Carrier Rate and Mutant Allele Frequency of GM1 Gangliosidosis in Miniature Shiba Inus (Mame Shiba): Population Screening of Breeding Dogs in Japan.

GM1 gangliosidosis is a progressive, recessive, autosomal, neurodegenerative, lysosomal storage disorder that affects the brain and multiple systemic organs due to an acid ß-galactosidase deficiency encoded by the GLB1 gene. This disease occurs in the Shiba Inu breed, which is one of the most popular traditional breeds in Japan, due to the GLB1:c.1649delC (p.P550Rfs*50) mutation. Previous surveys performed of the Shiba Inu population in Japan found a carrier rate of 1.02-2.94%. Currently, a miniature type of the Shiba Inu called "Mame Shiba", bred via artificial selection to yield smaller individuals, is becoming more popular than the standard Shiba Inu and it is now one of the most popular breeds in Japan and China. The GM1 gangliosidosis mutation has yet to be surveyed in the Mame Shiba population. This study aimed to determine the frequency of the mutant allele and carrier rate of GM1 gangliosidosis in the Mame Shiba breed. Blood samples were collected from 1832 clinically healthy adult Mame Shiba Inus used for breeding across 143 Japanese kennels. The genotyping was performed using a real-time PCR assay. The survey found nine carriers among the Mame Shibas, indicating that the carrier rate and mutant allele frequency were 0.49% and 0.00246, respectively. This study demonstrated that the mutant allele has already been inherited by the Mame Shiba population. There is a risk of GM1 gangliosidosis occurrence in the Mame Shiba breed if breeders use carriers for mating. Further genotyping surveys are necessary for breeding Mame Shibas to prevent the inheritance of this disease.

Development of novel potent ligands for GPR85, an orphan G protein-coupled receptor expressed in the brain.

GPR85 is a member of the G protein-coupled receptor and is a super-conserved receptor expressed in the brain sub-family (Super Conserved Receptor Expressed in Brain; SREB) with GPR27 and GPR173. These three receptors are "orphan receptors"; however, their endogenous ligands have not been identified. SREB has garnered the interest of many scientists because it is expressed in the central nervous system and is evolutionarily conserved. In particular, brain mass is reported to be increased and learning and memory are improved in GPR85 knockout mice (Matsumoto et al. 2008). In this study, we characterized newly synthesized compounds using a GPR85-Gsα fusion protein and the [35 S]GTPγS binding assay and identified novel GPR85 inverse-agonists with IC50 values of approximately 1 µM. To analyze the neurochemical character of the compounds and investigate the physiological significance of GPR85, we used cerebellar Purkinje cells expressing GPR85 and an electrophysiological technique. Based on the results, the inverse-agonist compound for GPR85 modulated potassium channel opening. Together with the results of previous gene analysis of GPR85, we expect that the development of the GPR85 ligand will provide new insights into a few types of neurological disorders.

Pretreatment Glasgow prognostic score as a predictor of outcomes in nivolumab-treated patients with advanced gastric cancer.

In Japan, South Korea, and Taiwan, nivolumab might provide overall survival benefits for patients with advanced gastric cancer. However, it is effective only in a limited number of patients. The Glasgow prognostic score is an indicator of the systematic inflammatory response and nutritional status. This study aimed to investigate the ability of the Glasgow prognostic score and other markers to predict the outcomes of patients treated with nivolumab. We reviewed the medical records of patients treated for advanced gastric cancer and who received nivolumab between February 2015 and June 2019 at Hyogo Cancer Center. The patients were categorized into two groups according to their Glasgow prognostic scores. Overall, 53.3% and 46.7% of the patients were assigned to groups with Glasgow prognostic scores of 0 and 1/2, respectively. The median durations of progression-free and overall survival of the participants were 2.3 and 5.7 months, respectively. The patients with a Glasgow prognostic score of 0 had significantly higher median overall survival than those with scores of 1 or 2 (16.4 vs. 4.2 months; p = 0.0006). This observation suggests that a pretreatment Glasgow prognostic score of 0 is associated with better outcomes, and this scoring system may be used as a predictor of outcomes in patients with advanced gastric cancer treated with nivolumab.

Clinical Factors of Delayed Perforation after Endoscopic Submucosal Dissection for Gastric Neoplasms.

BACKGROUND: Delayed perforation is a rare but severe complication of endoscopic submucosal dissection (ESD) for early gastric neoplasm (EGN). The aim of this study was to clarify clinical factors related to delayed perforation after ESD. METHODS: A total of 1158 consecutive patients with 1199 EGNs underwent ESD at our hospital between January 2000 and December 2015. Univariate analysis was used to identify clinicopathological factors related to delayed perforation. Moreover, duration of cautery needed for hemostasis was measured by comparison between perforated and nonperforated points in patients with delayed perforation. RESULTS: Delayed perforation occurred in 5 of 1158 consecutive patients with 1199 EGNs who underwent ESD (0.42%). All cases were diagnosed within 24 h after ESD and recovered with conservative management. On univariate analysis, location in the upper stomach was the factor most significantly associated with delayed perforation (P < 0.01). Duration of cautery needed for hemostasis was significantly longer at perforated points (9 s) than at nonperforated points (3.5 s) in five patients. CONCLUSIONS: Location in the upper stomach was the risk factor most prominently associated with delayed perforation after ESD for EGNs. In addition, delayed perforation appears associated with excessive electrocautery for hemostasis.

Polypoid leiomyosarcoma of the esophagus treated by endoscopic submucosal dissection.

We report a rare case of polypoid leiomyosarcoma of the esophagus that was treated by endoscopic submucosal dissection (ESD). A 63-year-old man with complaints of progressive dysphagia was referred to Hyogo Cancer Center for treatment of esophageal tumor. Esophagoscopy revealed a polypoid tumor 25 mm in diameter on the left side of the upper esophagus. Despite several biopsy specimens, the diagnosis could not be confirmed. Computed tomography showed a protruded, homogeneously enhancing mass in the upper esophagus, but no lymph node enlargement or metastasis. After 1.5 months, the esophagogram showed a filling defect 47 mm in diameter in the upper esophagus. Given this rapid tumor growth, en bloc resection was done by ESD for therapeutic diagnosis. After this treatment, the tumor seemed to grow larger, showing a short stalk and occupying the esophageal lumen. Histopathologically, the tumor comprised pleomorphic spindle cells with mitosis. Tumor invasion involved the lumina propria mucosae and contact with the muscularis mucosae, but not involving the submucosa. Immunohistochemical examination showed positive staining for smooth muscle actin and HHF35, but negative for desmin, caldesmon, CD34, c-kit, DOG1, ALK, S-100 protein and cytokeratin. These histopathological findings were compatible with a diagnosis of esophageal leiomyosarcoma derived from the muscularis mucosae.

Endoscopic submucosal dissection for early gastric cancer in anastomosis site after distal gastrectomy.

BACKGROUND: Detection of early gastric cancer (EGC) in the remnant stomach is increasing because of follow-up endoscopic surveillance programs. Endoscopic treatment appears to be desirable for EGC in the remnant stomach because it is less invasive than surgical resection. METHODS: In this retrospective study, to evaluate the feasibility of endoscopic submucosal dissection (ESD) for EGC in an anastomotic site, treatment results of ESD for EGC in an anastomotic site and in remnant stomach not involving an anastomotic site were compared. In total, 11 EGC lesions of anastomotic sites in 11 patients and 22 EGC lesions of remnant stomach not involving an anastomotic site in 21 patients were treated by ESD. RESULTS: All lesions were successfully treated by en bloc resection. There were three patients with perforations in the anastomotic site group. Although resected specimen size and tumor size were larger in the anastomotic site group than in the non-anastomotic site group (P < 0.01), the procedure duration was far longer in the anastomotic site group than in the non-anastomotic site group (P < 0.01). The speed of the procedure was faster in the non-anastomotic site group than in the anastomotic site group (P < 0.05). CONCLUSIONS: Although ESD for EGC in an anastomotic site is a time-consuming procedure and requires advanced techniques compared with ESD for EGC not involving an anastomotic site, a high en bloc resection rate was achieved. ESD by endoscopists with sufficient experience appears to be a feasible treatment for EGC in an anastomotic site.

[Assessment of treatment effect for acromegaly with sleep disordered breathing].

Acromegaly is caused by excessive secretion of growth hormone (GH) and presents with a variety of clinical manifestations, including facial disfigurement and abnormally large hands and feet, as well as diabetes mellitus, hypertension, and sleep-disordered breathing (SDB). Although SDB is known to be associated with serious symptoms, there have been few study reports, and no clear consensus has been reached regarding the method of assessment of individual treatments. We report herein on the results of surgical intervention with transsphenoidal surgery (TSS) for acromegaly and assessment of the treatment effect after the intervention. We studied 6 patients who received a diagnosis of acromegaly complicated with SDB and underwent TSS at our hospital. Polysomnography (PSG) was performed before and after TSS, and the polysomnograms were analyzed. We also examined changes in the levels of GH and insulin-like growth factor-1 (IGF-1) on blood biochemistry. In 6 cases of acromegaly with SDB, we were able to confirm endocrinologic improvement of TSS with blood biochemistry. However there was no meaningful improvement in the PSG index for SDB.

A novel serum metabolomics-based diagnostic approach to pancreatic cancer.

BACKGROUND: To improve the prognosis of patients with pancreatic cancer, more accurate serum diagnostic methods are required. We used serum metabolomics as a diagnostic method for pancreatic cancer. METHODS: Sera from patients with pancreatic cancer, healthy volunteers, and chronic pancreatitis were collected at multiple institutions. The pancreatic cancer and healthy volunteers were randomly allocated to the training or the validation set. All of the chronic pancreatitis cases were included in the validation set. In each study, the subjects' serum metabolites were analyzed by gas chromatography mass spectrometry (GC/MS) and a data processing system using an in-house library. The diagnostic model constructed via multiple logistic regression analysis in the training set study was evaluated on the basis of its sensitivity and specificity, and the results were confirmed by the validation set study. RESULTS: In the training set study, which included 43 patients with pancreatic cancer and 42 healthy volunteers, the model possessed high sensitivity (86.0%) and specificity (88.1%) for pancreatic cancer. The use of the model was confirmed in the validation set study, which included 42 pancreatic cancer, 41 healthy volunteers, and 23 chronic pancreatitis; that is, it displayed high sensitivity (71.4%) and specificity (78.1%); and furthermore, it displayed higher sensitivity (77.8%) in resectable pancreatic cancer and lower false-positive rate (17.4%) in chronic pancreatitis than conventional markers. CONCLUSIONS: Our model possessed higher accuracy than conventional tumor markers at detecting the resectable patients with pancreatic cancer in cohort including patients with chronic pancreatitis. IMPACT: It is a promising method for improving the prognosis of pancreatic cancer via its early detection and accurate discrimination from chronic pancreatitis.

Metabolomic analysis to discover candidate therapeutic agents against acute pancreatitis.

Novel and effective drugs against acute pancreatitis are required. Therefore, we examined the changes in the metabolite levels in the serum and pancreatic tissue of mice with cerulein- and arginine-induced pancreatitis using gas-chromatography/mass-spectrometry (GC/MS) and investigated whether these alterations affected the severity of acute pancreatitis. In the cerulein-induced pancreatitis model, 93 and 129 metabolites were detected in the serum and pancreatic tissue, respectively. In the L-arginine-induced acute pancreatitis model, 120 and 133 metabolites were detected in the serum and pancreatic tissue, respectively. Among the metabolites, the concentrations of tricarboxylic acid (TCA) cycle intermediates and amino acids were altered in pancreatitis, and in pancreatic tissue, the levels of the intermediates involved in the initial part of the TCA cycle were increased and those of the intermediates involved in the latter part of the TCA cycle were decreased. Some metabolites exhibited similar changes in both pancreatitis mouse models, e.g., the levels of glutamic acid and O-phosphoethanolamine were significantly decreased in the pancreatic tissue. Supplementation with glutamic acid and O-phosphoethanolamine attenuated the severity of cerulein-induced acute pancreatitis. Our results suggest that GC/MS-based metabolomics is capable of accurately representing the status of acute pancreatitis, leading to the discovery of therapeutic agents for pancreatitis.

Regulation of the metabolite profile by an APC gene mutation in colorectal cancer.

Mutation of the APC gene occurs during the early stages of colorectal cancer development. To obtain new insights into the mechanisms underlying the aberrant activation of the Wnt pathway that accompanies APC mutation, we carried out a gas chromatography-mass spectrometry-based semiquantitative metabolome analysis. In vitro experiments comparing SW480 cells expressing normal APC and truncated APC indicated that the levels of metabolites involved in the latter stages of the intracellular tricarboxylic acid cycle, including succinic acid, fumaric acid, and malic acid, were significantly higher in the SW480 cells expressing the truncated APC. In an in vivo study, we found that the levels of most amino acids were higher in the non-polyp tissues of APC(min/+) mice than in the normal tissues of the control mice and the polyp tissues of APC(min/+) mice. Ribitol, the levels of which were decreased in the polyp lesions of the APC(min/+) mice and the SW480 cells expressing the truncated APC, reduced the growth of SW480 cells with the APC mutation, but did not affect the growth of SW480 transfectants expressing full-length APC. The level of sarcosine was found to be significantly higher in the polyp tissues of APC(min/+) mice than in their non-polyp tissues and the normal tissues of the control mice, and the treatment of SW480 cells with 50 µM sarcosine resulted in a significant increase in their growth rate. These findings suggest that APC mutation causes changes in energetic metabolite pathways and that these alterations might be involved in the development of colorectal cancer.

A glycyrrhizin-containing preparation reduces hepatic steatosis induced by hepatitis C virus protein and iron in mice.

BACKGROUND/AIM: A European randomized trial showed biochemical effects of 6-month treatment with Stronger Neo-Minophagen C (SNMC), a glycyrrhizin-containing preparation, in patients with chronic hepatitis C, but its underlying mechanisms remain elusive. We reported previously that SNMC exhibits an anti-oxidative effect in hepatitis C virus (HCV) transgenic mice that develop marked hepatic steatosis with mitochondrial injury under iron overloading. Hepatic steatosis and iron overload are oxidative stress-associated pathophysiological features in chronic hepatitis C. The aim of this study was to investigate whether long-term treatment with SNMC could prevent the development of hepatic steatosis in iron-overloaded HCV transgenic mice. METHODS: C57BL/6 transgenic mice expressing the HCV polyprotein were fed an excess iron diet concomitantly with intraperitoneal injection of saline, SNMC, or seven-fold-concentrated SNMC thrice weekly for 6 months. RESULTS: Stronger Neo-Minophagen C inhibited the development of hepatic steatosis in a dose-dependent manner without affecting hepatic iron content, attenuated ultrastructural alterations of mitochondria of the liver, activated mitochondrial ß-oxidation with increased expression of carnitine palmitoyl transferase I and decreased the production of reactive oxygen species in the liver in iron-overloaded transgenic mice. However, SNMC hardly affected the unfolded protein response, which post-transcriptionally activates sterol regulatory element-binding protein 1, a transcription factor involved in lipid synthesis, even though we reported previously the activation of the unfolded protein response in the same iron-overloaded transgenic mice. CONCLUSIONS: These results suggest that SNMC prevents hepatic steatosis possibly by protecting mitochondria against oxidative stress induced by HCV proteins and iron overload.

ERAL1 is associated with mitochondrial ribosome and elimination of ERAL1 leads to mitochondrial dysfunction and growth retardation.

ERAL1, a homologue of Era protein in Escherichia coli, is a member of conserved GTP-binding proteins with RNA-binding activity. Depletion of prokaryotic Era inhibits cell division without affecting chromosome segregation. Previously, we isolated ERAL1 protein as one of proteins which were associated with mitochondrial transcription factor A by using immunoprecipitation. In this study, we analysed the localization and function of ERAL1 in mammalian cells. ERAL1 was localized in mitochondrial matrix and associated with mitoribosomal proteins including the 12S rRNA. siRNA knockdown of ERAL1 decreased mitochondrial translation, caused redistribution of ribosomal small subunits and reduced 12S rRNA. The knockdown of ERAL1 in human HeLa cells elevated mitochondrial superoxide production and slightly decreased mitochondrial membrane potential. The knockdown inhibited the growth of HeLa cells with an accumulation of apoptotic cells. These results suggest that ERAL1 is localized in a small subunit of the mitochondrial ribosome, plays an important role in the small ribosomal constitution, and is also involved in cell viability.

Hepatitis C virus protein and iron overload induce hepatic steatosis through the unfolded protein response in mice.

BACKGROUND/AIM: Hepatic iron overload and steatosis play critical roles in the progression of hepatitis C virus (HCV)-associated chronic liver disease. However, how these two pathophysiological features affect each other remains unknown. The aim of this study was to investigate how hepatic iron overload contributes to the development of hepatic steatosis in the presence of HCV proteins. METHODS: Male C57BL/6 transgenic mice expressing the HCV polyprotein and nontransgenic littermates were fed an excess-iron diet or a control diet. Mice in each group were assessed for the molecules responsible for fat accumulation in the liver. RESULTS: Hepatic iron levels were positively correlated with triglyceride concentrations in the liver for all mice. As compared with the livers of nontransgenic mice fed the control diet, the livers of transgenic mice fed the excess-iron diet showed a lower expression of carnitine palmitoyl transferase I, a higher expression of sterol-regulatory element-binding protein 1 and fatty acid synthetase and an activated unfolded protein response indicated by a higher expression of unspliced and spliced X-box DNA-binding protein 1 (XBP-1), phosphorylated eukaryotic initiation factor-2alpha (p-eIF2alpha), CCAAT/enhancer-binding protein homology protein (CHOP) and abundant autophagosomes concomitant with increased production of reactive oxygen species. Six-month treatment with the anti-oxidant N-acetyl cysteine dramatically reduced hepatic steatosis in transgenic mice fed the excess-iron diet through decreased expression of unspliced and spliced XBP-1, p-eIF2alpha, and CHOP. CONCLUSIONS: The iron-induced unfolded protein response appears to be one of the mechanisms responsible for fat accumulation in the liver in transgenic mice expressing the HCV polyprotein.

Hepatitis C virus-induced reactive oxygen species raise hepatic iron level in mice by reducing hepcidin transcription.

BACKGROUND & AIMS: Despite abundant clinical evidence, the mechanisms by which hepatic iron overload develops in patients with hepatitis C virus (HCV)-associated chronic liver disease remain unknown. The aim of this study was to investigate how hepatic iron overload develops in the presence of HCV proteins. METHODS: Male transgenic mice expressing the HCV polyprotein and nontransgenic control mice (C57BL/6) were assessed for iron concentrations in the liver, spleen, and serum and iron regulatory molecules in vivo and ex vivo. RESULTS: Transgenic mice had increased hepatic and serum iron concentrations, decreased splenic iron concentration, and lower hepcidin expression in the liver accompanied by higher expression of ferroportin in the duodenum, spleen, and liver. In response to hepatocellular iron excess, transferrin receptor 1 expression decreased and ferritin expression increased in the transgenic liver. Transgenic mice showed no inflammation in the liver but preserved the ability to induce hepcidin in response to proinflammatory cytokines induced by lipopolysaccharide. Hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein alpha (C/EBP) were down-regulated concomitant with increased expression of C/EBP homology protein, an inhibitor of C/EBP DNA binding activity, and with increased levels of reactive oxygen species in transgenic mice at the ages of 8 and 14 months. CONCLUSIONS: HCV-induced reactive oxygen species may down-regulate hepcidin transcription through inhibition of C/EBPalpha DNA binding activity by C/EBP homology protein, which in turn leads to increased duodenal iron transport and macrophage iron release, causing hepatic iron accumulation.

[Successful treatment for advanced hepatocellular carcinoma by hepatic arterial infusion of CDDP powder as second-line chemotherapy].

A 72-year-old man with type C liver cirrhosis had suffered from hepatocellular carcinoma (HCC) since April, 2001. HCC spread diffusely all over the right lobe of his liver, and the serum alpha-fetoprotein (AFP) value increased up to 42,696 ng/ml in June of 2004. He was implanted with a port-catheter system, and hepatic arterial infusion chemotherapy (HAIC) using low-dose of CDDP and 5-FU was started. However, it was not effective and after 4 months, the serum AFP level increased up to 755,030 ng/ml, ascites appeared, and gastro-esophageal varices also spread. No definite metastasis was detected, then we started to second-line chemotherapy. He was then given HAIC using CDDP powder for intraarterial use (CDDP 50 mg/m(2)/20 min, monthly). After 3 courses, the serum AFP level decreased to 9 10 ng/ml, and abdominal CT revealed that the main tumor had regressed and ascites had disappeared. After one more course, the serum AFP value decreased to 8 ng/ml and complete response was achieved on abdominal CT imaging. There was no major complication related to the chemotherapy. HAIC for advanced HCC using LFP has been reported to achieve favorable results, but no other regimens have been proved to the standard for HCC. HAIC using CDDP powder for advanced HCC may be beneficial as the second-line chemotherapy.