Establishment of Novel Protein Interaction Assays between Sin3 and REST Using Surface Plasmon Resonance and Time-Resolved Fluorescence Energy Transfer.

Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein-protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.

Novel Reporter System Monitoring IL-18 Specific Signaling Can Be Applied to High-Throughput Screening.

Very recently, the immunotherapies against cancer, autoimmune diseases, and infection have been feasible and promising. Thus, we have examined the possibility whether or not human gamma delta T cells can be applied for the novel immunotherapies. We previously established the cells stably maintaining NFkB-driven human secreted embryonic alkaline phosphatase (SEAP) expression. The cells can be used to determine the transcription activity of NFkB with high-standard dynamic range and accuracy. Because IL-18 is a kind of cytokines that enhances cytotoxicity and activity of human gamma delta T cells through NFkB activation, we have focused on the activity and signaling of IL-18. In this study, we modified the previous reporter cell that can determine the transcription activity of NFkB to express two subunits consisted of human IL-18 receptor. The modified cells secreted SEAP in response to treatment with human recombinant IL-18 in a concentration-dependent manner. We also observed the concentration-dependently enhancement of NFkB activity in the cells treated with mouse recombinant IL-18 although the affinity was lower compared to human recombinant IL-18. We also previously established the cells stably expressing and secreting human recombinant IL-18 and then validated whether or not the conditioned medium from the cells activate NFkB transcription activity using this assay. Our university has kept collecting many extracts from over 18,000 marine bacteria in our local sea around Omura bay-fungi, plants for Chinese herbal medicine, and so on-and also have kept gathering synthetic compounds from many Japanese chemists as drug libraries. Finally, in order to identify drugs mimicking IL-18 biological activity or possessing inhibitory effects on IL-18-induced NFkB, we demonstrated drug screening using number of extracts derived from marine bacteria and synthetic compounds.

Establishment of Novel High-Standard Chemiluminescent Assay for NTPase in Two Protozoans and Its High-Throughput Screening.

Toxoplasma gondii is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. Neospora caninum, another type of protozoa, is closely related to Toxoplasma gondii. Infections of the protozoa in animals also causes serious diseases such as Encephalomyelitis and Myositis-Polyradiculitis in dogs or abortion in cows. Both Toxoplasma gondii and Neospora caninum have similar nucleoside triphosphate hydrolases (NTPase), NcNTPase and TgNTPase-I in Neospora caninum and Toxoplasma gondii, respectively. These possibly play important roles in propagation and survival. Thus, we targeted the enzymes for drug discovery and tried to establish a novel high-standard assay by a combination of original biochemical enzyme assay and fluorescent assay to determine ADP content. We then validated whether or not it can be applied to high-throughput screening (HTS). Then, it fulfilled criterion to carry out HTS in both of the enzymes. In order to identify small molecules having inhibitory effects on the protozoan enzyme, we also performed HTS using two synthetic compound libraries and an extract library derived from marine bacteria and then, identified 19 compounds and 6 extracts. Nagasaki University collected many extracts from over 18,000 marine bacteria found in local Omura bay, and continues to compile an extensive collection of synthetic compounds from numerous drug libraries established by Japanese chemists.

Propensity score-matched analysis of the short-term outcomes of robotic versus laparoscopic surgery for rectal cancer.

PURPOSE: The advantages of robot-assisted rectal surgery (RARS) over conventional laparoscope-assisted rectal surgery (LARS) remain controversial. This study was performed to compare the short-term outcomes of RARS and LARS. METHODS: We retrospectively analyzed data of 207 patients who had undergone either RARS (n = 97) or LARS (n = 110) for rectal cancer (RC) from 2018 to 2020. A 1:1 matched propensity score-matched analysis was performed and the surgical outcomes of the two groups compared. RESULTS: After matching, a well-balanced cohort of 136 patients was analyzed (n = 68 in each group), and there was no significant difference in the median operative time. The RARS group had less intraoperative blood loss than the LARS group. There were no significant differences in length of postoperative hospital stay or complication rate between the two groups. In the subgroup of lower RC, defined as the inferior edge of the tumor being within the rectum distal to the peritoneal reflection, the rate of sphincter preservation was higher in the RARS group (81.8% vs. 44.4%, p = 0.021). CONCLUSION: This study shows that RARS is a safe and feasible approach for RC compared with LARS, RARS having the advantage of more often preserving the sphincter.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Neutrophil to Lymphocyte Ratio is a Predictive Factor of Malignant Potential for Intraductal Papillary Mucinous Neoplasms of the pancreas.

Intraductal papillary mucinous neoplasms (IPMNs) are cystic neoplasms with the potential for progression to pancreatic cancer. Accurate prediction of the malignant potential is challenging and a proper treatment strategy has not been well established. Preoperative neutrophil-to-lymphocyte ratio (NLR) is a biomarker of the malignant potential in patients with several types of malignancy. We explored malignant potential in patients with IPMN. The present study included 56 patients aged of 73 ± 9 years (mean ± standard deviation) who underwent curative resection for IPMN from 1996 to 2017. We analyzed the relationship between the characteristics including NLR and malignant component for predicting pathological results. The nonmalignant IPMN group (N = 21) included patients with low-grade dysplasia (LGD) and intermediate-grade dysplasia (IGD), and the malignant IPMN group (N = 35) included patients with high-grade dysplasia (HGD) and invasive carcinoma. In a univariate analysis, NLR ⩾ 2.2 (P = .001), prognostic nutritional index (PNI) < 45 (P = .016), CA 19-9 > 37 U/mL (P = .039), and cystic diameter ⩾ 30 mm (P = .010), and mural nodule (P = .010) were significantly different between the malignant IPMN and the nonmalignant IPMN groups. Multivariate analysis showed that high NLR (⩾2.2) (odds ratio 9.79; 95% confidence interval: 2.06-45.6), cystic diameter ⩾ 30 mm (4.65; 1.14-18.9), and mural nodule (4.91; 1.20-20.1) were independently predictive of malignant IPMN. These results suggest that preoperative NLR is a useful predictive biomarker for evaluating malignant potential in patients with IPMN.1.

Establishment of Novel Cells Stably Secreting Various Human IL-18 Recombinant Proteins.

BACKGROUND: The immunotherapies against cancer, autoinmmune diseases or infection are remarkable development. These days programmed cell death (PD)-1 antibody-induced immune checkpoint blockade or chimeric antigen receptor-T cells (CAR-T) have been shown to have eminent therapeutic effects on tumor development. We have focused on adoptive transfer with human gamma delta T cells for novel immunotherapies. Additionally, IL-18 is one of the cytokines that enhances cytokine secretion and cytotoxicity of human gamma delta T cells. METHOD: Thus, we established novel cell lines stably expressing and secreting various types of human recombinant IL-18 proteins to their culture supernatants using episomal vector. We also differentiated primary cultured human gamma delta T cells from peripheral blood mononuclear leukocytes to validate biological activity of the IL-18 proteins using measuring IFN-γ by ELISA. RESULTS AND CONCLUSION: Finally, we demonstrated that the supernatant could activate human gamma delta T cells using monitoring interferon gamma in culture medium.

Establishment of Novel Reporter Cells Stably Maintaining Transcription Factor-driven Human Secreted Alkaline Phosphatase Expression.

BACKGROUND: Transcriptional regulation is a very important and pivotal function in myriad biological responses. Thus, methods to determine transcriptional activity are required in not only basic medical research but also in drug discovery. We established novel reporter constructs using human secreted embryonic alkaline phosphatase (SEAP) and Epstein-Barr virus nuclear antigen (EBNA) 1, which can maintain constructs synchronized to host cell replication. METHODS: We established nuclear factor-kappa B (NFkB) or interferon regulatory factor (IRF) driven SEAP expression constructs and then, introduced them into culture cells. RESULTS: The cells maintain reporter constructs for a long period in the culture and produce SEAP into culture supernatant in response to each specific ligand such as lipopolysaccharide (LPS) and interferon- beta. Measuring SEAP with chemiluminescence makes it possible to get high standard dynamic range applying to high-throughput screening in drug discovery in both 96 and 384 well format. We can also use it to determine transcriptional activity in the cells transfected with expression plasmid or treated with various toll-like receptor (TLR) ligands in a concentration-dependent manner and time-dependent manner. Finally, we demonstrated drug screening using a number of natural products library. CONCLUSION: We for the first time established the two novel reporter cells and validated their quality and accuracy enough to carry out drug screening.

The effect of continuous arterial infusion of gabexate mesilate (FOY-007) on experimental acute pancreatitis.

The effectiveness of continuous arterial infusion of Gabexate Mesilate (FOY-007) on experimental acute pancreatitis was investigated. Acute necrotizing pancreatitis was induced by an injection of 10% Na-taurocholate (1ml/kg) into the main pancreatic duct of mongrel dogs. Animals were divided into three groups; Group A: non-treated control, Group B: after the induction of pancreatitis, injected with FOY-007 intravenously (5mg/kg/hr), Group C: after the induction of pancreatitis, injected with FOY-007 via the celiac artery. The changes in the values of amylase and lipase in serum and ascites etc. were examined. A histological examination was done and the FOY-007 concentration of the pancreas was measured. In both groups B and C, the serum levels of amylase and lipase reached significantly to low levels compared with those in group A. The extents of pancreatic parenchyma necrosis in each group were 36.1, 25.3 and 19.5%, respectively, and were significantly improved in group C. In addition, the FOY-007 levels in pancreas specimens in the intraarterial infusion group exceeded those in the intravenous infusion group by 32 times. The results suggest that continuous FOY-007 arterial infusion therapy is useful as a local treatment for severe acute pancreatitis.

Middle-preserving pancreatectomy for multifocal intraductal papillary mucinous neoplasms of the pancreas: report of a case.

Multifocal or continuous pancreatic lesion is identified frequently but finding an appropriate surgical approach is quite challenging. Total pancreatectomy is a useful procedure. However, postoperative endocrine and exocrine disturbance is inevitable. Recently, the safety and feasibility of parenchyma preserving pancreatectomy, including middle-preserving pancreatectomy (MPP), have been reported. MPP is a combined procedure of pancreaticoduodenectomy and distal pancreatectomy, while preserving the body of the pancreas, for cases of multifocal pancreatic lesions. So far, there have only been a few reports that have described MPP. We report a case of MPP for multifocal intraductal papillary mucinous neoplasms of the pancreas, describe the surgical procedure, and discuss the feasibility of MPP as parenchyma-preserving pancreatectomy with reference to the literature.