.

In small and large spinal dorsal root ganglion neurons, subtypes of voltage-gated sodium channels, such as NaV1.7, NaV1.8, and NaV1.9 are expressed with characteristically localized and may play different roles in pain transmission and intractable pain development. Selective stimulation of each specific subtype in vivo may elucidate its role of each subtype in pain. So far, this has been difficult with current technology. However, Optogenetics, a recently developed technique, has enabled selective activation or inhibition of specific neural circulation in vivo. Moreover, optogenetics had even been used to selectively excite NaV1.8-expressing dorsal root ganglion neurons to induce nocifensive behavior. In recent years, genetic modification technologies such as CRISPR/Cas9 have advanced, and various knock-in mice can be easily generated using such technology. We aimed to investigate the effects of selective optogenetic activation of NaV1.7-expressing afferents on mouse behavior. We used CRISPR/Cas9-mediated homologous recombination to generate bicistronic NaV1.7-iCre knock-in mice, which express iCre recombinase under the endogenous NaV1.7 gene promoter without disrupting NaV1.7. The Cre-driver mice were crossed with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7iCre/+;Ai32/+, NaV1.7iCre/iCre;Ai32/+, NaV1.7iCre/+;Ai32/Ai32, and NaV1.7iCre/iCre;Ai32/Ai32 mice. Compared with wild-type mice behavior, no differences were observed in the behaviors associated with mechanical and thermal stimuli exhibited by mice of the aforementioned genotypes, indicating that the endogenous NaV1.7 gene was not affected by the targeted insertion of iCre. Blue light irradiation to the hind paw induced paw withdrawal by mice of all genotypes in a light power-dependent manner. The threshold and incidence of paw withdrawal and aversive behavior in a blue-lit room were dependent on ChR2 expression level; the strongest response was observed in NaV1.7iCre/iCre;Ai32/Ai32 mice. Thus, we developed a non-invasive pain model in which peripheral nociceptors were optically activated in free-moving transgenic NaV1.7-ChR2 mice., Competing Interests: The authors have declared that no competing interests exist.

Pulsed radiofrequency (PRF) therapy is one of the most common treatment options for neuropathic pain, albeit the underlying mechanism has not been hitherto elucidated. In this study, we investigated the efficacy and mechanism of PRF therapy on resiniferatoxin (RTX)-induced mechanical allodynia, which has been used as a model of postherpetic neuralgia (PHN). Adult male rats were intraperitoneally injected with a vehicle or RTX. Furthermore, PRF current was applied on a unilateral sciatic nerve in all RTX-treated rats. On both ipsilateral and contralateral sides, the paw mechanical withdrawal thresholds were examined and L4-6 dorsal root ganglia (DRG) were harvested. In the DRG of rats with RTX-induced mechanical allodynia, Na V 1.7, a voltage-gated Na + channel, was upregulated following the enhancement of extracellular signal-regulated kinase phosphorylation. Early PRF therapy, which was applied 1 week after RTX exposure, suppressed this Na V 1.7 upregulation and showed an anti-allodynic effect; however, late PRF therapy, which was applied after 5 weeks of RTX exposure, failed to inhibit allodynia. Interestingly, late PRF therapy became effective after daily tramadol administration for 7 days, starting from 2 weeks after RTX exposure. Both early PRF therapy and late PRF therapy combined with early tramadol treatment suppressed Na V 1.7 upregulation in the DRG of rats with RTX-induced mechanical allodynia. Therefore, Na V 1.7 upregulation in DRG is related to the development of RTX-induced neuropathic pain; moreover, PRF therapy may be effective in the clinical management of patients with PHN via Na V 1.7 upregulation inhibition.

Dysfunction of the circadian clock has been implicated in the pathogenesis of cardiovascular disease. The CLOCK protein is a core molecular component of the circadian oscillator, so that mice with a mutated Clock gene (Clk/Clk) exhibit abnormal rhythms in numerous physiological processes. However, here we report that chronic kidney disease (CKD)-induced cardiac inflammation and fibrosis are attenuated in Clk/Clk mice even though they have high blood pressure and increased serum angiotensin II levels. A search for the underlying cause of the attenuation of heart disorder in Clk/Clk mice with 5/6 nephrectomy (5/6Nx) led to identification of the monocytic expression of G protein-coupled receptor 68 (GPR68) as a risk factor of CKD-induced inflammation and fibrosis of heart. 5/6Nx induces the expression of GPR68 in circulating monocytes via altered CLOCK activation by increasing serum levels of retinol and its binding protein (RBP4). The high-GPR68-expressing monocytes have increased potential for producing inflammatory cytokines, and their cardiac infiltration under CKD conditions exacerbates inflammation and fibrosis of heart. Serum retinol and RBP4 levels in CKD patients are also sufficient to induce the expression of GPR68 in human monocytes. Our present study reveals an uncovered role of monocytic clock genes in CKD-induced heart failure.

The perception of tastes is sensed by the receptors that stimulate sensory cells. We previously reported that TRPA1 and TRPV1 channels expressed in the oral cavity of mammals, are activated by the auto-oxidized product of epigallocatechin gallate (oxiEGCG), a major astringent catechin in green tea. Here, we investigated and compared the sensitivity of TRPA1 and TRPV1 from various animals to astringent polyphenols. We selected three polyphenols, oxiEGCG, tannic acid and myricetin. HEK293T cells expressing TRPA1 or TRPV1 from mammal, bird, reptile, amphibian, and fish, were analyzed for their activation by the Ca 2+ -imaging. We found the apparent diversity in the polyphenol-sensitivity among various animals. Mammalian TRPs showed relatively higher sensitivity to polyphenols, and especially, human TRPA1 and TRPV1 could be activated by all of three polyphenols at 20 μM. Reptile TRP channels, however, were insensitive to any polyphenols examined. Moreover, the polyphenol-sensitivity of zebrafish TRPA1 and TRPV1 was quite different from that of medaka TRP channels. Since many polyphenols are present in plants and the sensing of polyphenols using TRP channels in the oral cavity might cause astringent taste, the observed diversity of the polyphenol-sensitivity of TRP channels might be involved in the divergence in the food habit of various animals.

This research sets out to quantify the mortality and economic loss in individual Asian countries caused by the PM 2.5 emissions induced by the consumption of the world's five highest-consuming countries (US, China, Japan, Germany, UK). In 2010 alone, the economic impact of these five countries' consumption caused a loss of almost 45 billion US dollars due to the premature deaths of more than 1 million people in Asia, including 15 thousand children younger than 5 years old. The percentage ratio of economic loss to value-added driven by consumers via trade differed greatly among the impacted countries. For the US, the highest percentage loss was 4.1% in Laos, followed by 2.0% in Bangladesh, both markedly higher than the figures for the more developed countries, such as 0.21% for Japan and 0.18% for Korea. This reflects the inequitable value chain existing between consumer countries and impacted countries, and implies that developing countries are obtaining value-added in exchange for unintentionally increased health risks, delaying their development and potentially creating a vicious circle that hinders much-needed improvements in areas like poverty reduction and public health. This inequitable situation needs to be redressed through introduction of clean energy and other types of technological assistance to help achieve United Nations Sustainable Development Goals 7, 10 and 13. Such as move is essential if premature infant deaths are to be curtailed., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Even in coastal oceans where tidal currents are predominant, long-term mean currents are of great interest since they are responsible for the transport of materials over long timescales. Tides could significantly affect mean currents in long, narrow straits due to tide-topography interaction, but it is yet unclear how and to what extent tides control throughflows. Here, we focus on the throughflow in the Seto Inland Sea, Japan, which has enormous impacts on the marine environment while its long-term mean characteristics, even the flow direction, are not well described by observations. By using a state-of-the-art ocean model, we show that the simulated throughflow is eastward on annual average and its volume transport is considerably suppressed by tides. It is found that tides enhance mixing and induce time-mean eddies, and both work to reduce the throughflow. A westward throughflow was previously estimated based on an acoustic measurement. The discrepancy between this estimate and our result would be due to whether or not such eddies are taken into account. These findings imply that tides may also suppress the throughflow of the other straits around the world. Revealing such tidal effects may contribute to a better performance of oceanic and climate simulations.

Transmembrane (TM) proteins localized at the plasma membrane, such as transporters and receptors, play important roles in regulating the selective permeability of the blood-brain barrier (BBB). The purpose of the present study was to clarify the differences in the expression levels of TM proteins in the plasma membrane between two established human BBB model cell lines, hCMEC/D3 and HBMEC/ciβ, in order to assist researchers in selecting the most appropriate cell line for particular purposes. We first confirmed that plasma membranes could be enriched sufficiently for a quantitative proteomics study by using the Plasma Membrane Protein Extraction Kit provided by BioVision with a modified protocol. This method was applied to hCMEC/D3 and HBMEC/ciβ cells, and fractions were used for untargeted quantitative proteomics based on sequential window acquisition of all theoretical fragment-ion spectra. In the plasma membrane fractions, 345 TM proteins were quantified, among which 135 showed significant expression differences between the two cell lines. In hCMEC/D3 cells, amino acid transporters SNAT1, SNAT2, SNAT5, ASCT1, CAT1, and LAT1; adenosine 5'-triphosphate-binding cassette transporters P-gp and MRP4; and GLUT1 were more highly expressed. The transferrin receptor expression was also 4.56-fold greater in hCMEC/D3 cells. In contrast, HBMEC/ciβ cells expressed greater levels of IgG transporter neonatal Fc receptor, as well as tight-junction proteins PECAM1, JAM1, JAM3, and ESAM. Our results suggest that hCMEC/D3 cells have greater efflux transport, amino acid transport, and transferrin receptor-mediated uptake activities, whereas HBMEC/ciβ cells have greater IgG-transport activity and tight-junction integrity.

Antibiotics act on bacterial flora originally present in the intestine, and changes in the intestinal flora have various effects on the host. This study investigated changes in the protein levels of drug transporters and metabolizing enzymes in the small intestines of antibiotic-treated mice by proteomic analysis. After the oral administration of non-absorbable antibiotics (vancomycin and polymyxin B) for 5 days, 15 drug transporter or metabolizing enzyme proteins had significantly changed levels among 1780 proteins identified in small intestinal epithelial cells. Of these, the levels of peptide transporter 1 (Pept1), multidrug resistance protein 1a (Mdr1a), and multidrug resistance-associated protein 2 (Mrp2) were increased approximately 2-fold. In addition, the levels of two Cyp4f proteins were decreased and those of Cyp4b1, Ces1d, and three glutathione S-transferase (Gst) proteins were increased. Our results indicate that the oral administration of antibiotics changes the levels of proteins related to the absorption and metabolism of drugs in the small intestine, and suggest that substrate drugs of these proteins have a risk for indirect drug interactions with antibacterial drugs via the intestinal flora., (Copyright © 2019 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to developing benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). The mechanism of NF1-tumorigenesis or the curatives have not been established. Using unique trascriptome and proteome integration method, iPEACH (1), we previously identified translationally controlled tumor protein (TCTP) as a novel biological target for NF1-associated tumors (2). Here, we identified specific TCTP-interacting proteins by sequential affinity purification and data-independent mass spectrometry acquisition (AP-DIA/SWATH) to investigate the role of TCTP in NF1-associated malignant tumors. TCTP mainly interacts with proteins related to protein synthesis and especially to elongation factor complex components, including EF1A2, EF1B, EF1D, EF1G, and valyl-tRNA synthetase (VARS), in NF1-deficient malignant tumor cells. Interestingly, TCTP preferentially binds to EF1A2 (normally found only in neural and skeletal-muscle cells and several cancer cells), rather than EF1A1 despite the high homologies (98%) in their sequences. The docking simulation and further validations to study the interaction between TCTP and EF1A2 revealed that TCTP directly binds with EF1A2 via the contact areas of EF1A2 dimerization. Using unique and common sequences between EF1A2 and EF1A1 in AP-DIA/SWATH, we quantitatively validated the interaction of EF1A2 and TCTP/other elongation factors and found that TCTP coordinates the translational machinery of elongation factors via the association with EF1A2. These data suggest that TCTP activates EF1A2-dependent translation by mediating complex formation with other elongation factors. Inhibiting the TCTP-EF1A2 interaction with EF1A2 siRNAs or a TCTP inhibitor, artesunate, significantly down-regulated the factors related to protein translation and caused dramatic suppression of growth/translation in NF1-associated tumors. Our findings demonstrate that a specific protein translation machinery related to the TCTP-EF1A2 interaction is functionally implicated in the tumorigenesis and progression of NF1-associated tumors and could represent a therapeutic target., (© 2019 Kobayashi et al.)

Purpose: The aim of this work is to investigate the roles of solute carrier family 22 member 18 (SLC22A18) in lipid metabolism and in establishing the tumor phenotype of HepG2 cells., Methods: SLC22A18-knockdown HepG2 cells were established by stable transfection with shRNA. Protein expression levels were measured by quantitative proteomics and Western blot analysis. Cell growth was examined by cell counting kit. Accumulation of triglyceride-rich lipid droplets was measured by Oil-Red O staining. Cell migration and invasion were examined by Transwell assays., Results: SLC22A18-knockdown HepG2 cells accumulated triglyceride-rich lipid droplets and showed decreased expression levels of lysosomal/autophagic proteins, suggesting that lipid degradation is suppressed. Growth of HepG2 cells was decreased by SLC22A18 knockdown, but was restored by free fatty acid supplementation. In addition, SLC22A18 knockdown decreased the expression of insulin-like growth factor-binding protein 1 (IGFBP-1) and increased the invasion ability of HepG2 cells. Exogenous IGFBP-1 blocked the increase of invasion activity induced by SLC22A18 knockdown., Conclusion: Our results suggest that suppression of SLC22A18 decreased the supply of intracellular free fatty acids from triglyceride-rich lipid droplets by impairing the lysosomal/autophagy degradation pathway and reduced the invasive activity of HepG2 cells by decreasing IGFBP-1 expression.

The SLC22A18 gene, which encodes an orphan transporter, is located at the 11p15.5 imprinted region, an important tumor suppressor gene region. However, the role of SLC22A18 in tumor suppression remains unclear. Here, we investigated the involvement of SLC22A18 in cell growth, invasion, and drug resistance of MCF7 human breast cancer cell line. Western blot analysis indicated that SLC22A18 is predominantly expressed at intracellular organelle membranes. Quantitative proteomics showed that knockdown of SLC22A18 significantly altered the expression of 578 (31.0%) of 1867 proteins identified, including proteins related to malignancy and poor prognosis of breast cancer. SLC22A18 knockdown (1) increased MCF7 cell growth concomitantly with a >7-fold increase of annexin A8 (involved in cell growth and migration; a predictor of poor prognosis), (2) induced spherical morphology of MCF7 cells concomitantly with a nearly 3-fold increase of CD44 (involved in regulation of malignant phenotypes), and (3) increased chemosensitivity to vinca alkaloids concomitantly with a >80% reduction of doublecortin-like kinase 1 (involved in regulation of microtubule polymerization). Our results suggest that SLC22A18 may act as a tumor suppressor by regulating the expression levels of cell growth-related proteins, and vinca alkaloids might show therapeutic efficacy against low-SLC22A18-expressing breast cancer., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Antibiotic-caused changes in intestinal flora (dysbiosis) can have various effects on the host. Secondary bile acids produced by intestinal bacteria are ligands for specific nuclear receptors, which regulate glucose, lipid, and drug metabolism in the liver. The present study aimed to clarify the effect of changes in secondary bile acids caused by antibiotic-induced dysbiosis on the host physiology, especially glucose, lipid, and drug metabolism. After oral administration of non-absorbable antibiotics for 5 days, decreased amounts of secondary bile acid-producing bacteria in faeces and a reduction in secondary bile acid [lithocholic acid (LCA) and deoxycholic acid (DCA)] levels in the liver were observed. Serum glucose and triglyceride levels were also decreased, and these decreases were reversed by LCA and DCA supplementation. Quantitative proteomics demonstrated that the expression levels of proteins involved in glycogen metabolism, cholesterol, bile acid biosynthesis, and drug metabolism (Cyp2b10, Cyp3a25, and Cyp51a1) were altered in the liver in dysbiosis, and these changes were reversed by LCA and DCA supplementation. These results suggested that secondary bile acid-producing bacteria contribute to the homeostasis of glucose and triglyceride levels and drug metabolism in the host, and have potential as therapeutic targets for treating metabolic disease.

A key virulence factor of enterohaemorrhagic Escherichia coli (EHEC) is the bacteriophage-encoded Shiga toxin (Stx). Stxs are classified into two types, Stx1 and Stx2, and Stx2-producing strains are thought to cause more severe infections than strains producing only Stx1. Although O26 : H11 is the second most prevalent EHEC following O157 : H7, the majority of O26 : H11 strains produce Stx1 alone. However, Stx2-producing O26 strains have increasingly been detected worldwide. Through a large-scale genome analysis, we present a global phylogenetic overview and evolutionary timescale for E. coli O26 : H11. The origin of O26 has been estimated to be 415 years ago. Sequence type 21C1 (ST21C1), one of the two sublineages of ST21, the most predominant O26 : H11 lineage worldwide, emerged 213 years ago from one of the three ST29 sublineages (ST29C2). The other ST21 lineage (ST21C2) emerged 95 years ago from ST21C1. Increases in population size occurred in the late 20th century for all of the O26 lineages, but most remarkably for ST21C2. Analysis of the distribution of stx2-positive strains revealed the recent and repeated acquisition of the stx2 gene in multiple lineages of O26, both in ST21 and ST29. Other major EHEC virulence genes, such as type III secretion system effector genes and plasmid-encoded virulence genes, were well conserved in ST21 compared to ST29. In addition, more antimicrobial-resistance genes have accumulated in the ST21C1 lineage. Although current attention is focused on several highly virulent ST29 clones that have acquired the stx2 gene, there is also a considerable risk that the ST21 lineage could yield highly virulent clones.

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories.

Schizophrenia is a disabling mental illness associated with dysfunction of the prefrontal cortex, which affects cognition and emotion. The purpose of the present study was to identify altered molecular networks in the prefrontal cortex of schizophrenia patients by comparing protein expression levels in autopsied brains of patients and controls, using a combination of targeted and focused quantitative proteomics. We selected 125 molecules possibly related to schizophrenia for quantification by knowledge-based targeted proteomics. Among the quantified molecules, GRIK4 and MAO-B were significantly decreased in plasma membrane and cytosolic fractions, respectively, of prefrontal cortex. Focused quantitative proteomics identified 15 increased and 39 decreased proteins. Network analysis identified "GNA13-ERK1-eIF4G2 signaling" as a downregulated network, and proteins involved in this network were significantly decreased. Furthermore, searching downstream of eIF4G2 revealed that eIF4A1/2 and CYFIP1 were decreased, suggesting that downregulation of the network suppresses expression of CYFIP1, which regulates actin remodeling and is involved in axon outgrowth and spine formation. Downregulation of this signaling seems likely to impair axon formation and synapse plasticity of neuronal cells, and could be associated with development of cognitive impairment in the pathology of schizophrenia., Biological Significance: The present study compared the proteome of the prefrontal cortex between schizophrenia patients and healthy controls by means of targeted proteomics and global quantitative proteomics. Targeted proteomics revealed that GRIK4 and MAOB were significantly decreased among 125 putatively schizophrenia-related proteins in prefrontal cortex of schizophrenia patients. Global quantitative proteomics identified 54 differentially expressed proteins in schizophrenia brains. The protein profile indicates attenuation of "GNA13-ERK signaling" in schizophrenia brain. In particular, EIF4G2 and CYFIP1, which are located downstream of the GNA13-ERK network, were decreased, suggesting that the attenuation of this signal network may cause impairment of axon formation and synapse plasticity in the brain of schizophrenia patients. Our results provide a novel insight into schizophrenia pathology, and could be helpful for drug development., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Dysbiosis (alteration of intestinal flora) is associated with various host physiologies, including diseases. The purpose of this study was to clarify the effect of dysbiosis on protein expression levels in mouse liver and kidney by quantitative proteomic analysis, focusing in particular on drug-metabolizing enzymes and transporters in order to investigate the potential impact of dysbiosis on drug pharmacokinetics. Germ-free (GF) mice and antibiotics-treated mice were used as dysbiosis models. Expression levels of 825 and 357 proteins were significantly changed in the liver and kidney, respectively, of GF mice (vs specific-pathogen-free mice), while 306 and 178 proteins, respectively, were changed in antibiotics-treated mice (vs vehicle controls). Among them, 52 and 16 drug-metabolizing enzyme and transporter proteins were significantly changed in the liver and kidney, respectively, of GF mice, while 25 and 8, respectively were changed in antibiotics-treated mice. Expression of mitochondrial proteins was also changed in the liver and kidney of both model mice. In GF mice, Oatp1a1 was decreased in both the liver and kidney, while Sult1a1 and two Cyp enzymes were increased and Gstp1, four Cyp enzymes, three Ces enzymes, Bcrp1, and Oct1 were decreased in the liver. In antibiotics-treated mice, Cyp51a1 was increased and three Cyp enzymes, Bcrp1, and Bsep were decreased in the liver. Notably, the expression of Cyp2b10 and Cyp3a11 was greatly decreased in the liver of both models. Cyp2b activity in the liver microsomal fraction was also decreased. Our results indicate that dysbiosis changes the protein expression of multiple drug-metabolizing enzymes and transporters in the liver and kidney and may alter pharmacokinetics in the host.

The purpose of the present study was to examine simultaneously the absolute protein amounts of 152 membrane and membrane-associated proteins, including 30 metabolizing enzymes and 107 transporters, in pooled microsomal fractions of human liver, kidney, and intestine by means of SWATH-MS with stable isotope-labeled internal standard peptides, and to compare the results with those obtained by MRM/SRM and high resolution (HR)-MRM/PRM. The protein expression levels of 27 metabolizing enzymes, 54 transporters, and six other membrane proteins were quantitated by SWATH-MS; other targets were below the lower limits of quantitation. Most of the values determined by SWATH-MS differed by less than 50% from those obtained by MRM/SRM or HR-MRM/PRM. Various metabolizing enzymes were expressed in liver microsomes more abundantly than in other microsomes. Ten, 13, and eight transporters listed as important for drugs by International Transporter Consortium were quantified in liver, kidney, and intestinal microsomes, respectively. Our results indicate that SWATH-MS enables large-scale multiplex absolute protein quantification while retaining similar quantitative capability to MRM/SRM or HR-MRM/PRM. SWATH-MS is expected to be useful methodology in the context of drug development for elucidating the molecular mechanisms of drug absorption, metabolism, and excretion in the human body based on protein profile information., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transient receptor potential A1 (TRPA1) is the only member of the mouse, chick, and frog TRPA family, whereas 2 paralogs (zTRPA1a and zTRPA1b) are present in zebrafish. We herein investigated functional differences in the 2 zebrafish TRPA1s. HEK293T cells were used as heterologous expression systems, and the sensitivities of these cells to 4 chemical irritants (allyl isothiocyanate [AITC], caffeine, auto-oxidized epigallocatechin gallate [EGCG], and hydrogen peroxide [H2O2]) were compared with Ca(2+) imaging techniques. Sensitivities to the activators for AITC, oxidized EGCG, and H2O2 were higher in cells expressing zTRPA1a than in those expressing zTRPA1b, whereas caffeine appeared to activate both cells equally. We also characterized the thermal sensitivity of Xenopus oocytes expressing each TRPA1 electrophysiologically using a 2-electrode voltage clamp. Although endogenous currents induced by a cold stimulation were observed in control oocytes in some batches, oocytes expressing zTRPA1b showed significantly stronger cold- and heat-induced responses. However, significant thermal activation was not observed in oocytes expressing zTRPA1a. The results obtained using in vitro expression systems suggest that zTRPA1a is specialized for chemical sensing, whereas zTRPA1b responds to thermal stimuli. Furthermore, characterization of the chimeric molecule of TRPA1a and 1b revealed the importance of the N-terminal region in chemical and thermal sensing by zTRPA1s., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Transient receptor potential ankyrin 1 (TRPA1) is one of the main sensors for noxious stimuli in animals. Recent studies on the cloning and characterization of TRPA1 channels from several organisms showed the functional diversity of TRPA1 in sensing chemicals and temperature. Nociceptive receptors have been suggested to play important roles in adaptation to the environment by and survival strategies of animals; therefore, the sensitivity of various vertebrate TRPA1s needs to be examined in more detail. Here, we focused on fish TRPA1s and investigated the chemical sensing properties of pufferfish (Takifugu) TRPA1 (pfTRPA1). We determined how mammalian TRPA1 ligands activated pfTRPA1 using a Ca-imaging technique. The results obtained indicated that the sensitivity of pfTRPA1 to known TRPA1 ligands was lower than that of mammalian TRPA1s, except for the response ability to allyl isothiocyanate. We also investigated the effects of tannic acid, a type of polyphenol, by measuring ionic currents in Xenopus oocytes in a two-electrode voltage clamp. Although mouse TRPA1 was inhibited by tannic acid, pfTRPA1 channels were enhanced by the treatment with tannic acid. Taken together, these results suggest that pfTRPA1 is not a simple sensor with a lower sensitivity to chemical stimulation, but is actually a specialized sensor with unique properties.

The sensation of astringency is elicited by catechins and their polymers in wine and tea. It has been considered that catechins in green tea are unstable and auto-oxidized to induce more astringent taste. Here, we examined how mammalian transient receptor potential V1 (TRPV1) and TRPA1, which are nociceptive sensors, are activated by green tea catechins during the auto-oxidation process. Neither TRPV1 nor TRPA1 could be activated by any of the freshly prepared catechin. When one of the major catechin, epigallocatechin gallate (EGCG), was preincubated for 3h in Hank's balanced salt solution, it significantly activated both TRP channels expressed in HEK293 cells. Even after incubation, other catechins showed much less effects. Results suggest that only oxidative products of EGCG activate both TRPV1 and TRPA1. Dorsal root ganglion (DRG) sensory neurons were also activated by the incubated EGCG through TRPV1 and TRPA1 channels. Liquid chromatography-mass spectrometry revealed that theasinensins A and D are formed during incubation of EGCG. We found that purified theasinensin A activates both TRPV1 and TRPA1, and that it stimulates DRG neurons through TRPV1 and TRPA1 channels. Results suggested a possibility that TRPV1 and TRPA1 channels are involved in the sense of astringent taste of green tea., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Here, we investigated which taste ligand induces the CCK (cholecystokinin) release from intestinal STC-1 cells. We first developed a new assay to measure the release of CCK. The expression vector for CCK type A receptor (CCKAR) was permanently introduced into HEK293T cells and a cell line was established (CCKAR/HEK). Then, STC-1 cells were treated with taste ligands and the incubated buffer of STC-1 cells containing released CCK was applied to CCKAR/HEK cells.Since CCKAR couples to Gq-signaling, the CCK-induced receptor activation can be monitored by the method of Ca2+-imaging. Therefore, when CCK is released from STC-1 cells to culture medium with taste stimulation, Ca2+ activation of CCKAR/HEK should be observed. Among five different taste ligands (saccharin, Na-glutamate, NaCl, denatonium benzoate, HCl), only denatonium benzoate and HCl induced the release of CCK in STC-1 cells. Thus, we found that only specific taste ligands induce the CCK release, and that other three taste ligands cannot induce the release of CCK despite of their ability to elevate the intracellular Ca2+ level in STC-1 cells.

To determine the expression level of Shiga toxin (Stx) 2-related toxins (Stx2 and Stx2c) produced by each of 33 Stx-producing Escherichia coli (STEC) O157 strains, stx2 and stx2c mRNAs (stx2-related mRNA) were measured using real-time PCR with primers that recognize sequences common to stx2 and stx2c. The amount of Stx2 and Stx2c protein was measured using a reversed passive latex agglutination (RPLA) kit. Expression of stx2-related mRNA was significantly higher in STEC O157 strains carrying the stx2 gene (i.e., stx2, stx1/stx2, or stx2/stx2c) than in most strains that carried the stx2c gene but not the stx2 gene (i.e., stx2c or stx1/stx2c). RPLA might not measure the precise amount of each toxin variant; nevertheless, stx2-inclusive strains had 40-fold higher mean toxin titers than did strains that carried the stx2c gene but not the stx2 gene, with the exception of 1 stx2c strain. Interestingly, 1 stx2c strain that was isolated from a patient with severe hemorrhagic diarrhea had the highest stx2-related mRNA expression and the highest toxin titer of all 33 STEC O157 strains. Taken together, these findings indicated that measurement of stx2-related mRNA expression could reflect differences in production levels of toxins among STEC strains.

A characteristic astringent taste is elicited by polyphenols. Among the polyphenols, catechins and their polymers are the most abundant polyphenols in wine and tea. A typical green tea polyphenol is epigallocatechin gallate (EGCG). Currently, the mechanism underlying the sensation of astringent taste is not well understood. We observed by calcium imaging that the mouse intestinal endocrine cell line STC-1 responds to the astringent compound, EGCG. Among major catechins of green tea, EGCG was most effective at eliciting a response in this cell line. This cellular response was not observed in HEK293T or 3T3 cells. Further analyses demonstrated that the 67-kDa laminin receptor, a known EGCG receptor, is not directly involved. The Ca(2+) response to EGCG in STC-1 cells was decreased by inhibitors of the transient receptor potential A1 (TRPA1) channel. HEK293T cells transfected with the mouse TRPA1 (mTRPA1) cDNA showed a Ca(2+) response upon application of EGCG, and their response properties were similar to those observed in STC-1 cells. These results indicate that an astringent compound, EGCG, activates the mTRPA1 in intestinal STC-1 cells. TRPA1 might play an important role in the astringency taste on the tongue.

We found that a scaffold protein, spinophilin (SPL), can interact with M2 and M3-muscarinic acetylcholine receptors (mAChRs). As SPL can also bind to RGS8 by using the different region of SPL, we investigated the effects of SPL on the function of RGS8 regulating signals from M2 and M3 receptors. M2 receptor-mediated Gi-signaling was studied by monitoring G-protein-coupled inwardly rectifying K+ channels, and M3 receptor-mediated Gq-signaling was monitored by the increase of Ca2+-activated Cl(-) current. The expression of SPL could enhance the regulatory function of RGS8 on the M3-mAChR, but the acceleration function of RGS8 on the M2-mediated signaling could not be enhanced by SPL. Results showed that the recruitment of RGS8 to the receptor differentially affects the function of RGS8 among receptors.

Background: We histopathologically examined Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3)-positive hepatocellular carcinoma (HCC) and protein induced by vitamin K absence (PIVKA) II-positive HCC to clarify the efficacy of these markers for predicting a poor prognosis., Methods: Serum AFP-L3 and PIVKA II was measured in 110 HCC patients. AFP-L3 was measured by lectin-affinity electrophoresis coupled with antibody-affinity blotting, and PIVKA II by using a high-sensitivity kit. The growth type, capsule formation, capsule infiltration, portal vein invasion, intrahepatic metastasis and histological tumor grade were evaluated pathologically., Results: Thirty-eight (35%) HCC patients were AFP-L3-positive, and 63 (57%) were PIVKA II-positive. In AFP-L3-positive HCC, the frequencies of an infiltrative growth type (positive : negative = 66% : 42%, P = 0.027) and a poorly differentiated type (positive : negative = 32% : 6%, P < 0.001) were significantly higher than in AFP-L3-negative HCC. In PIVKA II-positive HCC, the frequencies of an infiltrative growth type (positive : negative = 62% : 28%, P < 0.001), vascular invasion (positive : negative = 63% : 26%, P < 0.001), and intrahepatic metastasis (positive : negative = 38% : 4%, P < 0.001) were significantly higher than in PIVKA II-negative HCC. In both AFP-L3- and PIVKA II-positive HCC, the frequency of a poorly differentiated growth type was significantly higher than in HCC positive for either AFP-L3 or PIVKA II or HCC negative for both AFP-L3 and PIVKA II (both positive : either positive : both negative = 37% : 12% : 0%; P = 0.014, P < 0.001, respectively)., Conclusions: AFP-L3 was related to progression from moderately differentiated to poorly differentiated HCC, whereas PIVKA II was more specific to vascular invasion. PIVKA II is therefore likely to be a useful indicator of vascular invasion.

Background and Aims: Scirrhous hepatocellular carcinoma (SHCC) is characterized by diffuse fibrosis of the tumor, however, its clinicopathological features are not fully clarified. This study aimed to clarify the clinicopathological features of SHCC., Methods: Among 546 consecutively resected HCC without preoperative anticancer therapies, 25 SHCC were selected for the study and compared with 521 cases without scirrhous as the control., Results: SHCC accounted for 4.6% of cases. On diagnostic imagings, SHCC was frequently misdiagnosed as cholangiocarcinoma (CC), combined HCC-CC or metastatic carcinoma. Overall survival rate was significantly higher than the control. The average (+/-SD) tumor size of SHCC was 3.4 +/- 1.8 cm without significant difference to the control. The majority of SHCC (88%) were located close to the liver capsule. SHCC was characterized by stellate fibrosis (84%), no encapsulation (100%), no necrosis and hemorrhage (100%), intratumoral portal tracts (80%), remarkable lymphocyte infiltration (84%), clear cell change (84%), and hyaline bodies (52%). The number of alpha-smooth muscle actin-positive myofibroblast-like cells (activated stellate cells) in the tumor was about three times more than that in the control. Regarding the developmental mechanism of scirrhous change, a close correlation with unique tumor location and activation of stellate cells was suggested., Conclusions: SHCC presents with characteristic clinicopathological features and the recognition of SHCC is important for both clinicians and pathologists.

Background/aims: This study aimed to evaluate the usefulness of (18)F-fluoro-2-deoxy-d-glucose positron emission tomography (PET) and PET plus computed tomography (CT) fusion images for the detection of extrahepatic metastases of hepatocellular carcinoma (HCC) and combined hepatocellular and cholangiocarcinoma (combined HCC/CC)., Methods: Twenty-one patients with HCC and combined HCC/CC were enrolled in the study from December 2004 to February 2005. In all patients, PET and CT of the chest to pelvis region were performed. The sensitivity of PET plus CT fusion images was compared with the sensitivity of PET, CT, and bone scintigraphy., Results: In 14 patients, a total of 58 extrahepatic metastases were diagnosed. The detection rate of PET plus CT fusion images, PET, CT, and bone scintigraphy was 98.2% (57 of 58 metastases), 89.6% (52 of 58 metastases), 91.2% (52 of 57 metastases), and 68.7% (11 of 16 bone metastases), respectively. No extrahepatic metastases were detected in the other seven patients. The detection rate of PET was 10/18 (55.6%) for intrahepatic lesions of HCC and combined HCC/CC., Conclusions: The fusion of PET plus CT images is useful in detecting extrahepatic metastases in HCC and combined HCC/CC patients.

Background/aims: Vascular endothelial growth factor-C (VEGF-C) is thought to be an important factor in tumor angiogenesis/lymphangiogenesis, but its role in hepatocellular carcinoma (HCC) has not yet been fully investigated., Methods: We immunohistochemically examined VEGF-C expression in surgically resected tissues of 90 HCC., Results: In the 78 HCC with a single histological grade, VEGF-C expression was significantly stronger in poorly differentiated HCC than in well- (P=0.003) or moderately differentiated HCC (P=0.0002). A 'nodule-in-nodule' case presented VEGF-A expression in the well-differentiated component and VEGF-C expression in the moderately-poorly differentiated component. According to nodular diameter, VEGF-C expression was significantly higher in nodules of 3.0 cm or larger (P=0.0263). Extrahepatic metastases seen in seven cases expressed VEGF-C. In 20 of the 28 cases who were able to be followed up, the frequency of intrahepatic recurrence tended to be higher and extrahepatic metastasis was significantly higher in the cases who had VEGF-C expression in the tumor casts of the intrahepatic portal/hepatic vein branches than other cases without the expression (P=0.0139). Disease-free survival time tended to be shorter in cases with VEGF-C expression in tumor casts of the portal/hepatic vein than in those without VEGF-C expression (P=0.053; log-rank test)., Conclusions: VEGF-C expression is related to the progression of HCC, and VEGF-C expression in tumor casts of the intrahepatic portal/hepatic vein is considered to be a factor indicating recurrence/metastasis sites.

We examined pathomorphologically small nodular lesions in hepatitis C virus-related cirrhosis (type C cirrhosis). Small nodular lesions seen in the non-cancerous areas of 128 consecutively resected hepatocellular carcinomas (HCCs) associated with type C cirrhosis were selected for the study. From these, 18 large regenerative nodules (LRNs), 14 low-grade dysplastic nodules (LDNs), 10 high-grade DNs (HDNs), and 12 well-differentiated HCCs were detected. The diameters of HDN (11.7+/-4.3 mm) and well-differentiated HCC (12.3+/-3.5) were significantly larger than those of LRN (8.1+/-1.0) and LDN (8.6+/-2.7). Fatty change was not seen in LRNs and LDNs, while it was found in 4 HDNs (40%) and 8 well-differentiated HCCs (75%). Iron deposits were found in 2 LRNs (11%), 5 LDNs (36%), and 3 HDNs (30%), but not in the well-differentiated HCCs. The number of Kupffer cells in LDN and HDN was increased compared with that in the surrounding regenerative nodules. Cell density and the degree of neovascularization were well correlated with the progression from DN to HCC. The lack of neovascularization supports the concept that LRN has no neoplastic potency and can be regarded as the morphologic marker in the differentiation of LRN from DN.

Background/aims: Currently, focal nodular hyperplasia (FNH)-like nodules in cirrhotic liver is spotlighted. Unique hypervascular nodules mimicking FNH-like nodule in alcoholic liver cirrhosis were clinicopathologically clarified., Methods: Six resected and six biopsy cases of small hypervascular nodules found in alcoholic cirrhosis were studied clinicopathologically., Results: All cases were male and consumed 90-150 g/day of ethanol for longer than 20 years, and hepatitis virus markers were negative. The nodules, 9-21 mm in diameter, were detected by ultrasonography during follow-up of alcoholic cirrhosis, and showed hypervascularity on angiography. Six patients were diagnosed as hepatocellular carcinoma and six were as hyperplastic nodule by biopsy, and the former six cases received partial hepatectomy. All of the resected nodules were completely or incompletely encapsulated. Histologically, all resected and biopsy nodules showed moderate increase of cell-density with an irregular trabecular pattern, and scar-like fibrosis with anomalous blood vessels, and unpaired arteries. All nodules showed marked or mild iron deposits in hepatocytes and/or kupffer cells, and a diffuse capillarization of the sinusoids., Conclusions: The nodules in the present series seem to fall in the same category as FNH-like nodules in cirrhotic liver, and should be taken account in screening programs including patients with alcoholic cirrhosis.

A new technique of laryngoplasty using the function of the suprahyoid muscles (hyoid transposition laryngoplasty) was reported. Total laryngectomy is carried out as usual and the tracheal stump is anastomosed directly to the hypopharynx. The hyoid bone is cut medially, then transpositioned and sutured to the posterolateral margins of the tracheal stump. With this procedure, the new glottis is pressed strongly to the tongue base during swallowing and aspiration is avoided. Postoperative swallowing and speech in the cases operated on with this method were satisfactory, and even nasal respiration was possible in some cases.