👤 Tadashige Nozaki

Canine hepatocellular carcinoma (HCC) requires further molecular characterization to identify diagnostic and therapeutic targets, and to establish whether dogs with this condition can model the human disease. Accordingly, we aimed to identify differentially expressed genes (DEGs) in canine HCC and evaluate cross-species transcriptomic dysregulation in canine and human HCC. Liver tissue samples from three dogs with HCC and three healthy dogs were subjected to next-generation sequencing, followed by RT-qPCR validation. Identified DEGs were then targeted in bioinformatics analyses (pathway enrichment, protein-protein interaction network, and hub gene analyses) for molecular characterization and comparison with human HCC datasets. We identified 975 DEGs (upregulated: 604; and downregulated: 371). Extracellular matrix-receptor interaction, focal adhesion, cell adhesion molecule, PI3K/Akt signaling, and cytokine/chemokine-related pathways were enriched. C1R, APOC3, C1QA, APOA1, C1QB, ACTG1, C1QC, CRP, ANXA5, and ANXA2 were identified as hub genes. Canine and human HCCs share 118 DEGs, highlighting conserved alterations in metabolic pathways, PI3K-Akt signaling, focal adhesion, and PPAR signaling pathways. Based on human HCC data, SPP1, NQO1, RRM2, APOA1, APOC3, ALDOB, and IGF1 were identified as prognosticators indicating poor overall survival. This study presents the first cross-species transcriptomic analysis of canine HCC, revealing significant molecular resemblances to human HCC, indicating it may be a promising comparative model for studying tumor biology, drug responses, and novel therapeutic interventions. Show less

Research indicates that impairment of instrumental activities of daily living (IADLs) leads to reduced physical activity (PA) in daily life. However, these studies often rely on subjective measures such as questionnaires and interviews to assess PA. This study examined the association between IADL frequency and objectively measured PA in stable individuals with cardiovascular disease (CVD). In this cross-sectional study, we included people with CVD who had been receiving outpatient care under stable conditions for at least 6 months. IADL frequency was assessed using the Frenchay Activities Index (FAI). PA was measured using accelerometers over 2 weeks to calculate the daily average number of steps, low-intensity PA (LPA), and moderate-to-vigorous-intensity PA (MVPA). A multivariate linear regression model analyzed the associations between the FAI scores (total and sub-items) and PA levels. This study included 1126 stable participants with CVD (median age, 74.0 years; 278 females). After adjusting for clinical confounding factors, a high FAI total score was significantly associated with higher levels of PA (number of steps per day, unstandardized coefficient [В] = 78.1, LPA per day, В = 0.7, and MVPA per day, В = 0.2). In the FAI subitems, 4 housework and 6 leisure activities were positively associated with the daily average number of steps and LPA, and 2 leisure activities were positively associated with daily MVPA. Greater IADL frequency was associated with higher objectively measured PA in stable participants with CVD. Leisure-related activities were associated with increased MVPA, suggesting that encouraging these activities may help promote meaningful PA engagement in this population. Show less

Developing strategies for the radiosensitization of cancer cells by the inhibition of genes, which harbor low toxicity to normal cells, will be useful for improving cancer radiotherapy. Here, we focused on a β-site of amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1; β-secretase, memapsin-2). By functional inhibition of this peptidase by siRNA, it has also recently been shown that the DNA strand break marker, γH2AX foci, increased, suggesting its involvement in DNA damage response. To investigate this possibility, we knocked down Show less

The epithelial-to-mesenchymal transition (EMT) is a critical process by which cancer cells acquire malignant features. However, the molecular mechanism and functional implications of EMT and the mesenchymal-to-epithelial transition (MET) in tumor progression remain elusive. In this study, we established two aggressive cancer cell lines from the human oral cancer cell line SAS, mesenchymal-like SAS-m4 and epithelial-like SAS-δ. SAS-δ is a revertant cell obtained by inducing MET in SAS-m4. SAS-δ, but not SAS-m4, exhibited abnormal cell growth, including piled-up overgrowth and invasive tumor formation in the tongues of nude mice, suggesting that SAS-δ represented more advanced cancer cells than the parental SAS cells. EMT-related transcriptional factor SLUG is phosphorylated at T208 and partly stabilized by the Hippo pathway kinases, LATS1 and LATS2. Depletion of SLUG promoted the invasive activity of SAS-δ by increasing the protein levels of LATS1/2 and the proportion of the phosphorylated form among total SLUG protein. Our results suggest that the LATS1/2-SLUG axis regulates the transition of SAS cells to the advanced stage via repeated switching between EMT and MET. Therefore, an anti-SLUG-pT208 antibody would be valuable not alone as a malignant tumor marker antibody but also as a prognostic tool for patients with malignant disease. Show less

In this study, we elucidated the architectures of two multisubunit complexes, the BBSome and exocyst, through a novel application of fluorescent fusion proteins. By processing lysates from cells co-expressing GFP and RFP fusion proteins for immunoprecipitation with anti-GFP nanobody, protein-protein interactions could be reproducibly visualized by directly observing the immunoprecipitates under a microscope, and evaluated using a microplate reader, without requiring immunoblotting. Using this 'visible' immunoprecipitation (VIP) assay, we mapped binary subunit interactions of the BBSome complex, and determined the hierarchies of up to four subunit interactions. We also demonstrated the assembly sequence of the BBSome around the centrosome, and showed that BBS18 (also known as BBIP1 and BBIP10) serves as a linker between BBS4 and BBS8 (also known as TTC8). We also applied the VIP assay to mapping subunit interactions of the exocyst tethering complex. By individually subtracting the eight exocyst subunits from multisubunit interaction assays, we unequivocally demonstrated one-to-many subunit interactions (Exo70 with Sec10+Sec15, and Exo84 with Sec10+Sec15+Exo70). The simple, versatile VIP assay described here will pave the way to understanding the architectures and functions of multisubunit complexes involved in a variety of cellular processes. Show less

Adenosine monophosphate-activated protein kinase (AMPK) is a sensor for cellular energy status. When the cellular energy level is decreased, AMPK is activated and functions to suppress energy-consuming processes, including protein synthesis. Recently, AMPK has received attention as an attractive molecular target for cancer therapy. Several studies have revealed that the activation of AMPK by chemical stimulators, such as metformin, induces apoptosis in a variety of hematologic malignant cells. From another perspective, these results suggest that the function of AMPK is impaired in hematologic tumor cells. However, the precise mechanisms by which this impairment occurs are not well understood. In melanoma cells, oncogenic BRAF constitutively activates the extracellular signal-regulated kinase (ERK) pathway and phosphorylates liver kinase B1, an upstream activator of 5' adenosine monophosphate-activated protein kinase (AMPK), resulting in the inactivation of liver kinase B1 and AMPK. In this study, we analyzed whether ERK is involved in the suppression of AMPK activity using established and primary human leukemia cells. We found an inverse correlation between the intensity of ERK activity and the degree of AMPK activation after stimulation with either glucose deprivation or metformin. We also found that the inhibition of ERK activity by U0126 restored AMPK activation after metformin treatment. Furthermore, a combined treatment with metformin and U0126 enhanced the antileukemic activity of metformin. Importantly, metformin induced ERK activation by suppressing the protein levels of dual specificity phosphatase 6, a negative regulator of ERK. This crosstalk between AMPK and ERK could diminish the antileukemic activity of metformin. Taken together, our present observations suggest a novel therapeutic strategy for improving the efficacy of metformin in treating leukemia. Show less

Although many of the factors and molecules closely associated with non-alcoholic steatohepatitis (NASH) have been reported, the role of inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) on the progression of NASH remains unclear. We therefore investigated the role of iNOS-derived NO in NASH pathogenesis with a long-term follow-up study using systemic iNOS-knockout mice under high-fat diet (HFD) conditions. iNOS-knockout and wild-type mice were fed a basal or HFD for 10 or 48 weeks. Lipid accumulation, fibrosis, and inflammation were evaluated, and various factors and molecules closely associated with NASH were analyzed. Marked fibrosis and inflammation (indicators of NASH) were observed in the livers of iNOS-knockout mice compared to wild-type mice after 48 weeks of a HFD; however, lipid accumulation in iNOS-knockout mice livers was less than in the wild-type. Increased expressions of various cytokines that are transcriptionally controlled by NF-kB in iNOS-deficient mice livers were observed during HFD conditions. iNOS-derived NO may play a protective role against the progression to NASH during an HFD by preventing fibrosis and inflammation, which are mediated by NF-kB activation in Kupffer cells. A lack of iNOS-derived NO accelerates progression to NASH without excessive lipid accumulation. Show less

The pathogenic amoeba Entamoeba histolytica is one of the causative agents of health hazards in tropical countries. It causes amoebic dysentery, colitis and liver abscesses in human. Iron is one of the essential nutritional resources for survival and chronic infection caused by the amoeba. The parasite has developed multiple ways to import, sequester and utilize iron from various iron-binding proteins from its host. In spite of its central role in pathogenesis, the mechanism of iron uptake by the parasite is largely unknown. Here, we carried out a systematic study to understand the role of some of the amoebic homologues of mammalian endocytic Rab GTPases (Rab5 and Rab21, Rab7A and Rab7B) in intracellular transport of human holo-transferrin by the parasite. Flow cytometry and quantitative microscopic image analysis revealed that Rab5 and Rab7A are required for the biogenesis of amoebic giant endocytic vacuoles (GEVs) and regulate the early phase of intracellular trafficking of transferrin. Rab7B is involved in the late phase, leading to the degradation of transferrin in the amoebic lysosome-like compartments. Using time-lapse fluorescence imaging in fixed trophozoites, we determined the kinetics of the vesicular transport of transferrin through Rab5-, Rab7A- and Rab7B-positive compartments. The involvement of Rab7A in the early phase of endocytosis by the parasite marks a significant divergence from its host in terms of spatiotemporal regulation by the Rab GTPases. Show less

The protozoan parasite Entamoeba histolytica causes a wide spectrum of intestinal infections. In severe cases, the trophozoites can breach the mucosal barrier, invade the intestinal epithelium and travel via the portal circulation to the liver, where they cause hepatic abscesses, which can prove fatal if left untreated. The host Extra Cellular Matrix (ECM) plays a crucial role in amoebic invasion by triggering an array of cellular responses in the parasite, including induction of actin rich adhesion structures. Similar actin rich protrusive structures, known as 'invadosomes', promote chemotactic migration of the metastatic cancer cells and non-transformed cells by remodeling the ECM. Recent studies showed a central role for Rab GTPases, the master regulators of vesicular trafficking, in biogenesis of invadosomes. Here, we showed that fibronectin, a major host ECM component induced actin remodeling in the parasite in a Rab21 dependent manner. The focalized actin structures formed were reminiscent of the mammalian invadosomes. By using various approaches, such as immunofluorescence confocal microscopy and scanning electron microscopy, along with in vitro invasion assay and matrix degradation assay, we show that the fibronectin induced formation of amoebic actin dots depend on the nucleotide status of the GTPase. The ECM components, fibronectin and collagen type I, displayed differential control over the formation of actin dots, with fibronectin positively and collagen type I negatively modulating it. The cell surface adhesion molecule Gal/GalNAc complex was also found to impose additional regulation on this process, which might have implication in collagen type I mediated suppression of actin dots. Show less

The orderly differentiation of cell lineages within gastric glands is regulated by a complicated interplay of local mucosal growth factors and hormones. Histamine secreted from enterochromaffin-like cells plays an important role in not only stimulated gastric acid secretion but also coordination of intramucosal growth and lineage differentiation. We have examined histidine-decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis, to evaluate the influence of histamine on differentiation of fundic mucosal lineages and the development of metaplasia following induction of acute oxyntic atrophy. Stomachs from HDC-deficient mice and wild-type mice were evaluated at 8 wk and 12 mo of age. DMP-777 was administrated orally to 6-wk-old mice for 1 to 14 days. Sections of gastric mucosa were stained with antibodies against Mist1, intrinsic factor, H/K-ATPase, trefoil factor 2 (TFF2), chromogranin A, and Ext1 and for the cell cycle marker phospho-histone H3. HDC-deficient mice at 8 wk of age demonstrated a prominent increase in chief cells expressing Mist1 and intrinsic factor. Importantly Mist1-positive mature chief cells were present in the midgland region as well as at the bases of fundic glands, indicating a premature differentiation of chief cells. Mice dually deficient for both HDC and gastrin showed a normal distribution of chief cells in fundic glands. Treatment of HDC-deficient mice with DMP-777 led to loss of parietal cells and an accelerated and exaggerated emergence of mucous cell metaplasia with the presence of dual intrinsic factor and TFF2-expressing cells throughout the gland length, indicative of the emergence of spasmolytic polypeptide-expressing metaplasia (SPEM) from chief cells. These findings indicate that histamine, in concert with gastrin, regulates the appropriate differentiation of chief cells from mucous neck cells as they migrate toward the bases of fundic glands. Nevertheless, histamine is not required for emergence of SPEM following acute oxyntic atrophy. Show less