Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

<p>Abstract</p> <p>Background</p> <p><it>Vibrio parahaemolyticus </it>is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of <it>V. parahaemolyticus</it>. Most <it>V. parahaemolyticus </it>isolates from the environment do not produce TDH or TRH. Total <it>V. parahaemolyticus </it>has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total <it>V. parahaemolyticus </it>using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of <it>Vibrio parahaemolyticus</it>.</p> <p>Results</p> <p>The assay provided markedly more sensitive and rapid detection of <it>V. parahaemolyticus </it>strains than conventional biochemical and PCR assays. The assay correctly identified 143 <it>V. parahaemolyticus </it>strains, but did not detect 33 non-<it>parahaemolyticus Vibrio </it>and 56 non-<it>Vibrio </it>strains. Sensitivity of the LAMP assay for direct detection of <it>V. parahaemolyticus </it>in pure cultures and in spiked shrimp samples was 5.3 × 10²CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 <it>V. parahaemolyticus </it>strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of <it>V. parahaemolyticus </it>required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.</p> <p>Conclusion</p> <p>The LAMP assay is a sensitive, rapid and simple tool for the detection of <it>V. parahaemolyticus </it>and will facilitate the surveillance for control of contamination of <it>V. parahaemolyticus </it>in seafood.</p

Intestinal Fibroblast/Myofibroblast TRP Channels in Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is characterized by the repeated cycles of inflammation and healing of the gut, which ultimately progress into intestinal fibrosis. Colonic fibroblast/myofibroblast’s functions such as transformation, proliferation, invasion, migration, stress fiber formation, collagen synthesis, and cytokine/chemokine secretion are well estimated. However, the detailed mechanism can rarely be found so far. Thus, we focused on transient receptor potential (TRP) protein super family activated by various physical/chemical stimulations based on the above-described recognitions and also conducted the following examinations for the potential roles in Ca2+ signal transduction in fibroblast/myofibroblasts cells, which play an important role in intestinal inflammation and tissue remodeling. This chapter not only facilitates the understanding about the new role of intestinal fibroblast/myofibroblasts TRP channel for regulating inflammation, fibrotic processes but also suggests a novel molecular target of IBD treatment in future

Clinical response in Japanese metastatic melanoma patients treated with peptide cocktail-pulsed dendritic cells

BACKGROUND: Metastatic, chemotherapy-resistant melanoma is an intractable cancer with a very poor prognosis. As to immunotherapy targeting metastatic melanoma, HLA-A2(+ )patients were mainly enrolled in the study in Western countries. However, HLA-A24(+ )melanoma patients-oriented immunotherapy has not been fully investigated. In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on metastatic melanoma patients with HLA-A2 or A24 genotype. METHODS: Nine cases of metastatic melanoma were enrolled into a phase I study of monocyte-derived dendritic cell (DC)-based immunotherapy. HLA-genotype analysis revealed 4 cases of HLA-A*0201, 1 of A*0206 and 4 of A*2402. Enriched monocytes were obtained using OptiPrep™ from leukapheresis products, and then incubated with GM-CSF and IL-4 in a closed serum-free system. After pulsing with a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-2, MAGE-3 and MART-1 or MAGE-1) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Finally, thawed DCs were washed and injected subcutaneously (s.c.) into the inguinal region in a dose-escalation manner. RESULTS: The mean percentage of DCs rated as lin(-)HLA-DR(+ )in melanoma patients was 46.4 ± 15.6 %. Most of DCs expressed high level of co-stimulatory molecules and type1 phenotype (CD11c(+)HLA-DR(+)), while a moderate number of mature DCs with CD83 and CCR7 positive were contained in DC products. DC injections were well tolerated except for transient liver dysfunction (elevation of transaminases, Grade I-II). All 6 evaluable cases except for early PD showed positive immunological responses to more than 2 melanoma peptides in an ELISPOT assay. Two representative responders demonstrated strong HLA-class I protein expression in the tumor and very high scores of ELISPOT that might correlate to the regression of metastatic tumors. Clinical response through DC injections was as follows : 1CR, 1 PR, 1SD and 6 PD. All 59 DC injections in the phase I study were tolerable in terms of safety, however, the maximal tolerable dose of DCs was not determined. CONCLUSIONS: These results suggested that peptide cocktail-treated DC-based immunotherapy had the potential for utilizing as one of therapeutic tools against metastatic melanoma in Japan

OMAE2011-49594 DEVELOPMENT OF 6% NICKEL STEEL FOR LNG STORAGE TANKS

ABSTRACT 9% Ni steel has been used for LNG storage tanks for more than four decades although 5.5% Ni steel (N-TUF CR196) was developed in the 1970&apos;s using a special heat treatment method named L-treatment. The reason why the actual application of 5.5% Ni steel has not been attained to LNG storage tanks is mainly because the requirement of fracture properties is not confirmed for the tanks. Under the circumstances of expanding demand for natural gas and double-integrity in LNG storage tanks, we restarted developing low Ni steel for LNG storage tanks by using both conventional and advanced techniques. For the application of low Ni steel to the present LNG storage tanks, both fracture initiation and propagation properties of base metal plates and welded joints should be concerned. The fracture initiation and propagation properties of base metal were compensated with the intercritical reheating process (L-treatment), and the propagation property was additionally enhanced by combining TMCP with L-treatment. In addition, the chemical composition adjustment and the homogenization treatment of solute elements were conducted for improving the fracture initiation and propagation properties of welded joints. 6% Ni steel plates were manufactured by the process of continuous casting, reheating, hot rolling, direct quenching (TMCP), L-treatment, and tempering, and their chemical composition was 0.05C-0.06Si-1.0Mn-6.3Ni-Cr-Mo. As the results of fracture property evaluation including large-scale fracture tests such as the duplex ESSO test and the wide plate tensile test, it was demonstrated that 6% Ni steel has good characteristics regarding brittle fracture initiation and propagation in base metal plates and welded joints

Integrative and theoretical research on the architecture of a biological system and its disorder

Uchida S., Asai Y., Kariya Y., et al. Integrative and theoretical research on the architecture of a biological system and its disorder. Journal of Physiological Sciences , (2019); https://doi.org/10.1007/s12576-019-00667-8.An organism stems from assemblies of a variety of cells and proteins. This complex system serves as a unit, and it exhibits highly sophisticated functions in response to exogenous stimuli that change over time. The complete sequencing of the entire human genome has allowed researchers to address the enigmas of life and disease at the gene- or molecular-based level. The consequence of such studies is the rapid accumulation of a multitude of data at multiple levels, ranging from molecules to the whole body, that has necessitated the development of entirely new concepts, tools, and methodologies to analyze and integrate these data. This necessity has given birth to systems biology, an advanced theoretical and practical research framework that has totally changed the directions of not only basic life science but also medicine. During the symposium of the 95th Annual Meeting of The Physiological Society of Japan 2018, five researchers reported on their respective studies on systems biology. The topics included reactions of drugs, ion-transport architecture in an epithelial system, multi-omics in renal disease, cardiac electrophysiological systems, and a software platform for computer simulation. In this review article these authors have summarized recent achievements in the field and discuss next-generation studies on health and disease

Superconductivity Series in Transition Metal Dichalcogenides by Ionic Gating

Functionalities of two-dimensional (2D) crystals based on semiconducting transition metal dichalcogenides (TMDs) have now stemmed from simple field effect transistors (FETs) to a variety of electronic and opto-valleytronic devices, and even to superconductivity. Among them, superconductivity is the least studied property in TMDs due to methodological difficulty accessing it in different TMD species. Here, we report the systematic study of superconductivity in MoSe2, MoTe2 and WS2 by ionic gating in different regimes. Electrostatic gating using ionic liquid was able to induce superconductivity in MoSe2 but not in MoTe2 because of inefficient electron accumulation limited by electronic band alignment. Alternative gating using KClO4/polyethylene glycol enabled a crossover from surface doping to bulk doping, which induced superconductivities in MoTe2 and WS2 electrochemically. These new varieties greatly enriched the TMD superconductor families and unveiled critical methodology to expand the capability of ionic gating to other materials

iPSC-Based Compound Screening and In Vitro Trials Identify a Synergistic Anti-amyloid β Combination for Alzheimer’s Disease

In the process of drug development, in vitro studies do not always adequately predict human-specific drug responsiveness in clinical trials. Here, we applied the advantage of human iPSC-derived neurons, which offer human-specific drug responsiveness, to screen and evaluate therapeutic candidates for Alzheimer’s disease (AD). Using AD patient neurons with nearly 100% purity from iPSCs, we established a robust and reproducible assay for amyloid β peptide (Aβ), a pathogenic molecule in AD, and screened a pharmaceutical compound library. We acquired 27 Aβ-lowering screen hits, prioritized hits by chemical structure-based clustering, and selected 6 leading compounds. Next, to maximize the anti-Aβ effect, we selected a synergistic combination of bromocriptine, cromolyn, and topiramate as an anti-Aβ cocktail. Finally, using neurons from familial and sporadic AD patients, we found that the cocktail showed a significant and potent anti-Aβ effect on patient cells. This human iPSC-based platform promises to be useful for AD drug development