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Mycobacterium bovis BCG as a Delivery System for the dtb Gene Antigen from Diphtheria Toxin

American Journal of Molecular Biology 2017年第4期7卷 176-189页

作者： Dilzamar V.Nascimento Odir A.Dellagostin Denise C.S.Matos Douglas McIntosh raphael hirata jr. Geraldo M.B.Pereira Ana Luíza Mattos-Guaraldi Geraldo R.G.Armoa Faculdade de Ciências Médicas Universidade do Estado do Rio de JaneiroRio de JaneiroBrazil Núcleo de Biotecnologia Universidade Federal de PelotasCampus UniversitárioPelotasBrazil Instituto de Tecnologia em Imunobiológicos Fundacao Oswaldo CruzRio de JaneiroBrazil Instituto Oswaldo Cruz Fundacao Oswaldo CruzRio de JaneiroBrazil Instituto de Veterinária Universidade Federal Rural do Rio de JaneiroSeropedicaRio de JaneiroBrazil

Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.

关键词： Recombinant BCG Diphtheria Toxin dtb Gene Park Williams 8(PW8) Corynebacterium diphtheriae rDTBPW8 pUS977 Vector

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Major Elements Geochemistry of Sedimentary Rocks from CorumbataíFormation, Santa Gertrudes Ceramic Pole, São Paulo, Brazil

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Geomaterials 2014年第1期4卷 11-17页

作者： Maria Margarita Torres Moreno Rogers raphael da Rocha Letícia hirata Godoy Ceramica Rochaforte Rio Claro Brazil Department of Petrology and Metalogeny Institute of Geosciences and Exact Sciences Paulista State University Rio Claro Brazil

We conducted a geochemical study of the major elements of 29 clay samples from 10 mining fronts, which are used for ceramic coatings at Santa Gertrudes Ceramic Pole, São Paulo, Brazil. This region is considered as the biggest ceramic pole in Latin America. The mines are located in Corumbataí Formation (Paraná Sedimentary Basin) and are mined in bench system. The samples were analyzed as a whole and along each profile to evaluate the variation of the concentration of major elements and weathering effects. The results show a differentiation into three groups: 1) carbonate clays;2) lower portion samples;3) upper portion samples, and properties that allow obtaining useful information about the use and selection of raw materials to the formulation of ceramic masses and for quality control of raw materials in order to achieve products of similar quality.

关键词： Clay Minerals Raw Material Ceramic Coatings Weathering

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Plasmid instability when the hsp60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

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American Journal of Molecular Biology 2013年第2期3卷 81-86页

作者： Dilzamar V.Nascimento Odir A.Dellagostin raphael hirata jr.Geraldo M.B.Pereira Ana Luiza Mattos-Guaraldi Geraldo R.Garcia Armoa Faculdade de Ciencias Médicas Universidade do Estado do Rio de JaneiroRio de JaneiroBrazil Centro de Biotecnologia Universidade Federal de PelotasCampus UniversitárioPelotasBrazil Instituto Oswaldo Cruz Fundacao Oswaldo CruzRio de JaneiroBrazil

The genetic modification of the live attenuated Mycobacterium bovis BCG to deliver a protective Corynebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in BCG, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant BCG transformant to express the dtb gene of C. diphtheriae we generated two new rBCG strains by transforming the Moreau substrain of BCG with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After transformation recombinant BCG clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rBCGs transformed with the construct carrying the weak PAN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.

关键词： BCG Vaccine Recombinant BCG Fragment B of Diphtheria Toxin Anti-Diphtheria Vaccine

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Hepatocellular Carcinoma: Risk Factors, Diagnosis, Staging and Treatment in a Referral Centre

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Journal of Cancer Therapy 2013年第2期4卷 384-393页

作者： raphael Raphe Willian J. Duca Paulo C. Arroyo jr. Rita C. da Silva Renato F. da Silva Postgrauduate Course Faculty of Medicine of Sao Jose do Rio Preto FAMERP Sao Jose do Rio Preto Brazil Unit of Surgery and Liver Transplant Department of Surgery Hospital de Base FUNFARME/FAMERP Sao Jose do Rio Preto Brazil Unit of Surgery and Liver Transplant Department of Medicine Hospital de Base FUNFARME/FAMERP Sao Jose do Rio Preto Brazil

Introduction: Hepatocellular carcinoma is the most common primary neoplasm of the liver and a significant cause of mortality in patients with cirrhosis. A retrospective cross-sectional study was performed to analyze epidemiological aspects related to risk factors, diagnosis, staging and first-line treatment in a closed population. Methods: The medical records of patients seen between November 1998 and May 2011 were revisited. Results: Of the 272 patients included in this study, 229 (84.2%) were male and the average age was 57.1 years (standard deviation 10.9 years). The most common etiology was hepatitis C virus infection in 145 (55.1%) patients, with this being the single cause in 88 (33.4%) patients. The largest masses ranged from 6 mm to 260 mm in diameter with a mean of 61.4 mm (standard deviation 41.5 mm). Only one mass was found in 145 (64.2%) cases, two masses in 26 (11.5%), three masses in 9 (4%) and 46 patients (20.3%) had multifocal disease. Early stage disease was diagnosed in 47 patients (22.0%), advanced stage in 65 (30.4%) and terminal stage in 32 (14.9%). Hepatocellular carcinoma was found by chance in 11%. Diagnosis was by means of imaging in 175 (68.1%) cases. The level of alpha-fetoprotein was measured in 209 patients, with 29.2% having levels lower than 20 ng/mL and 34.9% having levels above 400 ng/mL. Specific treatment was administered in 236 patients (86.8%) with hepatic chemoembolization in 127 (46.7%) and liver transplantation in 72 (26.5%);of these 33 (45.8%) received hepatic chemoembolization as a bridge to transplantation. Thirty-four patients (12.5%) received only supportive therapy. Conclusions: Patients are chiefly male and disease involvement generally occurs in the 5th decade of life. Cirrhosis was present in most patients and hepatitis C virus infection was the commonest etiologic agent. Only one imaging examination was required for diagnosis in most patients. The measurement of alpha-fetoprotein levels did not prove to be a good too

关键词： Hepatocellular Carcinoma Epidemiology Risk Factors Liver Cancer Hepatitis Alcohol

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Dual-Delay-Path Ring Oscillator with Self-Biased Delay Cells for Clock Generation

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Circuits and Systems 2023年第6期14卷 19-28页

作者： Agord de Matos Pinto jr.raphael Ronald Noal Souza Mateus Biancarde Castro Eduardo Rodrigues de Lima Leandro Tiago Manêra School of Electrical and Computer Engineering (FEEC) University of Campinas Campinas Brazil Eldorado Research Institute Campinas Brazil

This work summarizes the structure and operating features of a high-performance 3-stage dual-delay-path (DDP) voltage-controlled ring oscillator (VCRO) with self-biased delay cells for Phase-Locked Loop (PLL) structurebased clock generation and digital system driving. For a voltage supply VDD = 1.8 V, the resulting set of performance parameters include power consumption PDC = 4.68 mW and phase noise PN@1MHz = -107.8 dBc/Hz. From the trade-off involving PDC and PN, a system level high performance is obtained considering a reference figure-of-merit ( FoM = -224 dBc/Hz ). Implemented at schematic level by applying CMOS-based technology (UMC L180), the proposed VCRO was designed at Cadence environment and optimized at MunEDA WiCkeD tool.

关键词： Phase Locked Loop (PLL) Voltage-Controlled Ring Oscillators (VCRO) Dual-Delay-Path DDP Delay Cells

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Lafoensia pacari alleviates intestinal damage by modulating cyclooxygenase-2:In silico and in vivo evaluation in a colitis model

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World Journal of Gastroenterology 2023年第17期29卷 2628-2641页

作者： Gabrielle Caroline Peiter Thayene Kamyli Moesch Queiroz Edson Luiz Michalkiewicz jr.raphael Henrique Chappuis Jennefer Sousa Luz Luiz Henrique Casagrande Piovezani Cleison Ferreira Silva Matheus Nozomi Tsutumi Augusto Fernandes Chaves Rafael Messias Luiz Cinthia Façanha Wendel Ana Carla Zarpelon-Schutz Kádima Nayara Teixeira Programa Multicêntrico de Pós-graduação em Biquímica e Biologia Molecular-Setor Palotina Universidade Federal do ParanáPalotina 85.950-000ParanáBrazil Programa de Pós-graduação em Biotecnologia-Setor Palotina Universidade Federal do ParanáPalotina 85.950-000ParanáBrazil Campus Toledo Universidade Federal do ParanáToledo 85.919-899ParanáBrazil

BACKGROUND Inflammatory bowel diseases(IBD)are a worldwide health problem and mainly affect young people,consequently affecting the *** treatments are often associated with side effects,and new therapeutic options are *** centuries,plants have represented important substrates in the field of drug *** pacari(***)is a plant whose pharmaceutical potential has been described,and may have biological activity relevant to the treatment of IBD *** To investigate the activity of keto-alcoholic extracts of *** with respect to ameliorating the inflammatory and nociceptive symptoms of acute experimental colitis in *** Keto-alcoholic extracts of *** leaves and bark were administered to male andfemale Swiss mice weighing 25 g to 30 g(n=8 male mice and n=8 female mice).The effect of these extracts was observed in an acetic acid-induced acute experimental model of colitis with regard to antinociception/analgesia and inflammatory tissue *** macroscopic indices included the Wallace score and the colon weight obtained using a precision *** hyperalgesia was determined using an electronic *** related to overt pain was determined by quantifying the number of writhing instances within 20 min of administration of acetic *** docking was performed using human and murine cyclooxygenase-2(COX-2)with 3 flavonoids(ellagic acid,kaempferol,and quercetin)on the AutoDock Vina *** of variance followed by Tukey’s posttest was used with P<0.05 indicating *** In this murine model of colitis,administration of extracts from *** ameliorated acetic acidinduced writhing and colitis-associated inflammatory *** improvements may be attributable to the reduction in edema,inflammation(e.g.,ulcers,hyperemia,and bowel wall damage),and the intensity of abdominal *** keto-alcoholic extracts of *** leaves and bark administered at a

关键词： Antinociceptive activity Anti-inflammatory activity Cyclooxygenase-2 Flavonoids Inflammatory bowel disease Lafoensia pacari

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Alterations in the human epidermal growth factor receptor 2-phosphatidylinositol 3-kinase-v-Akt pathway in gastric cancer

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World Journal of Gastroenterology 2012年第45期18卷 6577-6586页

作者： Yasutaka Sukawa Hiroyuki Yamamoto Katsuhiko Nosho Hiroaki Kunimoto Hiromu Suzuki Yasushi Adachi Mayumi Nakazawa Takayuki Nobuoka Mariko Kawayama Masashi Mikami Takashi Matsuno Tadashi Hasegawa Koichi hirata Kohzoh Imai Yasuhisa Shinomura First Department of Internal Medicine Sapporo Medical University School of MedicineSapporo 60-8543Japan Department of Molecular Biology Sapporo Medical University School of MedicineSapporo 60-8543Japan First Department of Surgery Sapporo Medical University School of MedicineSapporo 60-8543Japan Department of Gastroenterology JR Sapporo HospitalSapporo 60-8543Japan Department of Surgery Sapporo Gekakinen HospitalSapporo 60-8543Japan Department of Surgical Pathology Sapporo Medical University HospitalSapporo 60-8543Japan Division of Cancer Research The Institute of Medical ScienceThe University of TokyoTokyo 108-8639Japan

AIM:To investigate human epidermal growth factor receptor 2(HER2)-phosphatidylinositol 3-kinase(PI3K)-vAkt murine thymoma viral oncogene homolog signaling ***:We analyzed 231 formalin-fixed,paraffinembedded gastric cancer tissue specimens from Japanese patients who had undergone surgical *** patients' age,sex,tumor location,depth of invasion,pathological type,lymph node metastasis,and pathological stage were determined by a review of the medical *** of HER2 was analyzed by immunohistochemistry(IHC) using the HercepTest TM *** criteria for HER2 positivity(0,1+,2+,and 3+) were *** that scored 3+ were considered *** of phospho Akt(pAkt) was also analyzed by *** were considered pAkt-positive when the percentage of positive tumor cells was 10% or ***3K,catalytic,alpha polypeptide(PIK3CA) mutations in exons 1,9 and 20 were analyzed by ***-Barr virus(EBV) infection was analyzed by in situ hybridization targeting EBV-encoded small RNA(EBER) with an EBER-RNA *** instability(MSI) was analyzed by polymerase chain reaction using the mononucleotide markers BAT25 and ***:HER2 expression levels of 0,1+,2+ and 3+ were found in 167(72%),32(14%),12(5%) and 20(8.7%) samples,***2 overexpression(IHC 3+) significantly correlated with intestinal histological type(15/20 vs 98 /205,P = 0.05).PIK3CA mutations were present in 20 cases(8.7%) and significantly correlated with MSI(10/20 vs 9/211,P < 0.01).The mutation frequency was high(21%) in T4 cancers and very low(6%) in T2 *** in exons 1,9 and 20 were detected in 5(2%),9(4%) and 7(3%) cases,*** new types of PIK3CA mutation,R88Q and R108H,were found in *** PIK3CA mutations were heterozygous missense singlebase substitutions,the most common being H1047R(6/20,30%) in *** cancers(8%) were EBV-positive and this positivity significantly correlated with a diffu

关键词： Human epidermal growth factor receptor 2 Phosphatidylinositol 3-kinase Catalytic Alpha polypep-tide Epstein-Barr virus Aid: Gastric cancer

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