Keiko Matsuura

The Hippo pathway has been implicated in the tumorigenesis of hepatocellular carcinoma (HCC). We aimed to determine the expression of Hippo pathway-related proteins in HCC using immunohistochemical staining and compare the data among patients with different underlying clinical background diseases. We collected 40 resected HCC formalin-fixed paraffin-embedded samples and categorized them into 4 groups according to pre-existing conditions [17 samples of alcohol consumption (HCC-A) and 6, 8, and 9 samples with hepatitis B virus infection (HCC-B), hepatitis C virus infection (HCC-C), and metabolic diseases (HCC-M), respectively]. Using immunohistochemistry, we investigated the Hippo pathway-related proteins YAP1, CADM1, and SOX9. Hypertension was the most common disease (72.5%) among patients with HCC, followed by diabetes mellitus (47.5%). Regarding the background liver disease, significant findings of steatohepatitis (SH) were observed in the HCC-A (35.3%) and HCC-M (88.9%) groups compared with those in the HCC-B (16.7%) and HCC-C (0%) groups. A significant finding of metabolic dysfunction-associated SH (MASH) was also observed in the HCC-M group (88.9%), and liver cirrhosis was predominantly observed in the HCC-C group (100%). Twenty-seven patients had Hippo-inactive status, and 13 patients had active status. SH was more predominant in the HCC-M group with Hippo inactivity (100%) than in the HCC-A group (50.0%). The average tumor size was significantly larger in the Hippo-inactive group than in the Hippo-active group. Hippo pathway-associated proteins could serve as potential therapeutic targets for MASH and MASH-related HCC if the Hippo pathway is inactivated.

Ovarian clear cell carcinoma (OCCC) is an aggressive epithelial subtype characterized by intrinsic chemoresistance and limited targeted therapies. Transcri

Stress urinary incontinence commonly arises with aging or following prostatectomy, yet its underlying mechanisms remain unclear. To address this, we investigated the role of metabolic pathways—particularly the tricarboxylic acid (TCA) cycle—in the differentiation of human external urethral sphincter myoblasts. Immortalized sphincter cells (US2-KD) were induced to differentiate over 192 hours. Metabolomic profiling using gas chromatography–mass spectrometry, along with pathway enrichment analysis, identified key metabolic changes. Inhibition of mitochondrial pyruvate transport with UK5099 markedly suppressed TCA cycle metabolites, including citrate, α-ketoglutarate, fumarate, and malate. This inhibition also significantly reduced MYH7 expression and intracellular adenosine triphosphate levels throughout the differentiation period. These results demonstrate that the TCA cycle plays a critical role in both energy metabolism and the differentiation of urethral sphincter myoblasts. This study is the first to suggest that impaired TCA cycle activity may contribute to the pathogenesis of Stress urinary incontinence and represents a potential therapeutic target. Our findings offer new insight into age-related metabolic decline associated with Stress urinary incontinence and support the development of therapies that combine metabolic modulation with regenerative approaches.

The variants of concern (VOCs) of SARS-CoV-2 have exhibited different phenotypic characteristics in clinical settings which are yet to be fully explored. This study aimed to characterize the viral replication features of major VOCs of SARS-CoV-2 and their association with pathogenicity. The Alpha, Delta, and Omicron variants of SARS-CoV-2 isolated from the COVID-19 patients in Japan were propagated in VeroE6/TMPRSS2 cells. The viral replication and pathological features were evaluated by laser and electron microscopy at different time points. The results revealed that the Delta variant dominantly infected the VeroE6/TMPRSS2 cells and formed increased syncytia compared to the Alpha and Omicron variants. Relatively large numbers of virions and increased immunoreactivities of the SARS-CoV-2 N-protein were detected in the endoplasmic reticulum and intracellular vesicles of Delta-infected cells. Interestingly, the N-protein and virions were detected in the nucleus of Delta-infected cells, while such properties were not observed in the case of Alpha and Omicron variants. In addition, early nuclear membrane damage followed by severe cellular damage was prominent in Delta-infected cells. A unique mutation (G215C) in the N-protein of the Delta variant is thought to be associated with severe cell damage. In conclusion, this study highlights the distinct replicative and pathogenic characteristics of the Delta variant of SARS-CoV-2 compared to the Alpha and Omicron variants, shedding light on the potential mechanisms underlying its increased pathogenicity.

Sustained exposure to acetaldehyde, the major metabolite of ethanol, may influence psychomotor performance even after the breath ethanol level significantly drops several hours following ethanol consumption. We examined the relationship between psychomotor function and changes in exhaled ethanol and acetaldehyde concentrations after consuming a low dose (0.33 g/kg) of barley shochu, a traditional Japanese distilled alcohol beverage, at the point when the exhaled ethanol concentrations dropped below 78,000 parts per billion (0.15 mg/L), the standard threshold for driving under the influence of alcohol in Japan. We assessed how the genetic polymorphisms of rs671 G/G homozygous (*1/*1) and G/A heterozygous (*1/*2) of ALDH2 influenced the kinetics of ethanol and acetaldehyde in exhaled air and psychomotor dynamics using the Digit Symbol Substitution Test (DSST), Critical Flicker Fusion Test (CFFT), and visual analogue scale (VAS) up to 12 h after shochu or water intake. There was no significant difference in DSST and CFFT scores depending on genotype; however, the time required for the DSST to attain the level prior to drinking was longer in the ALDH2 *1/*2 group than in the *1/*1 group. In the VAS test, facial flushing and mood elevation tended to be higher in the *1/*2 group after shochu consumption. VAS scores for mood elevation and facial flushing correlated with acetaldehyde concentration in exhaled breath. These results indicate that DSST recovery tends to be slower and mood elevation higher in the ALDH2 *1/*2 group even when exposed to a low dose of alcohol.

Males of Enterobius (Colobenterobius) serratus Hasegawa et al., 2003 (Nematoda: Oxyuridae) are described for the first time based on six individuals collected from the feces of proboscis monkeys, Nasalis larvatus, in the Lower Kinabatangan Wildlife Sanctuary, Sabah, Malaysian Borneo. The males show identical cephalic morphology to females, being readily distinguishable from their congeners by the serrated inner margins of the lips. The bicolored esophageal corpus, long thin spicule and developed spicular pouch with paired muscular bands are also remarkable characteristics, presumably shared by other Asian members of the subgenus.

We have previously reported that Salvador homologue 1 (SAV1), a component of the Hippo pathway, is significantly down‐regulated in high‐grade clear cell renal cell carcinoma (ccRCC) due to 14q copy number loss, and that this down‐regulation contributes to the proliferation and survival of renal tubular epithelial cells through activation of Yes‐associated protein 1 (YAP1), a downstream target of the Hippo pathway. However, the impact of SAV1 loss on the proliferation and survival of kidney cells in vivo remained to be determined. To address this issue, we generated kidney‐specific Sav1‐knockout (Cdh16‐Cre;Sav1^fl/fl) mice. Sav1 deficiency enhanced the proliferation of renal tubular epithelial cells in Cdh16‐Cre;Sav1^fl/fl mice, accompanied by nuclear localization of Yap1, suggesting suppression of the Hippo pathway. Sav1 deficiency in renal tubules also caused structural and cellular abnormalities of the epithelial cells, including significant enlargement of their nuclei. Furthermore, Cdh16‐Cre;Sav1^fl/fl mice developed both glomerular and tubular cysts. Although lining cells of the glomerular cysts showed no atypia, those of the tubular cysts showed variations in cell size and nuclear shape, which became more severe as the mice aged. In aged Cdh16‐Cre;Sav1^fl/fl mice, we observed focal disruption of proximal tubules and perivascular lymphocytic infiltration. In conclusion, Sav1 is required for the maintenance of growth, nuclear size and structure of renal tubules under physiological conditions, and its deficiency leads to the acquisition of enhanced proliferation of renal epithelial cells through suppression of Hippo signalling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Previously, we reported that genomic loss of 14q occurs more frequently in high‐grade than in low‐grade clear cell renal cell carcinomas (ccRCCs), and has a significant impact on the levels of expression of genes located in this region, suggesting that such genes may be involved in the malignant transformation of ccRCCs. Here, we found that six of the genes located in the minimal common region of 14q loss were significantly downregulated in high‐grade ccRCCs due to copy number loss. Using a dataset from The Cancer Genome Atlas Research Network, we found that downregulation of one of these six genes, WDR20, was significantly associated with poorer outcome in patients with ccRCC, suggesting that WDR20 downregulation may be involved in the malignant transformation of ccRCCs. In functional assays, exogenous WDR20 significantly inhibited the growth of RCC cell lines and induced apoptosis. Interestingly, the phosphorylation levels of ERK and protein kinase B/AKT, which reportedly contribute to the malignant phenotype of RCC cells, were clearly reduced by exogenous expression of WDR20. Thus, our data suggest that downregulation of WDR20 due to 14q loss may be involved in the malignant transformation of ccRCCs, in part through activation of the ERK and protein kinase B/AKT pathways.

This study was conducted to clarify the genomic profiles of metastatic clear cell renal cell carcinomas (ccRCCs) and identify the genes responsible for development of metastasis. We analyzed the genomic profiles of 20 cases of primary ccRCC and their corresponding metastases using array‐based comparative genomic hybridization, and identified 32 chromosomal regions in which gene copy number alterations were detected more frequently in metastases than in the primary tumors. Among these 32 regions, 9p24.1‐p13.3 loss was the most statistically significant alteration. Furthermore, we found that patients with 9p24.1‐p13.3 loss in primary tumors exhibited significantly lower rates of recurrence‐free and cancer‐specific survival, suggesting that 9p loss in the primary tumor is a potential biomarker predicting early recurrence of metastasis. Interestingly, the genomic profiles of primary tumors with 9p loss resembled those of their corresponding metastases, though 9p loss was accumulated in the metastases derived from the primary tumors without 9p loss. Comparison of the mRNA expression levels revealed that 2 of 58 genes located at 9p24.1‐p13.3 were downregulated due to gene copy number loss in ccRCCs. An overexpression study of these two genes in ccRCC cell lines revealed that downregulation of NDUFB6 due to loss at 9p24.1‐p13.3 may confer a growth advantage on metastatic ccRCC cells. These results were confirmed by analyzing the data of 405 cases of ccRCC obtained from The Cancer Genome Atlas (TCGA). On the basis of our present data, we propose that NDUFB6 is a possible tumor suppressor of metastatic ccRCCs.

Background: Previously, using miRNA microarray, we have found that miR-29c is significantly downregulated in advanced gastric carcinoma. In the present study, we investigated whether miR-29c functions as a tumor-suppressor miRNA in gastric carcinoma cells. For this purpose, we verified the downregulation of miR-29c in gastric carcinoma tissues, and assessed the biological effect of miR-29c on gastric carcinoma cells. Results: In miR-29c-transfected cells, both proliferation and colony formation ability on soft agar were significantly decreased. Although apoptosis was not induced, BrdU incorporation and the proportion of cells positive for phospho-histone H3 (S10) were significantly decreased in miR-29c-transfected cells, indicating that miR-29c may be involved in the regulation of cell proliferation. To explain the mechanism of growth suppression by miR-29c, we explored differentially expressed genes (>2-fold) in miR-29c-transfected cells in comparison with negative control transfected cells using microarray. RCC2, PPIC and CDK6 were commonly downregulated in miR-29c-transfected MKN45, MKN7 and MKN74 cells, and all of the genes harbored miR-29c target sequences in the 3’-UTR of their mRNA. RCC2 and PPIC were actually upregulated in gastric carcinoma tissues, and therefore both were identified as possible targets of miR-29c in gastric carcinoma. To ascertain whether downregulation of RCC2 and/or PPIC is involved in the growth suppression by miR-29c, we transfected siRNAs against RCC2 and PPIC into MKN45 and determined cell viability, the rate of BrdU incorporation, and caspase activity. We found that RCC2-knockdown decreased both cell viability and BrdU incorporation without any increase of caspase activity, while PPIC-knockdown did not, indicating that downregulation of RCC2 may be at least partly responsible for the growth suppression by miR-29c. Conclusions: Our findings indicate that miR-29c may have tumor-suppressive functions in gastric carcinoma cells, and that its decreased expression may confer a growth advantage on tumor cells via aberrant expression of RCC2.