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Retinoic acid (RA) is an active metabolite of vitamin A, which is an essential signaling molecule involved in cell fate decisions, such as differentiation, proliferation, and apoptosis, in a wide variety of cell types. Accumulated data have demonstrated that expression of RA-metabolizing enzymes, CYP26A1, B1, and C1 (cytochrome P450, family 26A1, B1, and C1, respectively), protects cells and tissues from exposure to RA through restriction of RA access to transcriptional machinery by converting RA to rapidly excreted derivatives. CYP26 enzymes play similar but separate roles in limiting the consequences of fluctuations in nutritional vitamin A. Recently, we found that RA depletion caused by expression of CYP26A1 promotes malignant behaviors of tumor cells derived from various tissues, implicating CYP26A1 as a candidate oncogene. We also showed that the expression levels of CYP26 enzymes are elevated in various types of cancer. We have provided evidence for oncogenic and cell survival properties of CYP26 enzymes, indicating that these molecules are possible therapeutic targets for CYP26-expressing malignancies.

Since its discovery in late 2019, severe acute respiratory syndrome coronavirus 2 has spread around the world, causing millions of deaths due to coronavirus disease 2019 (COVID-19). Numerous clinical and post-mortem investigations of COVID-19 cases have found myriad clinical and pathological manifestations of the disease. In this report, we present three autopsy cases in which, despite weaning from extracorporeal membrane oxygenation (ECMO), extensive intestinal epithelial shedding, probably due to ischemia, was followed by massive watery diarrhea and the spread of infection via the portal vein due to bacterial translocation, which resulted in cholangitis lenta. Thrombophilia was attributed to ECMO usage and COVID-19-related vascular endothelial damage. These cases provide instructive findings showing that the loss of the intestinal barrier may be the underlying cause of severe watery diarrhea and liver failure in COVID-19 patients, especially with ECMO usage.

Recent studies have revealed that aberrant expression of tight junction (TJ) proteins is a hallmark of various solid tumors and it is recognized as a useful therapeutic target. Claudin-6 (CLDN6), a member of the family of TJ transmembrane proteins, is an ideal therapeutic target because it is not expressed in human adult normal tissues. In this study, we found that CLDN6 is highly expressed in uterine cervical adenocarcinoma (ADC) and that high CLDN6 expression was correlated with lymph node metastasis and lymphovascular infiltration and was an independent prognostic factor. Shotgun proteome analysis revealed that cell-cell adhesion-related proteins and drug metabolism-associated proteins (aldo-keto reductase [AKR] family proteins) were significantly increased in CLDN6-overexpressing cells. Furthermore, overexpression of CLDN6 enhanced cell-cell adhesion properties and attenuated sensitivity to anticancer drugs including doxorubicin, daunorubicin, and cisplatin. Taken together, the results indicate that aberrant expression of CLDN6 enhances malignant potentials and drug resistance of cervical ADC, possibly due to increased cell-cell adhesion properties and drug metabolism. Our findings provide an insight into a new therapeutic strategy, a CLDN6-targeting therapy, against cervical ADC.

The tumor microenvironment plays an essential role in the development and progression of colorectal cancer (CRC). We recently reported that crosstalk between CRC cells and tumor-associated macrophages (TAMs) via serum amyloid A1 (SAA1) promotes invasion by T1 CRCs. In the present study, we aimed to clarify the role of neutrophils in early CRCs.Immunohistochemical analysis of CD66b, chemokine CXC motif ligand 8 (CXCL8 or interleukin-8, IL-8) and matrix metalloproteinase-9 (MMP-9) was performed using primary T1 CRCs (n = 49). The HL-60 human promyelocytic leukemia cell line and THP-1 human monocytic leukemia cell line were used to obtain neutrophil-like and macrophage-like cells, respectively. Boyden chamber assays were used to analyze cell migration and invasion, and quantitative RT-PCR was used to analyze gene expression.Immunohistochemical analysis revealed accumulation of neutrophils at the SAA1-positive invasive front of T1 CRCs. Experiments using HL-60 cells suggested that treatment with SAA1 induced neutrophil migration and expression of CXCL8 and MMP-9 in neutrophils and that neutrophils promote CRC cell migration and invasion. Immunohistochemistry confirmed accumulation of CXCL8- or MMP-9-positive neutrophils at the SAA1-positive invasive front of T1 CRCs. Moreover, co-culture experiments using CRC, THP-1 and HL-60 cells suggested that CRC cells activated by macrophages upregulate CXCL8 and MMP-9 in neutrophils.Our results suggest that interplay between macrophages and CRC cells leads to recruitment of neutrophils to the invasive front of T1 CRCs and that SAA1 secreted by CRC cells activate neutrophils to promote invasion.

Vitamin D is an essential nutrient for the human body that plays an important role in homeostasis and contributes to cell fate decision including proliferation, differentiation and cell viability. Accumulated epidemiological data suggest a relationship between vitamin D deficiency and carcinogenesis in various organs. However, the underlying mechanism remains to be further clarified. In this study, we showed that vitamin D metabolizing enzyme CYP24A1 promotes the oncogenic property of breast carcinoma cells. In addition, expression of CYP24A1 is elevated in invasive breast carcinoma and decreases the overall survival. Importantly, CYP24A1 suppression significantly enhances cell death sensitivity to two pharmacologically different acting anticancer drugs, cisplatin and gefitinib. The results of our study suggest the possibility of CYP24A1-inhibiting therapy for breast malignancy.

The CXCL12/CXCR4 axis plays a pivotal role in the progression of various malignancies, including oral squamous cell carcinoma (OSCC). In this study, we aimed to clarify the biological and clinical significance of CXCL12 in the tumor microenvironment of OSCCs.Publicly available single-cell RNA-sequencing (RNA-seq) datasets were used to analyze CXCL12 expression in head and neck squamous cell carcinomas (HNSCC). Immunohistochemical analysis of CXCL12, α-smooth muscle antigen (α-SMA), fibroblast activation protein (FAP) and CD8 was performed in a series of 47 surgically resected primary tongue OSCCs. Human skeletal muscle cells were co-cultured with or without OSCC cells, after which CXCL12 expression was analyzed using quantitative reverse-transcription PCR.Analysis of the RNA-seq data suggested CXCL12 is abundantly expressed in stromal cells within HNSCC tissue. Immunohistochemical analysis showed that in grade 1 primary OSCCs, CXCL12 is expressed in both tumor cells and muscle cells. By contrast, grade 3 tumors were characterized by disruption of muscle structure and reduced CXCL12 expression. Quantitative analysis of CXCL12-positive areas within tumors revealed that reduced CXCL12 expression correlated with poorer overall survival. Levels of CXCL12 expression tended to inversely correlate α-SMA expression and positively correlate with infiltration by CD8+ lymphocytes, though these relations did not reach statistical significance. CXCL12 was significantly upregulated in muscle cells co-cultured with OSCC cells.Our results suggest that tongue OSCC cells activate CXCL12 expression in muscle cells, which may contribute to tumor progression. However, CXCL12 is reduced in advanced OSCCs due to muscle tissue destruction.

Characterization of inter-regional interactions in brain is essential for understanding the mechanism relevant to normal brain function and neurological disease. The recently developed flexible micro (μ)-electrocorticography (μECoG) device is one prominent method used to examine large-scale cortical activity across multiple regions. The sheet-shaped μECoG electrodes arrays can be placed on a relatively wide area of cortical surface beneath the skull by inserting the device into the space between skull and brain. Although rats and mice are useful tools for neuroscience, current μECoG recording methods in these animals are limited to the parietal region of cerebral cortex. Recording cortical activity from the temporal region of cortex in mice has proven difficult because of surgical barriers created by the skull and surrounding temporalis muscle anatomy. Here, we developed a sheet-shaped 64-channel μECoG device that allows access to the mouse temporal cortex, and we determined the factor determining the appropriate bending stiffness for the μECoG electrode array. We also established a surgical technique to implant the electrode arrays into the epidural space over a wide area of cerebral cortex covering from the barrel field to olfactory (piriform) cortex, which is the deepest region of the cerebral cortex. Using histology and computed tomography (CT) images, we confirmed that the tip of the μECoG device reached to the most ventral part of cerebral cortex without causing noticeable damage to the brain surface. Moreover, the device simultaneously recorded somatosensory and odor stimulus-evoked neural activity from dorsal and ventral parts of cerebral cortex in awake and anesthetized mice. These data indicate that our μECoG device and surgical techniques enable the recording of large-scale cortical activity from the parietal to temporal cortex in mice, including somatosensory and olfactory cortices. This system will provide more opportunities for the investigation of physiological functions from wider areas of the mouse cerebral cortex than those currently available with existing ECoG techniques.

Aberrant expression of tight junction proteins has recently been focused on in the cancer research field. We previously showed that claudin-1 is aberrantly expressed from an early stage of uterine cervical adenocarcinoma and contributes to malignant potentials. To elucidate the molecular mechanisms underlying tumor-promoting roles of claudin-1, we established and analyzed claudin-1 knockout cells. Knockout of claudin-1 suppressed conventional tight junctional functions, barrier and fence functions, and expression of cell adhesion-associated proteins including E-cadherin. Comparative proteome analysis revealed that expression of claudin-1 affected expression of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling. Interactome analysis of the identified proteins revealed that E-cadherin and focal adhesion kinase play central roles in the claudin-1-dependently affected protein network. Moreover, knockout of claudin-1 significantly suppressed microvilli formation and activity of Ezrin/Radixin/Moesin. Taken together, the results indicate that expression of claudin-1 affects not only conventional tight junction function but also expression and activity of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling, to contribute to malignant potentials and microvilli formation in cervical adenocarcinoma cells.

Recent studies have shown that aberrant expression of tight junction proteins (TJP) contributes to malignant potential of various cancers. In the present study, we investigated the expression of junctional adhesion molecule‐A (JAM‐A), one of the transmembrane TJP, in uterine cervical adenocarcinoma and the significance of its expression for malignancy. Immunohistochemistry on human surgical specimens showed that JAM‐A was aberrantly expressed in neoplastic regions including adenocarcinoma in situ (AIS). Knockout of JAM‐A significantly suppressed cell proliferation and colony‐forming and migration abilities. We also showed that an antibody specific to an extracellular region of JAM‐A reduced cell proliferation ability and that loss of JAM‐A increased drug sensitivity of cervical adenocarcinoma cells. Based on a comprehensive proteome analysis, we found that poliovirus receptor (PVR/CD155) was regulated by JAM‐A and formed a physical interaction with JAM‐A. In human surgical specimens, PVR/CD155 expression was significantly correlated with some clinicopathological features and prognosis of cervical adenocarcinoma. Interestingly, most of the PVR/CD155‐positive cases expressed a high level of JAM‐A, and patients with the expression pattern of PVR/CD155 positive/JAM‐A high had significantly shorter periods of relapse‐free survival (P = .00964) and overall survival (P = .0204) than those for the other patients. Our observations suggest that aberrant expression of JAM‐A promotes malignancy of uterine cervical adenocarcinoma by regulation of PVR/CD155, and JAM‐A is therefore a potential therapeutic target for this malignancy., Aberrant expression of junctional adhesion molecule‐A (JAM‐A), one of the transmembrane tight junction proteins, contributes to the malignant potential of uterine cervical adenocarcinoma. We also show that loss of JAM‐A attenuated drug resistance of cervical adenocarcinoma cells and that an anti‐JAM‐A antibody inhibited cell proliferation, indicating that JAM‐A is a potential therapeutic target of the malignancy. Moreover, we show that a novel interaction between JAM‐A and poliovirus receptor (PVR/CD155) is associated with worse prognosis of cervical adenocarcinoma.

Desmoplastic reaction (DR) and inflammation are significant pathological manifestations of tumorigenesis in several cancers. However, the correlation between these stromal reactions and cervical adenocarcinoma has been poorly documented. This investigation elucidated whether DR is a prognostic indicator in early cervical adenocarcinoma patients. Fifty-nine patients with early stage cervical adenocarcinoma (stages I/II) were included in the study. DR was divided into three groups, mature, intermediate, and immature, based on the presence of myxoid stroma and hyalinized keloid-like collagen. Inflammatory cell responses were classified as mild, moderate, and severe. Those stromal reactions were separately evaluated in the invasion front stroma and intratumoral stroma. In both the intratumor and invasion front stroma, intermediate/immature DR was correlated with tumor size, T stage, N stage, lymphovascular invasion, and parametrial infiltration (p 0.001 to p 0.05). In addition, in the intratumoral stroma, intermediate/immature DR led to short relapse-free survival and overall survival (p 0.001). In the invasion front stroma, inflammatory cell responses were associated with DR immaturity and FIGO stage (p 0.01). These results suggest that the classification of DR maturity is a potential prognostic biomarker in early stage cervical adenocarcinoma patients. DR can be evaluated by routine HE staining without immunohistochemistry, making it convenient and economical in clinical practice.

We report a case of a 65-year-old patient with hypertension, dyslipidemia, type 2 diabetes, chronic kidney disease, and hyperuricemia, who showed an improvement in lifestyle-induced metabolic syndrome on the administration of 7.5 g of Kangen-karyu extract per day for 6 months. The levels of serum total cholesterol, low-density lipoprotein-cholesterol, and triglycerides were decreased. The systolic/diastolic blood pressure was decreased following administration. Other parameters such as estimated glomerular filtration rate, creatinine, uric acid, aspartate transaminase, alanine aminotransferase, γ-glutamyl transpeptidase, and creatine phosphokinase were improved by the administration of Kangen-karyu extract. At that time, the physical and subjective symptoms had partially disappeared. We present evidence supporting the use of Kangen-karyu extract against metabolic syndrome.

Recent studies have revealed that metabolic reprogramming is closely associated with epithelial‐mesenchymal transition (EMT) during cancer progression. Aldolase A (ALDOA) is a key glycolytic enzyme that is highly expressed in several types of cancer. In this study, we found that ALDOA is highly expressed in uterine cervical adenocarcinoma and that high ALDOA expression promotes EMT to increase malignant potentials, such as metastasis and invasiveness, in cervical adenocarcinoma cells. In human surgical specimens, ALDOA was highly expressed in cervical adenocarcinoma and high ALDOA expression was correlated with lymph node metastasis, lymphovascular infiltration, and short overall survival. Suppression of ALDOA expression significantly reduced cell growth, migration, and invasiveness of cervical cancer cells. Aldolase A expression was partially regulated by hypoxia‐inducible factor‐1α (HIF‐1α). Shotgun proteome analysis revealed that cell‐cell adhesion‐related proteins were significantly increased in ALDOA‐overexpressing cells. Interestingly, overexpression of ALDOA caused severe morphological changes, including a cuboidal‐to‐spindle shape shift and reduced microvilli formation, coincident with modulation of the expression of typical EMT‐related proteins. Overexpression of ALDOA increased migration and invasion in vitro. Furthermore, overexpression of ALDOA induced HIF‐1α, suggesting a positive feedback loop between ALDOA and HIF‐1α. In conclusion, ALDOA is overexpressed in cervical adenocarcinoma and contributes to malignant potentials of tumor cells through modulation of HIF‐1α signaling. The feedback loop between ALDOA and HIF‐1α could become a therapeutic target to improve the prognosis of this malignancy., Aldolase A (ALDOA) overexpression caused epithelial‐mesenchymal transition‐like morphological alterations in cervical adenocarcinoma cells (A‐C). ALDOA‐overexpressing cells showed increased stress fiber formation (D‐F, red) and reduced E‐cadherin expression (D‐F, green) and microvilli formation (G).

Claudins are major components of tight junctions that maintain cell polarity and intercellular adhesion. The dynamics of claudins in cancer cells have attracted attention as a therapeutic target. During carcinogenesis, claudin expression is generally downregulated; however, overexpression of claudin-18.2 has been observed in several types of cancers. Upregulated and mislocalized claudin-18.2 expression in cancer cells has been suggested as a therapeutic target. Research on claudin-18.2 has revealed its involvement in carcinogenesis. Clinical trials using zolbetuximab, a monoclonal antibody targeting claudin-18.2, for patients with advanced cancer yielded positive results with few high-grade adverse events; thus, it is expected to be a novel and effective therapeutic. Here, we review current insights into the role that claudin-18.2 plays in basic cancer research and clinical applications. A better understanding of these roles will facilitate the development of new treatment strategies for cancer patients with poor prognoses.

Submucosal invasion and lymph node metastasis are important issues affecting treatment options for early colorectal cancer (CRC). In this study, we aimed to unravel the molecular mechanism underlying the invasiveness of early CRCs. We performed RNA‐sequencing (RNA‐seq) with poorly differentiated components (PORs) and their normal counterparts isolated from T1 CRC tissues and detected significant upregulation of serum amyloid A1 (SAA1) in PORs. Immunohistochemical analysis revealed that SAA1 was specifically expressed in PORs at the invasive front of T1b CRCs. Upregulation of SAA1 in CRC cells promoted cell migration and invasion. Coculture experiments using CRC cell lines and THP‐1 cells suggested that interleukin 1β (IL‐1β) produced by macrophages induces SAA1 expression in CRC cells. Induction of SAA1 and promotion of CRC cell migration and invasion by macrophages were inhibited by blocking IL‐1β. These findings were supported by immunohistochemical analysis of primary T1 CRCs showing accumulation of M1‐like/M2‐like macrophages at SAA1‐positive invasive front regions. Moreover, SAA1 produced by CRC cells stimulated upregulation of matrix metalloproteinase‐9 in macrophages. Our data suggest that tumor‐associated macrophages at the invasive front of early CRCs promote cancer cell migration and invasion through induction of SAA1 and that SAA1 may be a predictive biomarker and a useful therapeutic target., We identified that serum amyloid A1 (SAA1) is upregulated in the poorly differentiated regions at the invasive front of T1 stage colorectal cancer (CRC). We found that tumor‐associated macrophages accumulated at the invasive front, where they stimulated SAA1 upregulation in CRC cells via interleukin 1β and promoted CRC cell migration and invasion. We also show that SAA1 secreted by CRC cells stimulates matrix metalloproteinase‐9 upregulation in macrophages at the invasive front.

To elucidate neural mechanisms underlying oscillatory phenomena in brain function, we have developed optogenetic tools and statistical methods. Specifically, opto-current-clamp induced oscillation reveals intrinsic frequency preferences in the neural circuits by oscillatory resonance. Furthermore, resonance or entrainment to intrinsic frequency is state-dependent. When resonance phenomena go beyond a certain range, it could even induce epileptic seizure in highly reproducible manner. We are able to study how seizures start, develop, and stop in neural circuits. Therefore, the optogenetics-induced oscillatory activation is a powerful tool in neuroscience research.

To elucidate the expression mechanisms of brain functions, we have developed an ultrathin fluorescence endoscope imaging system (U-FEIS) that can image cells in the brain at any depth while minimizing the invasion. The endoscope part of U-FEIS consists of a GRIN lens and a 10,000-pixel image fiber with a diameter of 450 μm. The specialized microscope of U-FEIS is within 30 cm square and includes lenses and optical filters optimized for the endoscope. Using U-FEIS, we successfully visualized neurons expressing GFP with single-cell resolution and recorded the multineuronal activities in vitro and in vivo. U-FEIS can also perform imaging and optical stimulation simultaneously. Therefore, U-FEIS should be a powerful optical tool in neuroscience research.

In the application of advanced neuroscience techniques including optogenetics to small awake animals, it is often necessary to restrict the animal's movements. A spherical treadmill is a beneficial option that enables virtual locomotion of body- or head-restrained small animals. Besides, it has a wide application range, including virtual reality experiments. This chapter describes the fundamentals of a spherical treadmill for researchers who want to start experiments with it. First, we describe the physical aspect of a spherical treadmill based on the simple mechanical analysis. Next, we explain the basics of data logging and preprocessing for behavioral analysis. We also provide simple computer programs that work for the purpose.

We have developed a Si opt-electro multifunctional neural probe with multiple waveguides and embedded optical fiber for highly accurate optical stimulation. The Si opt-electro multifunctional neural probe had 16 recording sites, three optical waveguides, and metal cover for suppressing light leakage. The other opt-electro multifunctional neural probe had an optical fiber in the trench of the probe shank, which leads to fewer damages to tissues. We evaluated the electrochemical properties of the recording sites and confirmed that the neural probe had suitable characteristics for neural recording. We also demonstrated the optical stimulation to the neurons expressing ChR2 using our probe. As a result, we succeeded in multisite optical stimulation and observed that no light leakage from the optical waveguides because of the metal cover. From in vivo experiments, we successfully recorded optically modulated local field potential using the fabricated Si neural probe with optical waveguides. Moreover, we applied current source density analysis to the recorded LFPs. As a result, we confirmed that the light-induced membrane current sinks in the locally stimulated area. The Si opto-electro multifunctional neural probe is one of the most versatile tools for optogenetics.

In the application of advanced neuroscience techniques including optogenetics to small awake animals, it is often necessary to restrict the animal’s movements. A spherical treadmill is a beneficial option that enables virtual locomotion of body- or head-restrained small animals. Besides, it has a wide application range, including virtual reality experiments. This chapter describes the fundamentals of a spherical treadmill for researchers who want to start experiments with it. First, we describe the physical aspect of a spherical treadmill based on the simple mechanical analysis. Next, we explain the basics of data logging and preprocessing for behavioral analysis. We also provide simple computer programs that work for the purpose.

To elucidate the expression mechanisms of brain functions, we have developed an ultrathin fluorescence endoscope imaging system (U-FEIS) that can image cells in the brain at any depth while minimizing the invasion. The endoscope part of U-FEIS consists of a GRIN lens and a 10,000-pixel image fiber with a diameter of 450 μm. The specialized microscope of U-FEIS is within 30 cm square and includes lenses and optical filters optimized for the endoscope. Using U-FEIS, we successfully visualized neurons expressing GFP with single-cell resolution and recorded the multineuronal activities in vitro and in vivo. U-FEIS can also perform imaging and optical stimulation simultaneously. Therefore, U-FEIS should be a powerful optical tool in neuroscience research.

To elucidate neural mechanisms underlying oscillatory phenomena in brain function, we have developed optogenetic tools and statistical methods. Specifically, opto-current-clamp induced oscillation reveals intrinsic frequency preferences in the neural circuits by oscillatory resonance. Furthermore, resonance or entrainment to intrinsic frequency is state-dependent. When resonance phenomena go beyond a certain range, it could even induce epileptic seizure in highly reproducible manner. We are able to study how seizures start, develop, and stop in neural circuits. Therefore, the optogenetics-induced oscillatory activation is a powerful tool in neuroscience research

The epithelial-mesenchymal transition (EMT) is an essential step in cancer progression. Epithelial cells possess several types of cell-cell junctions, and tight junctions are known to play important roles in maintaining the epithelial program. EMT is characterized by a loss of epithelial markers, including E-cadherin and tight junction proteins. Somewhat surprisingly, the evidence is accumulating that upregulated expression of tight junction proteins plays an important role in the EMT of cancer cells. Tight junctions have distinct tissue-specific and cancer-specific regulatory mechanisms, enabling them to play different roles in EMT. Tight junctions and related signaling pathways are attractive targets for cancer treatments; signal transduction inhibitors and monoclonal antibodies for tight junction proteins may be used to suppress EMT, invasion, and metastasis. Here we review the role of bicellular and tricellular tight junction proteins during EMT. Further investigation of regulatory mechanisms of tight junctions during EMT in cancer cells will inform the development of biomarkers for predicting prognosis as well as novel therapies.

Cervical adenocarcinomas are believed to lose estrogen response on the basis of no expression of a nuclear estrogen receptor such as ERα in clinical pathology. Here, we demonstrated that cervical adenocarcinoma cells respond to a physiological concentration of estrogen to upregulate claudin-1, a cell surface molecule highly expressed in cervical adenocarcinomas. Knockout of claudin-1 induced apoptosis and significantly inhibited proliferation, migration, and invasion of cervical adenocarcinoma cells and tumorigenicity in vivo. Importantly, all of the cervical adenocarcinoma cell lines examined expressed a membrane-bound type estrogen receptor, G protein–coupled receptor 30 (GPR30/GPER1), but not ERα. Estrogen-dependent induction of claudin-1 expression was mediated by GPR30 via ERK and/or Akt signaling. In surgical specimens, there was a positive correlation between claudin-1 expression and GPR30 expression. Double high expression of claudin-1 and GPR30 predicts poor prognosis in patients with cervical adenocarcinomas. Mechanism-based targeting of estrogen/GPR30 signaling and claudin-1 may be effective for cervical adenocarcinoma therapy.