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ABSTRACT Infection of C57BL/6 wild-type mice with Leishmania major 5-ASKH or Friedlin strains results in relatively similar pathogenicity with self-healing lesions within weeks. Parasite clearance depends on nitric oxide production by activated macrophages in response to cytokines produced mainly by CD4+ Th1 cells. In contrast, C57BL/6 Rag2 knockout mice, which lack T and B lymphocytes, show distinct pathologies during infection with these strains. Despite of the similar parasite number, the 5-ASKH infection induced severe inflammation rather than the Friedlin. To determine the immunological factors behind this phenomenon, we infected C57BL/6 Rag2 knockout mice with these two strains and compared immune cell kinetics and macrophage activation status. Compared with the Friedlin strain, the 5-ASKH strain elicited increased pathology associated with the accumulation of CD11bhigh, Ly6Ghigh neutrophils by week four and increased the expression of macrophage activation markers. We then analyzed the differentially expressed transcripts in infected bone marrow-derived macrophages by RNA sequencing. It showed upregulation of multiple inflammatory transcripts, including Toll-like receptor 1/2 (TLR1/2), CD69, and CARD14, upon 5-ASKH infection. Our findings suggest that different L. major strains can trigger distinct macrophage activation, contributing to the disease outcome observed in the absence of lymphocytes but not in the presence of lymphocytes. IMPORTANCE Disease manifestations of cutaneous leishmaniasis (CL) range from self-healing cutaneous lesions to chronic forms of the disease, depending on the infecting Leishmania sp. and host immune protection. Previous works on mouse models of CL show the distinct pathogenicity of Leishmania major strains in the absence of lymphocytes. However, the mechanisms of this pathology remain uncovered. In the trial to understand the immunological process involved in lymphocyte-independent pathology, we have found a specific induction of macrophages by different L. major strains that affect their ability to mount innate responses leading to neutrophilic pathology when lymphocytes are ablated.

Spatial analysis of RNAseq data is important. Here the authors report a method for transcriptome profiling combined with photo-isolation chemistry to allow determination of expression profiles specifically from photo-irradiated regions of interest which they use in mouse brains and embryonic tissues.

Abstract Background Heterozygous mutations in the transcription factor GATA2 result in a wide spectrum of clinical phenotypes, including monocytopenia and Mycobacterium avium complex (MAC) infection (MonoMAC) syndrome. Patients with MonoMAC syndrome typically are infected by disseminated nontuberculous mycobacteria, fungi, and human papillomavirus, exhibit pulmonary alveolar proteinosis during late adolescence or early adulthood, and manifest with decreased content of dendritic cells (DCs), monocytes, and B and natural killer (NK) cells. Case presentation A 39-year-old woman was diagnosed with MonoMAC syndrome postmortem. Although she was followed up based on the symptoms associated with leukocytopenia that was disguised as sarcoidosis with bone marrow involvement, she developed disseminated nontuberculous mycobacterial infection, fungemia, and MonoMAC syndrome after childbirth. Genetic testing revealed a heterozygous missense mutation in GATA2 (c.1114G > A, p.A372T). Immunohistochemistry and flow cytometry showed the disappearance of DCs and decreased frequency of NK cells in the bone marrow, respectively, after childbirth. Conclusions To the best of our knowledge, this is the first study reporting that MonoMAC syndrome can be exacerbated after childbirth, and that immunohistochemistry of bone marrow sections to detect decreased DC content is useful to suspect MonoMAC syndrome.

Summary: Photo-isolation chemistry (PIC) enables isolation of transcriptome information from locally defined areas by photo-irradiation. Here, we present an optimized PIC protocol for formalin-fixed frozen and paraffin mouse sections and fresh-frozen mouse sections. We describe tissue section preparation and permeabilization, followed by in situ reverse transcription using photo-caged primers. We then detail immunostaining and UV-mediated uncaging to the target areas, followed by linear amplification of uncaged cDNAs, library preparation, and quantification. This protocol can be applied to various animal tissue types.For complete details on the use and execution of this protocol, please refer to Honda et al. (2021).

Abstract Background Regimens combining pemetrexed (PEM) and immune checkpoint inhibitors (ICIs) targeting programmed cell death-1 (PD-1) or programmed death-ligand 1 (PD-L1) are widely used for the treatment of advanced non-squamous non-small-cell lung cancer (NSq-NSCLC). Recently, PEM was shown to induce immunogenic cell death (ICD) and to enhance immune-regulatory genes. Some patients demonstrate an extremely long-term response to PEM. It is possible that the continued response in these patients is dependent on not only the pharmacological induction of cytotoxic cell death but also antitumor immunity. However, factors that can predict outcomes associated with long-term PEM administration using blood test results have not yet been elucidated. We investigated the clinical characteristics and predictive factors in patients with advanced NSq-NSCLC who underwent long-term PEM maintenance therapy. Methods In total, 504 patients with advanced NSq-NSCLC who received PEM combination therapy/monotherapy (n = 414) or paclitaxel (PTX) combination therapy (n = 90) between January 2010 and November 2019 were recruited; 381 patients were retained for the final analysis. Patients treated with PEM (n = 301) were divided into subgroups according to the total cycles of PEM (≥ 17 [n = 25] for the long-term administration group and ≤ 16 [n = 276] for the intermediate/short-term group) and compared with another population (n = 80) treated with PTX combination regimen. We investigated clinical features and predictive biomarkers, focusing on immune-regulatory factors, absolute lymphocyte count (ALC), neutrophil-to-lymphocyte ratio (NLR), and PD-1 and PD-L1 expression, to predict long-term response to PEM. Results The long-term PEM administration group exhibited a higher ALC and a lower NLR than the shorter-term group did. Both these markers displayed greater association with progression-free survival and overall survival in the PEM combination therapy group than in the PTX combination therapy group. Increased PD-1 lymphocytes were associated with the long-term PEM response group, as PD-L1 expression in tumors was associated with a high incidence of immune-related adverse effects following ICI administration. Conclusions ALC, NLR, and PD-1 expression are PEM-mediated predictive biomarkers that are indirectly related to tumor immunity and can provide useful predictive information on the long-term response to PEM in patients with NSq-NSCLC.

In the pathogenesis of depression, heredity is believed to be a major factor. However, the mechanism by which heredity contributes to the onset of depression is not fully understood. Wistar Kyoto (WKY) rats have been used as an animal model for depression because of their increased depression-like behavior compared to Wistar (WIS) rats. In the present study, pups crossbred from WKY × WIS rats were used to evaluate locomotor activity in an open field test (OFT) and depression-like behavior in a forced swimming test (FST), with a focus on amino acid metabolism. Pups in the WKY♂ × WKY♀ group showed lower locomotor activity in the OFT and higher depression-like behavior in the FST than those in the WIS♂ × WIS♀ group. In addition, multiple regression analysis showed that the paternal strain had a greater effect than the maternal strain on locomotor activity and depression-like behavior in OFT and FST, respectively. Several amino acids in the brainstem, hippocampus, and striatum were significantly decreased through the influence of the WKY paternal strain, but not the WKY maternal strain. Based on these data from comparing WKY and WIS rats, we hypothesize that the hereditary effects of the WKY paternal strain on behavioral tests are partially caused by dysregulation of the amino acid metabolism in the brain.

Introduction We report a rare case of renal cell carcinoma with vaginal metastasis that recurred with rapid progression and was resistant to sunitinib and nivolumab. Case presentation A 68‐year‐old woman presented with renal cell carcinoma and vaginal metastasis. Multiple lung metastasis appeared 3 months after simultaneous radical nephrectomy and hysterectomy with vaginal resection. Despite the treatment with sunitinib and nivolumab, the patient died 7 months after surgery. Immunohistochemical staining of primary and metastatic tumor specimens was CD8 and programmed death ligand‐1 negative. Conclusion Although vaginal metastasis of renal cell carcinoma is rare, lack of CD8 and programmed death ligand‐1 expression may cause nivolumab resistance and may be useful markers in patients with metastatic renal cell carcinoma.

The estimation of larval dispersal on an ecological timescale is significant for conservation of marine species. In 2018, a semi-population outbreak of crown-of-thorns sea star, Acanthaster cf. solaris, was observed on a relatively isolated oceanic island, Ogasawara. The aim of this study was to assess whether this population outbreak was caused by large-scale larval recruitment (termed secondary outbreak) from the Kuroshio region. We estimated larval dispersal of the coral predator A. cf. solaris between the Kuroshio and Ogasawara regions using both population genomic analysis and simulation of oceanographic dispersal. Population genomic analysis revealed overall genetically homogenized patterns among Ogasawara and other Japanese populations, suggesting that the origin of the populations in the two regions is the same. In contrast, a simulation of 26-year oceanographic dispersal indicated that larvae are mostly self-seeded in Ogasawara populations and have difficulty reaching Ogasawara from the Kuroshio region within one generation. However, a connectivity matrix produced by the larval dispersal simulation assuming a Markov chain indicated gradual larval dispersal migration from the Kuroshio region to Ogasawara in a stepping-stone manner over multiple years. These results suggest that the 2018 outbreak was likely the result of self-seeding, including possible inbreeding (as evidenced by clonemate analysis), as large-scale larval dispersal from the Kurishio population to the Ogasawara population within one generation is unlikely. Instead, the population in Ogasawara is basically sustained by self-seedings, and the outbreak in 2018 was also most likely caused by successful self-seedings including possible inbreeding, as evidenced by clonemate analysis. This study also highlighted the importance of using both genomic and oceanographic methods to estimate larval dispersal, which provides significant insight into larval dispersal that occurs on ecological and evolutionary timescales.

Long-term operational stability and high-efficiency neuron stimulation are key to the development of retinal prostheses. In this research, a retinal device with a chemically inert and flexible substrate is introduced, which satisfies these requirements. We have developed a honeycomb-type retinal device that has a high-performance electrode for suprachoroidal transretinal stimulation. The device structure, in which conjunctional bioceramic substrates are embedded with large numbers of stimulating electrodes, provides high-resolution electrical stimulation. The custom CMOS microchip precisely controls the stimulation delivery of the electrodes to initiate artificial vision, offering a partial remedy for retinal ophthalmic diseases. The CMOS chip design was optimized to drastically reduce the number of input wirings. A high-performance stimulating electrode based on iridium oxide was fabricated using a unique solution process called chemical bath deposition (CBD). The honeycomb-type retinal device, equipped with CBD-derived iridium oxide electrodes, was used to evaluate the electrodes’ and device’s performances in vitro.

Accumulation of damaged mitochondria is implicated in a number of neurodegenerative disorders, including Parkinson's disease. Therefore, the machinery for mitochondrial quality control is important for the prevention of such diseases. It has been reported that Parkin‐ and p62/sequestosome 1 (SQSTM1)‐mediated clustering and subsequent elimination of damaged mitochondria (termed mitophagy) are critical for maintaining the quality of mitochondria under stress induced by uncoupling agents such as carbonyl cyanide m‐chlorophenyl hydrazone. However, the molecular mechanisms underlying mitochondrial translocation to the perinuclear region during mitophagy have not been adequately addressed to date. In this study, we found that BCL2‐associated athanogene 6 (BAG6; also known as BAT3 or Scythe) is required for this process. Indeed, RNA interference‐mediated depletion of endogenous BAG6 prevented Parkin‐dependent relocalization of mitochondrial clusters to the perinuclear cytoplasmic region, whereas BAG6 knockdown did not affect the translocation of Parkin and p62/SQSTM1 to the depolarized mitochondria and subsequent aggregation. These results suggest that BAG6 is essential for cytoplasmic redistribution, but not for clustering, of damaged mitochondria.

Background: Betaine is known to act against various biological stresses and its levels were reported to be decreased in schizophrenia patients. We aimed to test the role of betaine in schizophrenia pathophysiology, and to evaluate its potential as a novel psychotherapeutic. Methods: Using Chdh (a gene for betaine synthesis)-deficient mice and betaine-supplemented inbred mice, we assessed the role of betaine in psychiatric pathophysiology, and its potential as a novel psychotherapeutic, by leveraging metabolomics, behavioral-, transcriptomics and DNA methylation analyses. Findings: The Chdh-deficient mice revealed remnants of psychiatric behaviors along with schizophrenia-related molecular perturbations in the brain. Betaine supplementation elicited genetic background-dependent improvement in cognitive performance, and suppressed methamphetamine (MAP)-induced behavioral sensitization. Furthermore, betaine rectified the altered antioxidative and proinflammatory responses induced by MAP and in vitro phencyclidine (PCP) treatments. Betaine also showed a prophylactic effect on behavioral abnormality induced by PCP. Notably, betaine levels were decreased in the postmortem brains from schizophrenia, and a coexisting elevated carbonyl stress, a form of oxidative stress, demarcated a subset of schizophrenia with “betaine deficit-oxidative stress pathology”. We revealed the decrease of betaine levels in glyoxylase 1 (GLO1)-deficient hiPSCs, which shows elevated carbonyl stress, and the efficacy of betaine in alleviating it, thus supporting a causal link between betaine and oxidative stress conditions. Furthermore, a CHDH variant, rs35518479, was identified as a cis-expression quantitative trait locus (QTL) for CHDH expression in postmortem brains from schizophrenia, allowing genotype-based stratification of schizophrenia patients for betaine efficacy. Interpretation: The present study revealed the role of betaine in psychiatric pathophysiology and underscores the potential benefit of betaine in a subset of schizophrenia. Fund: This study was supported by the Strategic Research Program for Brain Sciences from AMED (Japan Agency for Medical Research and Development) under Grant Numbers JP18dm0107083 and JP19dm0107083 (TY), JP18dm0107129 (MM), JP18dm0107086 (YK), JP18dm0107107 (HY), JP18dm0107104 (AK) and JP19dm0107119 (KH), by the Grant-in-Aid for Scientific Research on Innovative Areas from the MEXT under Grant Numbers JP18H05435 (TY), JP18H05433 (AH.-T), JP18H05428 (AH.-T and TY), and JP16H06277 (HY), and by JSPS KAKENHI under Grant Number JP17H01574 (TY). In addition, this study was supported by the Collaborative Research Project of Brain Research Institute, Niigata University under Grant Numbers 2018–2809 (YK) and RIKEN Epigenetics Presidential Fund (100214–201801063606-340120) (TY). Keywords: Schizophrenia, Chdh, DNA methylation, Postmortem brain, Carbonyl stress, iPS cell

Recent studies have lent support to the possibility that inflammation is associated with the pathology of schizophrenia. In the study of measurement of inflammatory mediators, which are markers of inflammation, elevated inflammatory cytokine levels in the brain and blood have been reported in patients with schizophrenia. Several postmortem brain studies have also reported changes in the expression of inflammatory cytokines. However, it is not clear how these elevated inflammatory cytokines interact with other inflammatory mediators, and their association with the pathology of schizophrenia. We comprehensively investigated the expression of 30 inflammatory mediators in the superior temporal gyrus (STG) of 24 patients with schizophrenia and 26 controls using a multiplex method. Overall, inflammatory mediator expression in the STG was mostly unchanged. However, the expression of interleukin (IL)1-α and interferon-gamma-inducible protein (IP)-10 was decreased [IL-1α, median (IQR), 0.51 (0.37–0.70) vs. 0.87 (0.47–1.23), p = 0.01; IP-10, 13.99 (8.00–36.64) vs. 30.29 (10.23–134.73), p = 0.05], whereas that of IFN-α was increased [2.34 (1.84–4.48) vs. 1.94 (1.39–2.36), p = 0.04] in schizophrenia, although these alterations did not remain significant after multiple testing. Clustering based on inflammatory mediator expression pattern and analysis of upstream transcription factors using pathway analysis revealed that the suppression of IL-1α and IP-10 protein expression may be induced by regulation of a common upstream pathway. Neuroinflammation is important in understanding the biology of schizophrenia. While neuroimaging has been previously used, direct observation to determine the expression of inflammatory mediators is necessary. In this study, we identified protein changes, previously unreported, using comprehensive protein analysis in STG. These results provide insight into post-inflammatory alternation in chronic schizophrenia.

Sulawesi, an island located in a biogeographical transition zone between Indomalaya and Australasia, is famous for its high levels of endemism. Ricefishes (family Adrianichthyidae) are an example of taxa that have uniquely diversified on this island. It was demonstrated that habitat fragmentation due to the Pliocene juxtaposition among tectonic subdivisions of this island was the primary factor that promoted their divergence; however, it is also equally probable that habitat fusions and resultant admixtures between phylogenetically distant species may have frequently occurred. Previous studies revealed that some individuals of Oryzias sarasinorum endemic to a tectonic lake in central Sulawesi have mitochondrial haplotypes that are similar to the haplotypes of O. eversi, which is a phylogenetically related but geologically distant (ca. 190 km apart) adrianichthyid endemic to a small fountain. In this study, we tested if this reflects ancient admixture of O. eversi and O. sarasinorum. Population genomic analyses of genome-wide single-nucleotide polymorphisms revealed that O. eversi and O. sarasinorum are substantially reproductively isolated from each other. Comparison of demographic models revealed that the models assuming ancient admixture from O. eversi to O. sarasinorum was more supported than the models assuming no admixture; this supported the idea that the O. eversi-like mitochondrial haplotype in O. sarasinorum was introgressed from O. eversi. This study is the first to demonstrate ancient admixture of lacustrine or pond organisms in Sulawesi beyond 100 km. The complex geological history of this island enabled such island-wide admixture of lacustrine organisms, which usually experience limited migration.

Protamine, an antagonizing agent to heparin, is indispensable for dogs undergoing cardiopulmonary bypass. Protamine-induced hypotension (PIH) during cardiac anesthesia has been reported in humans. The purpose of this study was to describe the hemodynamic effect of protamine administration in dogs during cardiac surgery in clinical cases. Study design: Retrospective, clinical, cohort study. A total of 14 client-owned dogs who suffered heart failure due to medically uncontrolled myxomatous mitral valve disease (MMVD) were included in this study. The severity of MMVD was classified according to American College of Veterinary Internal Medicine staging (ACVIM: stage B2, C, D) and dogs undergoing mitral valve surgery. Records with clinical data for dogs treated between July 2019 to August 2020 were examined for age, sex, breed, body weight, concurrent diseases, hospitalization, anesthetic record, and mortality within 3 months after the operation. PIH was defined as mean arterial pressure (MAP) lowered by 20% of that before protamine infusion. To evaluate the effect of protamine on hemodynamic variables, each of the other values was compared with values at the beginning of protamine infusion. MAP decreased by 41.0 and 45.7% in two dogs (14.3%) compared with pressure before protamine infusion. Others did not show obvious alteration in hemodynamic variables. Epinephrine treatment alleviated hypotension in one dog. Another dog with systemic hypotension concomitant with elevated central venous pressure did not respond to epinephrine treatment and a reboot of extracorporeal circulation was required. Reheparinization and reinstitution of cardiopulmonary bypass successfully resuscitate the second dog. In conclusion, clinicians should alert the incidence of severe hypotension even with slow protamine infusion following canine cardiac surgery. This study also provides two effective treatments for catastrophic hypotension during protamine infusion.

Chronic pain and sleep have a bidirectional relationship that promotes a vicious circle making chronic pain more difficult to treat. Therefore, pain and sleep should be treated simultaneously. In our previous study, we suggested that hyperactivation of ascending serotonergic neurons could cause secondary sleep disturbance in chronic pain. This study aimed to demonstrate the effects of a tricyclic antidepressant (amitriptyline) and a selective 5-hydroxy-tryptamine 2A (5-HT2A) antagonist (MDL 100907) that adjust serotonergic transmission, on secondary sleep disturbance induced in a preclinical chronic pain model. We produced a chronic neuropathic pain model by partial sciatic nerve ligation in mice, analyzed their electroencephalogram (EEG) and electromyogram (EMG) using the SleepSign software, and evaluated the sleep condition of the pain model mice after administration of amitriptyline or MDL 100907. Amitriptyline improved thermal hyperalgesia and the amount of sleep, especially non-REM sleep. Time change of normalized power density of δ wave in the nerve ligation group with amitriptyline administration showed a normal pattern that was similar to sham mice. In addition, MDL 100907 normalized sleep condition similar to amitriptyline, without improvement in pain threshold. In conclusion, amitriptyline could improve sleep quantity and quality impaired by chronic pain. 5-HT2A receptor antagonism could partially contribute to this sleep improvement, but is not associated with pain relief.

A 73-year-old-male patient with hypertension and grade 3B chronic kidney disease underwent radical nephrectomy in the year 2000. As the renal function gradually worsened, the patient was started on hemodialysis in 2003. Eleven years after nephrectomy, the metastases were pointed out. The patient received sunitinib as first-line therapy. Thereafter, as the metastatic lesions became larger, we initiated nivolumab therapy (3 mg/m2 every 2 weeks). After 52 weeks, the metastatic lesions continued showing complete response (CR). No adverse effects were observed during nivolumab treatment.

Purpose: The purpose of this study was to show the effectiveness of combining intra-arterial infusion and systemic chemotherapy with concurrent radiotherapy for treatment of stage IV mandibular gingival cancer.Materials and Methods: A total of 23 patients with mandibular gingival cancer were treated with either docetaxel by intra-arterial infusion followed by systemic chemoradiotherapy with cisplatinum and 5-fluorouracil as a monthly regimen, or with docetaxel and cisplatinum by intra-arterial infusion followed by systemic chemoradiotherapy with 5-fluorouracil as a weekly or biweekly regimen. Tumor responses, locoregional control, overall survival, disease-specific survival, and adverse events were evaluated.Results: Of the 23 patients enrolled in the study, 22 completed the treatment. With regard to clinical stages, 82 % were diagnosed as IVA and 18 % IVB. Complete and partial response was observed in 82 and 18 %, respectively. Five-year overall survival, disease-specific survival, and locoregional control were 51, 70, and 72 %, respectively. No statistically significant difference was seen between the monthly regimen and the weekly plus biweekly regimen, although the latter resulted in longer survival and 88 % control.Conclusion: Combined intra-arterial infusion and systemic chemoradiotherapy may be an effective treatment for patients with stage IV mandibular gingival cancer. [ABSTRACT FROM AUTHOR]

Phosphoinositides (PIs) play important roles in the structure and function of the brain. Associations between PIs and the pathophysiology of schizophrenia have been studied. However, the significance of the PI metabolic pathway in the pathology of schizophrenia is unknown. We examined the expression of PI signaling-associated proteins in the postmortem brain of schizophrenia patients. Protein expression levels of phosphatidylinositol 4-phosphate 5-kinase type-1 gamma (PIP5K1C), phosphatidylinositol 4-kinase alpha (PIK4CA, also known as PIK4A), phosphatase and tensin homolog deleted from chromosome 10 (PTEN), protein kinase B (Akt), and glycogen synthase kinase 3β (GSK3β) were measured using enzyme-linked immunosorbent assays and multiplex fluorescent bead-based immunoassays of the prefrontal cortex (PFC) of postmortem samples from 23 schizophrenia patients and 47 normal controls. We also examined the association between PIK4CA expression and its genetic variants in the same brain samples. PIK4CA expression was lower, whereas Akt expression was higher, in the PFC of schizophrenia patients than in that of controls; PIP5K1C, PTEN, and GSK3β expression was not different. No single-nucleotide polymorphism significantly affected protein expression. We identified molecules involved in the pathology of schizophrenia via this lipid metabolic pathway. These results suggest that PIK4CA is involved in the mechanism underlying the pathogenesis of schizophrenia and is a potential novel therapeutic target.

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman disease. Although capsid formation and maturation in the alpha-herpesvirus herpes simplex virus 1 are well understood, these processes in KSHV remain unknown. The KSHV ORF7, encoding the viral terminase (DNA cleavage and packaging protein), is thought to contribute to capsid formation; however, functional information is lacking. Here, we investigated the role of ORF7 during KSHV lytic replication by generating two types of ORF7 knock-out (KO) mutants (frameshift-induced and stop codon-induced ORF7 deficiency), KSHV BAC16, and its revertants. The results revealed that both ORF7-KO KSHVs showed significantly reduced viral production but there was no effect on lytic gene expression and viral genome replication. Complementation assays showed virus production from cells harboring ORF7-KO KSHV could be recovered by ORF7 overexpression. Additionally, exogenously expressed ORF7 partially induced nuclear relocalization of the other terminase components, ORF29 and ORF67.5. ORF7 interacted with both ORF29 and ORF67.5, whereas ORF29 and ORF67.5 failed to interact with each other, suggesting that ORF7 functions as a hub molecule in the KSHV terminase complex for interactions between ORF29 and ORF67.5. These findings indicate that ORF7 plays a key role in viral replication, as a component of terminase.

Among polyphenol compounds, the flavan-3-ol structure, which is the basic unit of green tea catechins and the galloyl groups contained in green tea catechins are known to exhibit various functions. In this paper, we discuss how to concentrate highly functional polyphenol compounds by exploiting the interaction between gelatin and the catechol structures. First, we confirmed the interaction between heat-stabilized gelatin and flavan-3-ol derivatives, including synthesized compounds. When green tea leaf extract containing a large amount of flavan-3-ol derivatives was incubated with gelatin, most of the polyphenol compounds it contained were adsorbed. Because the compounds adsorbed on gelatin could not be eluted, DPPH radical and ABTS radical scavenging activity tests were conducted using the as-prepared gelatin–polyphenol complex. Radical scavenging activity was observed when the compounds were adsorbed on gelatin and heating at 90 °C for 5 min did not have a significant effect on their activity. These results suggest that functional polyphenols can be efficiently concentrated using heat-stabilized gelatin and retain their functionality while adsorbed.

Deoxynojirimycin (DNJ) is the archetypal iminosugar, in which the configuration of the hydroxyl groups in the piperidine ring truly mimic those of d-glucopyranose; DNJ and derivatives have beneficial effects as therapeutic agents, such as anti-diabetic and antiviral agents, and pharmacological chaperones for genetic disorders, because they have been shown to inhibit α-glucosidases from various sources. However, attempts to design a better molecule based solely on structural similarity cannot produce selectivity between α-glucosidases that are localized in multiple organs and tissues, because the differences of each sugar-recognition site are very subtle. In this study, we provide the first example of a design strategy for selective lysosomal acid α-glucosidase (GAA) inhibitors focusing on the alkyl chain storage site. Our design of α-1-C-heptyl-1,4-dideoxy-1,4-imino-l-arabinitol (LAB) produced a potent inhibitor of the GAA, with an IC50 value of 0.44 µM. It displayed a remarkable selectivity toward GAA (selectivity index value of 168.2). A molecular dynamic simulation study revealed that the ligand-binding conformation stability gradually improved with increasing length of the α-1-C-alkyl chain. It is noteworthy that α-1-C-heptyl-LAB formed clearly different interactions from DNJ and had favored hydrophobic interactions with Trp481, Phe525, and Met519 at the alkyl chain storage pocket of GAA. Moreover, a molecular docking study revealed that endoplasmic reticulum (ER) α-glucosidase II does not have enough space to accommodate these alkyl chains. Therefore, the design strategy focusing on the shape and acceptability of long alkyl chain at each α-glucosidase may lead to the creation of more selective and practically useful inhibitors.

We investigated the mechanisms associated with projected early-summer low-temperature extremes in Japan at the end of the 21st century by means of a well-developed nonhydrostatic regional climate model under the A1B scenario provided by the Intergovernmental Panel on Climate Change-Special Report on Emission Scenario. The projected surface air temperature reveals that even in a climate warmer than that at present, extremely low daily minimum temperatures in early summer are comparable to those in the present climate at several locations. At locations where future low temperatures are remarkable, the temperature drop at night is larger in the future than at present. This temperature drop results from mainly two heat fluxes: upward longwave radiation and latent heat flux. In the future climate, upward longwave radiation increases owing to high temperature at the surface around the time of the sunset. In addition, the upward flux of latent heat increases owing to low relative humidity just above the surface. These dryer conditions are associated with lower relative humidity at 850 hPa, suggesting the effects of synoptic systems. These two fluxes act to reduce the surface temperature, and hence surface air temperature.

Berry fruits are well known to contain large amounts of polyphenol compounds. Among them, flavan-3-ol derivatives are a group of secondary metabolism compounds currently attracting a great deal of attention owing to their health benefits. Not only the fruits, but also the leaves of raspberry plants, are highly esteemed for tea making around the world and are largely used for food. In this report, we discuss the results of our study on the effect of light and temperature on polyphenol accumulation in raspberry leaves. When raspberry was cultivated in a plant factory unit and light intensity, wavelength, and temperature were varied, the amount of total polyphenol increased under blue light. Quantitative determination of (+)-catechin, (–)-epicatechin, procyanidin B4, flavan-3-ol trimer, which are flavan-3-ol derivatives, was carried out using HPLC, whereby we confirmed their increase under blue light. Semi-quantitative RT-PCR showed correlation between chalcone synthase (CHS) gene expression and the amounts of the compounds measured in the leaves.

The reaction of 2,4-diethoxy-6-trifluoromethyl-3,4-dihydro-2H-pyran (1) with aromatic compounds in refluxing acetonitrile in the presence of p-toluenesulfonic acid gave the mixture of 4-aryl-2-trifluoromethyl-4H-pyrans (3) and 6-aryl-1,1,1-trifluorohexa-3,5-dien-2-ones (4). In contrast, the same reaction carried out in trifluoroacetic acid at ambient temperature afforded 4-aryl-2-ethoxy-6-trifluoromethyl-3,4-dihydro-2H-pyrans (2) selectively. These two types of reactions giving quite different products under each condition were studied on the basis of DFT calculations. Moreover, the proposed mechanism for the reaction of 5-trifluoroacetyl-6-trifluoromethyl-3,4-dihydro-2H-pyran (5) with aromatic compounds affording butadiene derivatives (6) exclusively was also discussed based on the calculations and comparison with the reactivity of pyrylium intermediate (7).

Introduction: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems are RNA-mediated adaptive immune systems that actagainst invading genetic elements such as phages or plasmids. CRISPR/Cas systems exist in nearly half of bacteria. Mycoplasma salivarium is a commensal species of the oropharynx. The American Type Culture Collection maintains five M. salivarium strains: ATCC 14277, 23064, 23557, 29803, and 33130. The genome sequence of ATCC 23064 revealed that it has an incomplete CRISPR/Cas system. However, the genome sequences of the remaining strains have not been analyzed., Methods: We performed polymerase chain reaction-amplicon sequencing and de novo genome sequencing to evaluate the presence of the CRISPR/Cas system in four strains., Results: Only ATCC 29803 possessed cas1, cas2, cas9 , and csn2 genes, a CRISPR array, and tracrRNA. The sequences of most components were identical between the CRISPR/Cas systems of ATCC 29803 and ATCC 23064, whereas the spacer sequences and a region of the cas9 gene were different. Unlike the CRISPR/Cas system of ATCC 23064, the cas9 gene of ATCC 29803 was not disrupted by the presence of stop codons., Conclusion: ATCC 29803 possesses genomic components required to express the type II-A CRISPR/Cas system, which potentially functions as an RNA-guided endonuclease., Competing Interests: No potential conflict of interest was reported by the authors., (© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Bacteria in genus Mycoplasma spp. are the smallest and simplest form of freely replicating bacteria, with 16 species known to infect humans. In the mouth, M. salivarium is the most frequently identified species. Mycoplasma spp. are parasites with small genomes. Although most of the Mycoplasma spp. that infect humans remain attached to the host cell surface throughout their life cycle, we have previously reported the presence of Mycoplasma salivarium in the epithelial cells of oral leukoplakia and oral lichen planus. However, the mechanism underlying the pathogenicity of M. salivarium has remained unclear. Further studies are needed to identify the process of infection of human cells and the stages in the life cycle of M. salivarium . Electron microscopy (EM) is the method of choice for morphological investigation of Mycoplasma spp. in cells or tissues. This study was performed to clarify and detail the ultrastructure of M. salivarium in tissue biopsies of oral mucosal leukoplakia, using three EM methods: (1) a standard EM processing method; (2) an ultracryotomy and immunolabeling method; and (3) the LR White resin post-embedding and immunolabeling method. This study included five oral leukoplakia tissue samples showing hyperplasia and hyperkeratosis. Although there was some variation in ultrastructural appearances between the three EM methods used, there were four ultrastructural appearances that are believed to reflect the stages of the M. salivarium life cycle in the epithelial cells of the oral mucosa: (1) small, electron-dense cellular-like structures or elementary bodies of M. salivarium ; (2) large structures of M. salivarium ; (3) M. salivarium organisms in cell division; (4) the sequence of events in the life cycle of M. salivarium that includes: (a) elementary bodies of M. salivarium deep in the oral mucosal epithelium; (b) replication by binary fission and daughter cell division from the elementary bodies; (c) maturation or degeneration of M. salivarium in the epithelial cells mainly in the upper part of the epithelium; and (d) death of the organisms in the granular and/or keratinized layer. These ultrastructural images may provide a useful reference for the identification of M. salivarium in diagnostic cytology or biopsy material.

Background: Oral lichen planus (OLP) is a T-cell-mediated inflammatory disease; however, its exact etiology is unknown. Hyperkeratosis is often observed in OLP lesions. Previous studies have revealed the localization of Mycoplasma salivarium in the epithelial cells of oral leukoplakia with hyperkeratosis. Herein, we investigated the presence of M. salivarium in OLP tissue by immunohistochemistry to determine the causative factor of OLP., Methods: Forty-one formalin-fixed, paraffin-embedded samples obtained from 31 patients with OLP were examined. Ten samples of normal-appearing oral mucosa were used as controls. Immunohistochemistry (IHC) was performed using anti-M. salivarium monoclonal antibodies., Results and Conclusions: Mycoplasma salivarium was detected in the epithelium and lymphocyte infiltrate area in 24 of 41 OLP samples (58.5%). The bacteria were intracellularly localized in epithelial cells, while it was unclear whether they were also localized in lymphocyte cells or in the extracellular spaces among the lymphocytes in the subepithelial lymphocyte infiltrate area. Little or no staining was observed in the epithelium in the normal-appearing mucosa samples. Sawtooth rete ridge formation was observed in 21 OLP samples (51.2%), and a significant positive correlation between sawtooth rete ridge formation and IHC positivity was demonstrated. However, the role of M. salivarium in the epithelium and lamina propria of OLP tissue remains unknown., (© 2017 The Authors Journal of Oral Pathology & Medicine Published by John Wiley & Sons Ltd.)

Molecular mechanism underlying the invasion of oral cancer cells remains to be clarified. We previously demonstrated that transforming growth factor-β1 (TGF-β1) induces the expression of mesenchymal markers in human oral squamous cell carcinoma HSC-4 cells. Intriguingly, the expression of the epithelial-mesenchymal transition-related transcription factor Slug was also significantly upregulated upon TGF-β1 stimulation. However, the mechanism by which Slug transduces the TGF-β1-induced signal to enhance the invasiveness of HSC-4 cells is poorly understood. Proteomic analysis revealed that the expression of matrix metalloproteinase (MMP)-10 was upregulated in TGF-β1-stimulated cells. Additionally, a Boyden chamber assay revealed that the TGF-β1-induced increase in invasiveness of HSC-4 cells was significantly inhibited by MMP-10 small interfering RNA (siRNA). Intriguingly, Slug siRNA suppressed TGF-β1-induced expression of MMP-10. These results suggest that TGF-β1 induces invasion in HSC-4 cells through the upregulation of MMP-10 expression in a Slug-dependent manner. On the other hand, Slug siRNA suppressed TGF-β1-induced Wnt-5b expression. Wnt-5b significantly induced MMP-10 expression, whereas Wnt-5b siRNA suppressed the TGF-β1-induced increase in invasiveness, suggesting that TGF-β1-induced expression of MMP-10 and the resulting upregulation of invasiveness are mediated by Wnt-5b. Overall, these results suggest that TGF-β1 stimulates HSC-4 cell invasion through the Slug/Wnt-5b/MMP-10 signalling axis., (© The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Background: Mycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia is the most prevalent potentially malignant disorder of the oral mucosa; its etiology has not been defined. Our previous study with DNA-binding fluorescent dye suggested the presence of mycoplasmas in the epithelial cells of leukoplakia tissue., Objective: Our aim was to detect M. salivarium in the epithelial cells of leukoplakia by immunohistochemistry., Design: We produced a polyclonal antibody (PAb) reactive to Mycoplasma by injecting a rabbit with M. salivarium cells (ATCC 23064) mixed with complete Freund's adjuvant and a monoclonal antibody specific to M. salivarium by injecting M. salivarium cells (ATCC 23557) mixed with complete Freund's adjuvant into the footpads of a rat. Then, we attempted to detect M. salivarium in the epithelium of leukoplakia tissues by immunohistochemistry., Results: We obtained an antimycoplasma rabbit PAb reactive to all seven Mycoplasma species used in this study. Three hybridoma clones producing monoclonal antibodies specific to M. salivarium were obtained, and an M. salivarium-specific monoclonal antibody, designated 7-6H, was established. Immunohistochemistry with these antibodies revealed M. salivarium in the epithelial cells of leukoplakia with hyperplasia and hyperkeratosis on histology. PCR and sequencing verified the presence of M. salivarium DNA in the epithelial cells of leukoplakia., Conclusion: Intracellular M. salivarium was identified in the epithelial cells of leukoplakia., (© 2014 The Authors. Journal of Oral Pathology & Medicine Published by John Wiley & Sons Ltd.)

Temporomandibular joint (TMJ) disorders (TMD) are usually diagnosed based on the patient's clinical findings and the results of image investigations; however, understanding of the inflammatory process in TMJ is difficult. In addition, many of the TMJ disease types share common principal symptoms. Therefore, TMJ diseases in the early stage can be misdiagnosed with TMD. It is hypothesized that cytopathologic examination of the joint lavage fluids is useful in interpreting the TMD-associated inflammatory process from a cellular aspect. The aim of this study was to assess the TMJ lavage fluid cytopathologically in TMD patients. Thirty-nine patients, clinically diagnosed as TMD, were included in the present study. Clinical symptoms of the patients were recorded. Forty-four samples of TMJ lavage fluid were collected and paraffin-embedded cell sections were made by cell block tissue array method. Cytologic conditions in upper articular cavity of TMJ were cytopathologically diagnosed and were compared with the clinical symptoms of each patient. Cell components were detected in 22 of the 44 analyzed joint lavage fluids. There was a correlation between cytopathologic findings and clinical symptoms. Variety of cytopathology and inflammatory conditions in patients with similar clinical symptoms were also found. The results suggested that cytopathologic examination of the joint lavage fluids from TMD patients is helpful for gaining an understanding of the inner local conditions of TMJ at the cellular level., (Copyright © 2013 Wiley Periodicals, Inc.)

Peripheral odontoma is rare, and only two cases of congenital peripheral odontoma have been reported. Congenital oral fibroma is also rare. We describe a unique case of congenital peripheral developing odontoma accompanied by congenital teratomatous fibroma in an infant. Both tumors were difficult to detect on radiography. Two small masses were seen in the median anterior portion of the palatal mucosa of a 9-month-old boy. The masses had been present since birth and were surgically removed at age 28 months, when one of the masses had grown to a diameter of 8 mm. Histopathologic examination showed a fibrous lesion and a tooth germ-like rounded lesion composed of dental papilla, enamel organ, dentin, and cementum. Although congenital odontoma is rare, it should be considered when selecting appropriate treatment, as early radiographic detection is difficult.

Background: The product of the Wilms' tumor gene, WT1 protein, is a tumor antigen for various kinds of cancer, and WT1 peptide-based cancer immunotherapy is widely anticipated as a new possibility for cancer treatment. The aim of this study was to investigate the expression of WT1 from quantitative and morphological perspectives in oral squamous cell carcinoma (OSCC), the most widespread malignant neoplasm of the oral cavity., Methods: Six OSCC cell lines and tissue sections from 29 OSCC patients were analyzed. To detect WT1 expression, reverse transcription-polymerase chain reaction analysis (RT-PCR), real-time PCR, Western blots, and immunofluorescence flow cytometry for WT1 were performed on the cell lines, and immunohistochemistry and fluorescence in situ hybridization (FISH) were performed on the tissue sections., Results: WT1 mRNA was found overexpressed in one of the six OSCC cell lines, with expression levels higher than that seen in human leukemia cell line (K562). Immunohistochemical analysis of tissue sections showed overexpression of WT1 protein in two patients, concentrated mainly in the cytoplasm of the outer one to three cell layers of the cancer nests. This was consistent with the expression of WT1 mRNA observed by FISH. Meanwhile, WT1 was not detected on normal oral epithelium. WT1 protein was detected on actively proliferating cancer nests and even on elongated epithelial ridge where new droplet-cancer-nests were being formed and starting infiltration toward subepithelial layer., Conclusions: The results suggest that WT1 plays an important role in the pathogenesis of some types of OSCC, particularly in proliferation of the cancer cells., (© 2012 John Wiley & Sons A/S.)

The periodontal ligament (PDL) is a fibrous connective tissue located between the tooth root and the alveolar bone. We previously demonstrated that a single cell-derived culture of primarily cultured PDL fibroblasts has the potential to construct an endothelial cell (EC) marker-positive blood vessel-like structure, suggesting that the fibroblastic lineage cells in ligament tissue could act as the endothelial progenitor cells (EPCs), which regenerate to construct a vascular system around the damaged ligament tissue. Moreover, we showed that EPC-like fibroblasts expressed not only EC markers but also smooth muscle cell (SMC) markers. Generally, an interaction between ECs and SMCs regulates blood vessel development and remodeling, and is required for the formation of a mature and functional vascular network. However, the mechanism underlying the SMC differentiation of the ligament-derived EPC-like fibroblasts remains to be clarified. In this study, we showed that suppression of fibroblast growth factor 1 (FGF-1)-induced extracellular signal-regulated kinase 1/2 (ERK1/2) signaling with the MAPK/ERK kinase (MEK) inhibitor U0126 completely abolished the FGF-1-induced proliferation of the ligament-derived EPC-like fibroblasts. In addition, U0126 treatment of FGF-1-stimulated ligament-derived EPC-like fibroblasts significantly induced the SMC differentiation of the cells. Thus, FGF-1-induced ERK1/2 signaling not only promoted the proliferation of the ligament-derived EPC-like fibroblasts, but also suppressed the SMC differentiation of the cells, suggesting that FGF-1 controls the construction of a vascular network around the ligament tissue by regulating the proliferation and SMC differentiation of the EPC-like cells through ERK-mediated signaling.

Background: An in vitro cell culture system of dental epithelium is useful for the investigations of cellular differentiation and function of ameloblast in amelogenesis and of regenerative therapy in human tooth. However, there have been no immortalized human dental epithelial ameloblastic-lineage cell lines, which proliferate indefinitely and additionally produce enamel matrix proteins., Methods: We transfected two retroviral constructs of human telomerase reverse transcriptase (hTERT) cDNA and mouse cyclin-dependent kinase 4 (cdk4) cDNA into the primary ameloblastoma cells and isolated immortalized human dental epithelial cell lines of HAM1, HAM2 and HAM3. The three cell lines were examined by electron microscopy, assay of senescence-associated β-galactosidase activity, mRNA expression and immuno-reactivity of dental epithelial marker cell molecules and enamel matrix proteins., Results: They showed undifferentiated phenotypes in monolayer culture and did not have any β-galactosidase activity. The transcripts of dental epithelial cell markers of Msx2, Jagged1, Notch1, Sp3, Sp6, keratin 14 and keratin 18 were confirmed. In addition, mRNA and protein expression of ameloblastin and enamelin were also detected in three cell lines. All cells in the three cell lines were keratin 14- and 18-positive and some elongated cells were Jagged1-positive. Msx2-positive nuclei were noted in only HAM2 cells., Conclusion: We established three cell lines by transfection of hTERT and cdk4 cDNAs, which were characterized as dental epithelial progenitor cells containing ameloblast-lineage cell phenotype., (© 2010 John Wiley & Sons A/S.)

We studied 1809 patients with oral cancer who visited and were treated, in 2002, at the 148 institutions certified as training facilities by the Japanese Society of Oral and Maxillofacial Surgeons. Of these institutions, 39 are dental university hospitals, 44 are medical university hospitals, 64 are general hospitals, and for 1 institution, the classification was not known. The patients consisted of 1071 (59.2%) males and 738 (40.8%) females (male: female ratio, 1.45:1), who had a average age of 65.2 years. The tongue (40.2%) was the most common site affected, followed by the gingiva (32.7%), buccal mucosa (10.1%), and oral floor (9.0%). There were 6 cases of multiple intraoral cancers. On histopathological examinations, squamous cell carcinoma (88.7%) was the most common type found, followed by adenoid cystic carcinoma (2.1%), and mucoepidermoid carcinoma (1.7%). Cases classified as T2N0 were the most common (32.1%), followed by T1N0 (21.4%), T4N0 (8.0%), and T2N1 (7.6%). Distant metastasis occurred in 17 patients (1.0%). Nonepithelial tumors, among which malignant melanoma was the most common type, accounted for 1.8% of the tumors. The sizes of the nonepithelial malignant tumors ranged from 1.0 to 7.0 cm, with an average size of 3.7 cm.

Background: Arteriovenous malformations (AVMs) of the mandible are relatively rare and potentially life-threatening lesions. Treatment is usually difficult. This study presents a case with high-flow AVM of the mandible in which most of the AVM were occluded by transvenous coil embolization., Methods: Transvenous embolization using several size 57 microcoils and 3 Gianturco coils was performed through a right femoral vein access. The small residual AVM was occluded by superselective transarterial injection of cyanoacrylate., Results: Angiography after embolization showed almost complete obliteration of AVM. Panoramic radiograph 2 years after treatment confirmed reossification. There was no recurrence of the symptoms in a follow-up evaluation 2 years later., Conclusion: Transvenous coil embolization may be a safer and more effective method in the treatment of mandibular AVM., (Copyright 1999 John Wiley & Sons, Inc. Head Neck 21: 574-577, 1999.)

Behçet's disease is associated with the HLA-B51 antigen. However, it has not yet been clarified if the HLA-B51 gene itself is the susceptibility gene related to this disease or if it is some other non-HLA gene in linkage disequilibrium with HLA-B51. Therefore, we screened one of the HSP70 genes, HUM70t (HSP70-Hom), around the class III region and the microsatellite sequence located between the HLA-B and TNF genes for genetic polymorphism in BD. A comparison between patients with BD and healthy controls revealed no significant difference in the frequency of the HUM70t polymorphism. In the microsatellite sequence, Tau-a, in the region between the HLA-B and TNF genes, the frequency of 14 repetitions of GT was increased significantly and that of 11 repetitions was decreased significantly in the patient group. Further, the allelic distributions of the B51 antigen-associated microsatellite polymorphism differed significantly between patients and healthy controls, and in the B51 antigen-negative subjects, analysis of the microsatellite polymorphism also revealed a significant difference in the haplotype frequency between the patient and control groups. These results suggest that the HLA-B51 gene may not be the primary locus responsible for BD, and implicate some other gene(s) located between the TNF and HLA-B genes.

Three water-soluble derivatives of naphthalene endoperoxide were prepared to examine the toxicity of singlet oxygen towards Escherichia coli. On incubation without irradiation, these endoperoxides produce singlet oxygen thermolytically in a dose-dependent manner. The amount of singlet oxygen produced per unit time can be controlled by varying both the incubation temperature and the number of methyl substituents of the naphthalene endoperoxide derivatives. 3-(1,4-Dihydro-1,4-epidioxy-4-methyl-1-naphthyl)propionic acid (EP-1), one of the derivatives, inhibited E. coli growth dose- and incubation temperature-dependently and caused E. coli lethality. Furthermore, EP-1 did not induce superoxide dismutase or catalase in E. coli even when the cells were incubated in nutritionally rich medium containing trypticase/soy/yeast extract. Singlet oxygen, one of the reactive oxygen species, did not act as a signal for induction of superoxide dismutase and catalase, in contrast to the actions of superoxide and hydrogen peroxide.

Behçet's disease is known to be associated with HLA-B51. To address the possibility that a non-human leukocyte antigen (HLA) gene closely linked to the HLA-B gene, such as tumor necrosis factor (TNF)-alpha, TNF-beta, or ECl (the locus that determines the susceptibility to alloreactive natural killer [NK] cells), is involved in the susceptibility to Behçet's disease, NcoI and EcoRI restriction fragment length polymorphisms in the TNF-beta gene and the susceptibility to lysis by alloreactive NK cells were investigated in Behçet's patients. In our NcoI restriction fragment length polymorphism (RFLP) analysis in the TNF-beta gene, the frequency of the NcoI 5.5 kb homozygote was decreased considerably in the patients, especially those with the ocular lesions, in relation to the healthy controls. However, no significant difference was observed between these groups in the EcoRI RFLP band distribution in this gene or the in susceptibility to lysis by alloreactive NK cells. These results indicated that a non-HLA gene located around the TNF gene region centromic of the HLA-B gene was a candidate to control the genetic susceptibility to Behçet's disease.

During stereotaxic surgery anatomical structures and their extent on trajectories were identified by neural noise (field potential) obtained by semi-microelectrode technique. Locations of anatomical structures (the white mater, caudate nucleus, thalamus, subthalamic field and nuclei in the subthalamic field) were fed into microcomputer (NEC PC-8001) as 3 dimensional correlates and stored in minifloppy disc system (NEC PC-8031). Data with satisfactory recordings from 48 trajectories from 30 patients (18 parkinsonism, 5 central pain, 7 others) entered this study. Microcomputer was so programmed that locations of the trajectories and electrophysiologically identified anatomical structures at that location in various coronal, sagittal and horizontal planes were displayed. Accumulation of this display from various groups of patients automatically created electrophysiological atlas. For comparison of thus created electrophysiological atlas with anatomical atlas display was made on various sections of the Schaltenbrand and Bailey's Atlas which were also stored in the mimifloppy disc system. Electrophysiological topography of the thalamus and adjacent structures was reasonably similar to anatomical topography with minor, but significant individual variations. In most cases the ventral border of the thalamus was located in the close vicinity of CA-CP line, however, in some cases the thalamus was located deep into the subthalamic field. This was thought to be due to the difficulty in identifying the ventral border of the thalamus by this technique and in such occasions other neurophysiological method for identifying the ventral border of the thalamus, i.e., thalamocortical evoked potential and other stimulation technique, were necessary. Medial aspect of the V. im. nucleus emitted neural noise of different characteristics which in current program was recognized as the subthalamic field. This implies that the medial and lateral aspects of the V. im. nucleus are cytoarchitecturally different and that it is possible to differentiate the medial and lateral aspects of the V. im. nucleus by this technique. The lateral thalamic border, i.e., thalamocapsular border, also showed relatively wide range of individual variations. When various parameters including age, sex, diagnosis and width of the 3rd ventricles were examined for possible cause of these variations, a tendency was noted that the thalamocapsular border was medially placed when the width of the 3rd ventricle was small. In this system it is possible to display sequentially electrophysiologically identified structures along any trajectory. This display method, which was called "tract study," was very usefull for comprehensive understanding of location of trajectory in relation to individually varying anatomical structures...