A Crucial Role of Bone Morphogenetic Protein Signaling in the Wound Healing Response in Acute Liver Injury Induced by Carbon Tetrachloride

Background. Acute liver injury induced by administration of carbon tetrachloride (CCl4) has used a model of wound repair in the rat liver. Previously, we reported transient expression of bone morphogenetic protein (Bmp) 2 or Bmp4 at 6–24 h after CCl4 treatment, suggesting a role of BMP signaling in the wound healing response in the injured liver. In the present study, we investigated the biological meaning of the transient Bmp expression in liver injury. Methods. Using conditional knockout mice carrying a floxed exon in the BMP receptor 1A gene, we determined the hepatic gene expressions and proliferative activity following CCl4-treated liver. Results. We observed retardation of the healing response in the knockout mice treated with CCl4, including aggravated histological feature and reduced expressions of the albumin and Tdo2 genes, and a particular decrease in the proliferative activity shown by Ki-67 immunohistochemistry. Conclusion. Our findings suggest a crucial role of BMP signaling in the amelioration of acute liver injury

Development of a novel automatic ascites filtration and concentration equipment with multi‐ring‐type roller pump units for cell‐free and concentrated ascites reinfusion therapy

Cell‐free and concentrated ascites reinfusion therapy (CART) is an effective therapy for refractory ascites. However, CART is difficult to perform as ascites filtration and concentration is a complicated procedure. Moreover, the procedure requires the constant assistance of a clinical engineer or/and the use of an expensive equipment for the multi‐purpose blood processing. Therefore, we developed a CART specialized equipment (mobility CART [M‐CART]) that could be used safely with various safety measures and automatic functions such as automatic washing of clogged filtration filter and self‐regulation of the concentration ratio. Downsizing, lightning of the weight, and automatic processing in M‐CART required the use of newly developed multi‐ring‐type roller pump units. This equipment was approved under Japanese regulations in 2018. In performing 41 sessions of CART (for malignant ascites, 22 sessions; and hepatic ascites, 19 sessions) using this equipment in 17 patients, no serious adverse event occurred. An average of 4494 g of ascites was collected and the total amount of ascites was processed in all the sessions without any trouble. The mean weight of the processed ascites was 560 g and the mean concentration ratio was 8.0. The ascites were processed at a flow rate of 50 mL/min. The mean ascites processing time was 112.5 minutes and a 106.5‐minutes (95.2%) ascites processing was performed automatically. The operator responded to alarms or support information 3.2 times on average (3.1 minutes, 2.1% of ascites processing time). Human errors related to ascites processing were detected by M‐CART at 0.4 times per session on average and were appropriately addressed by the operator. The frequencies of automatic washing of clogged filtration filter and self‐regulation of the concentration ratio were 31.7% and 53.7%, respectively. The mean recovery rates (recovery dose) of protein, albumin, and immunoglobulin G were 72.9%, 72.9%, and 71.2% (65.9 g, 34.9 g, and 13.2 g), respectively. Steroids were administered in 92.7% of the sessions to prevent fever and the mean increase in body temperature was 0.53°C. M‐CART is a compact and lightweight automatic CART specialized equipment that can safely and easily process a large quantity of ascites without the constant assistance of an operator

Research and development of exclusive equipment for cell-free and concentrated ascites reinfusion therapy (CART) by medical-industrial, hospital-university, and multifarious worker cooperation

Cell-free and concentrated ascites reinfusion therapy（CART）is an effective and safe therapy for patients with refractory ascites or pleural effusion. CART was initially indicated for cirrhotic ascites, and has come to be widely used for malignant ascites. Recently, cancer therapy that applies cancer cells obtained by filtration process is considered, and CART attracts attention as one of the important therapies to support future cancer therapy. However, the numbers of CART in Japan is not sufficient because the equipment for CART is high price and large. Additionally, the specialized medical staff such as clinical engineers is necessary for CART because of complicated operation. Therefore, we think that development of next-generation type equipment for CART that can be performed safely, easily, and reliably is necessary. We could develop the exclusive equipment for CART according to the project management by multifarious worker cooperation in five years

Evidence for an Essential Deglycosylation-Independent Activity of PNGase in Drosophila melanogaster

BACKGROUND: Peptide:N-glycanase (PNGase) is an enzyme which releases N-linked glycans from glycopeptides/glycoproteins. This enzyme plays a role in the ER-associated degradation (ERAD) pathway in yeast and mice, but the biological importance of this activity remains unknown. PRINCIPAL FINDINGS: In this study, we characterized the ortholog of cytoplasmic PNGases, PNGase-like (Pngl), in Drosophila melanogaster. Pngl was found to have a molecular weight of approximately 74K and was mainly localized in the cytosol. Pngl lacks a CXXC motif that is critical for enzymatic activity in other species and accordingly did not appear to possess PNGase activity, though it still retains carbohydrate-binding activity. We generated microdeletions in the Pngl locus in order to investigate the functional importance of this protein in vivo. Elimination of Pngl led to a serious developmental delay or arrest during the larval and pupal stages, and surviving mutant adult males and females were frequently sterile. Most importantly, these phenotypes were rescued by ubiquitous expression of Pngl, clearly indicating that those phenotypic consequences were indeed due to the lack of functional Pngl. Interestingly, a putative "catalytic-inactive" mutant could not rescue the growth-delay phenotype, indicating that a biochemical activity of this protein is important for its biological function. CONCLUSION: Pngl was shown to be inevitable for the proper developmental transition and the biochemical properties other than deglycosylation activity is important for its biological function

Knockdown of 15-bp Deletion-Type v-raf Murine Sarcoma Viral Oncogene Homolog B1 mRNA in Pancreatic Ductal Adenocarcinoma Cells Repressed Cell Growth In Vitro and Tumor Volume In Vivo

Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second-most common cause of death within the next 10 years. Due to the limited efficacy of available therapies, the survival rate of PDAC patients is very low. Oncogenic BRAF mutations are one of the major causes of PDAC, specifically the missense V600E and L485–P490 15-bp deletion mutations. Drugs targeting the V600E mutation have already been approved by the United States Food and Drug Administration. However, a drug targeting the deletion mutation at L485–P490 of the BRAF gene has not been developed to date. The BxPC-3 cell line is a PDAC-derived cell line harboring wild-type KRAS and L485–P490 deleted BRAF genes. These cells are heterozygous for BRAF, harboring both wild-type BRAF and BRAF with the 15-bp deletion. In this study, siRNA was designed for the targeted knockdown of 15-bp deletion-type BRAF mRNA. This siRNA repressed the phosphorylation of extracellular-signal-regulated kinase proteins downstream of BRAF and suppressed cell growth in vitro and in vivo. Furthermore, siRNAs with 2′-O-methyl modifications at positions 2–5 reduce the seed-dependent off-target effects, as confirmed by reporter and microarray analyses. Thus, such siRNA is a promising candidate therapy for 15-bp deletion-type BRAF-induced tumorigenesis