Structural Changes in Stx1 Engineering Monoclonal Antibody Improves Its Functionality as Diagnostic Tool for a Rapid Latex Agglutination Test

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Daniela Luz

2018-02-01

Full Text Available Stx1 toxin is one of the AB5 toxins of Shiga toxin-producing Escherichia coli (STEC responsible for foodborne intoxication during outbreaks. The single-chain variable fragment (scFv is the most common recombinant antibody format; it consists of both variable chains connected by a peptide linker with conserved specificity and affinity for antigen. The drawbacks of scFv production in bacteria are the heterologous expression, conformation and stability of the molecule, which could change the affinity for the antigen. In this work, we obtained a stable and functional scFv-Stx1 in bacteria, starting from IgG produced by hybridoma cells. After structural modifications, i.e., change in protein orientation, vector and linker, its solubility for expression in bacteria was increased as well as the affinity for its antigen, demonstrated by a scFv dissociation constant (KD of 2.26 × 10−7 M. Also, it was able to recognize purified Stx1 and cross-reacted with Stx2 toxin by ELISA (Enzyme-Linked Immunosorbent Assay, and detected 88% of Stx1-producing strains using a rapid latex agglutination test. Thus, the scFv fragment obtained in the present work is a bacteria-produced tool for use in a rapid diagnosis test, providing an alternative for STEC diagnosis.

Efecto citotóxico de la toxina shiga tipo 2 y su subunidad b en células epiteliales tubulares renales humanas en cultivo Cytotoxic effect of Shiga toxin type 2 and its B subunit on human renal tubular epithelial cell cultures

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Virginia Pistone Creydt

2005-04-01

Full Text Available Escherichia coli enterohemorrágica productora de toxina Shiga (Stx causa diarrea acuosa, colitis hemorrágica y síndrome urémico hemolítico (SUH. En Argentina, el SUH es la principal causa de insuficiencia renal en niños. El objetivo de este trabajo fue estudiar la toxicidad de Stx tipo 2 (Stx2 y su subunidad B (Stx2B en células epiteliales tubulares renales humanas (CERH, en presencia y ausencia de factores inflamatorios. Los efectos citotóxicos se evaluaron como alteración de la funcionalidad del epitelio; daños histológicos; viabilidad celular; síntesis de proteínas y apoptosis celular. Los resultados muestran que Stx2 regula el pasaje de agua a través de CERH a tiempos menores de 1h de incubación. A tiempos mayores, hasta 72 hs, el estudio de la morfología, la viabilidad, la síntesis de proteínas y la apoptosis demostró que las CERH fueron sensibles a la acción citotóxica de Stx2 y Stx2B de una manera dosis y tiempo dependiente. Estos efectos fueron potenciados por lipopolisacáridos bacterianos (LPS, IL-1b, y butirato.Shiga toxin (Stx-producing E.coli causing watery diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome (HUS. In Argentina, HUS is the most common cause of acute renal failure in children. The purpose of the present study was to examine the cytotoxicity of Stx type 2 (Stx2 and its B subunit (Stx2B on human renal tubular epithelial cells (HRTEC, in the presence and absence of inflammatory factors. Cytotoxic effects were assessed in terms of functionality of the epithelium, histological damage, cell viability, protein synthesis and cellular apoptosis. Results show that Stx2 regulates the passage of water through the HRTEC within an incubation period of 1h. Within longer periods, up to 72 hours, the study of morphology, viability, protein synthesis and apoptosis shows that HRTEC were sensitive to the cytotoxic action of Stx2 and Stx2B in a dose- and time-dependent manner. These effects were potentiated by

Membrane invagination induced by Shiga toxin B-subunit

DEFF Research Database (Denmark)

Pezeshkian, W.; Hansen, Allan Grønhøj; Johannes, Ludger

2016-01-01

-atom molecular dynamics and Monte Carlo simulations we show that the molecular architecture of STxB enables the following sequence of events: the Gb3 binding sites on STxB are arranged such that tight avidity-based binding results in a small increment of local curvature. Membrane-mediated clustering of several...... toxin molecules then creates a tubular membrane invagination that drives toxin entry into the cell. This mechanism requires: (1) a precise molecular architecture of the STxB binding sites; (2) a fluid bilayer in order for the tubular invagination to form. Although, STxB binding to the membrane requires...... specific interactions with Gb3 lipids, our study points to a generic molecular design principle for clathrin-independent endocytosis of nanoparticles....

An efficient co-expression and purification system for the complex of Stx4 and C-terminal domain of Synip

International Nuclear Information System (INIS)

Tian Wei; Ma Cong; Liu Yingfang; Xu Tao

2008-01-01

Synip and Stx4 complex plays a key role in GLUT4 vesicle trafficking and fusion with plasma membrane. The interaction of Synip with Stx4 prevents interaction of VAMP2 located in GLUT4 vesicle with Stx4 in basal state. Insulin induces the dissociation of the Synip and Stx4 complex, and then triggers VAMP2 to interact with Stx4 to form the SNARE complex, thus promoting the vesicle fusion. In this report, we adopt a novel system for co-expression of the Synip and Stx4 by using two common vectors pGEX6p-1 and pET28a(+) to investigate their expression, purification, and interaction. Through this co-expression system, we successfully co-expressed the Synip and Stx4 complex with high yield, and co-purified at an approximate 1:1 molar ratio with high purity (95%). We also demonstrate that the 1-28 residues of Stx4 are dispensable for interaction with Synip using this co-expression system

The Mobilome; A Major Contributor to Escherichia coli stx2-Positive O26:H11 Strains Intra-Serotype Diversity

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Sabine Delannoy

2017-09-01

Full Text Available Shiga toxin-producing Escherichia coli of serotype O26:H11/H- constitute a diverse group of strains and several clones with distinct genetic characteristics have been identified and characterized. Whole genome sequencing was performed using Illumina and PacBio technologies on eight stx2-positive O26:H11 strains circulating in France. Comparative analyses of the whole genome of the stx2-positive O26:H11 strains indicate that several clones of EHEC O26:H11 are co-circulating in France. Phylogenetic analysis of the French strains together with stx2-positive and stx-negative E. coli O26:H11 genomes obtained from Genbank indicates the existence of four clonal complexes (SNP-CCs separated in two distinct lineages, one of which comprises the “new French clone” (SNP-CC1 that appears genetically closely related to stx-negative attaching and effacing E. coli (AEEC strains. Interestingly, the whole genome SNP (wgSNP phylogeny is summarized in the cas gene phylogeny, and a simple qPCR assay targeting the CRISPR array specific to SNP-CC1 (SP_O26-E can distinguish between the two main lineages. The PacBio sequencing allowed a detailed analysis of the mobile genetic elements (MGEs of the strains. Numerous MGEs were identified in each strain, including a large number of prophages and up to four large plasmids, representing overall 8.7–19.8% of the total genome size. Analysis of the prophage pool of the strains shows a considerable diversity with a complex history of recombination. Each clonal complex (SNP-CC is characterized by a unique set of plasmids and phages, including stx-prophages, suggesting evolution through separate acquisition events. Overall, the MGEs appear to play a major role in O26:H11 intra-serotype clonal diversification.

The Mobilome; A Major Contributor to Escherichia coli stx2-Positive O26:H11 Strains Intra-Serotype Diversity.

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Delannoy, Sabine; Mariani-Kurkdjian, Patricia; Webb, Hattie E; Bonacorsi, Stephane; Fach, Patrick

2017-01-01

Shiga toxin-producing Escherichia coli of serotype O26:H11/H- constitute a diverse group of strains and several clones with distinct genetic characteristics have been identified and characterized. Whole genome sequencing was performed using Illumina and PacBio technologies on eight stx2 -positive O26:H11 strains circulating in France. Comparative analyses of the whole genome of the stx2 -positive O26:H11 strains indicate that several clones of EHEC O26:H11 are co-circulating in France. Phylogenetic analysis of the French strains together with stx2 -positive and stx -negative E. coli O26:H11 genomes obtained from Genbank indicates the existence of four clonal complexes (SNP-CCs) separated in two distinct lineages, one of which comprises the "new French clone" (SNP-CC1) that appears genetically closely related to stx -negative attaching and effacing E. coli (AEEC) strains. Interestingly, the whole genome SNP (wgSNP) phylogeny is summarized in the cas gene phylogeny, and a simple qPCR assay targeting the CRISPR array specific to SNP-CC1 (SP_O26-E) can distinguish between the two main lineages. The PacBio sequencing allowed a detailed analysis of the mobile genetic elements (MGEs) of the strains. Numerous MGEs were identified in each strain, including a large number of prophages and up to four large plasmids, representing overall 8.7-19.8% of the total genome size. Analysis of the prophage pool of the strains shows a considerable diversity with a complex history of recombination. Each clonal complex (SNP-CC) is characterized by a unique set of plasmids and phages, including stx -prophages, suggesting evolution through separate acquisition events. Overall, the MGEs appear to play a major role in O26:H11 intra-serotype clonal diversification.

Phage-mediated Shiga toxin (Stx) horizontal gene transfer and expression in non-Shiga toxigenic Enterobacter and Escherichia coli strains.

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Khalil, Rowaida K S; Skinner, Craig; Patfield, Stephanie; He, Xiaohua

2016-07-01

Enterobacter cloacae M12X01451 strain recently identified from a clinical specimen produces a new Stx1 subtype (Stx1e) that was not neutralized by existing anti-Stx1 monoclonal antibodies. Acquisition of stx by Ent. cloacae is rare and origin/stability of stx1e in M12X01451 is not known. In this study, we confirmed the ability of Stx1a- and Stx1e-converting phages from an Escherichia coli O157:H7 strain RM8530 and M12X01451 respectively to infect several E. coli and Ent. cloacae strains. stx1e was detected in 97.5% and 72.5% of progenies of strains lysogenized by stx1e phage after 10 (T10) and 20 (T20) subcultures, versus 65% and 17.5% for stx1a gene. Infection of M12X01451 and RM8530 with each other's phages generated double lysogens containing both phages. stx1a was lost after T10, whereas the stx1e was maintained even after T20 in M12X01451 lysogens. In RM8530 lysogens, the acquired stx1e was retained with no mutations, but 20% of stx1a was lost after T20 ELISA and western blot analyses demonstrated that Stx1e was produced in all strains lysogenized by stx1e phage; however, Stx1a was not detected in any lysogenized strain. The study results highlight the potential risks of emerging Stx-producing strains via bacteriophages either in the human gastrointestinal tract or in food production environments, which are matters of great concern and may have serious impacts on human health. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Paralytic shellfish toxins, including deoxydecarbamoyl-STX, in wild-caught Tasmanian abalone (Haliotis rubra).

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Harwood, D Tim; Selwood, Andrew I; van Ginkel, Roel; Waugh, Craig; McNabb, Paul S; Munday, Rex; Hay, Brenda; Thomas, Krista; Quilliam, Michael A; Malhi, Navreet; Dowsett, Natalie; McLeod, Catherine

2014-11-01

For the first time wild-caught Tasmanian abalone, Haliotis rubra, have been reported to contain paralytic shellfish toxins (PSTs). This observation followed blooms of the toxic dinoflagellate Gymnodinium catenatum. No illnesses were reported, but harvesting restrictions were enforced in commercial areas. Abalone were assayed using HPLC-FLD methodology based on AOAC official method 2005.06. An uncommon congener, deoxydecarbamoyl-STX (doSTX), was observed in addition to regulated PSTs as unassigned chromatographic peaks. A quantitative reference material was prepared from contaminated Tasmanian abalone viscera and ampouled at 54.2 μmol/L. The LD50 of doSTX via intraperitoneal injection was 1069 nmol/kg (95% confidence limits 983-1100 nmol/kg), indicating it is nearly 40 times less toxic than STX. A toxicity equivalence factor of 0.042 was generated using the mouse bioassay. Levels of PSTs varied among individuals from the same site, although the toxin profile remained relatively consistent. In the foot tissue, STX, decarbamoyl-STX and doSTX were identified. On a molar basis doSTX was the dominant congener in both foot and viscera samples. The viscera toxin profile was more complex, with other less toxic PST congeners observed and was similar to mussels from the same site. This finding implicates localised dinoflagellate blooms as the PST source in Tasmanian abalone. Copyright © 2014 Elsevier Ltd. All rights reserved.

STX140, but not paclitaxel, inhibits mammary tumour initiation and progression in C3(1/SV40 T/t-antigen transgenic mice.

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Florence Meyer-Losic

Full Text Available Despite paclitxael's clinical success, treating hormone-refractory breast cancer remains challenging. Paclitaxel has a poor pharmacological profile, characterized by a low therapeutic index (TIX caused by severe dose limiting toxicities, such as neutropenia and peripheral neuropathy. Consequently, new drugs are urgently required. STX140, a compound previously shown to have excellent efficacy against many tumors, is here compared to paclitaxel in three translational in vivo breast cancer models, a rat model of peripheral neuropathy, and through pharmacological testing. Three different in vivo mouse models of breast cancer were used; the metastatic 4T1 orthotopic model, the C3(1/SV40 T-Ag model, and the MDA-MB-231 xenograft model. To determine TIX and pharmacological profile of STX140, a comprehensive dosing regime was performed in mice bearing MDA-MD-231 xenografts. Finally, peripheral neuropathy was examined using a rat plantar thermal hyperalgesia model. In the 4T1 metastatic model, STX140 and paclitaxel significantly inhibited primary tumor growth and lung metastases. All C3(1/SV40 T-Ag mice in the control and paclitaxel treated groups developed palpable mammary cancer. STX140 blocked 47% of tumors developing and significantly inhibited growth of tumors that did develop. STX140 treatment caused a significant (P<0.001 survival advantage for animals in early and late intervention groups. Conversely, in C3(1/SV40 T-Ag mice, paclitaxel failed to inhibit tumor growth and did not increase survival time. Furthermore, paclitaxel, but not STX140, induced significant peripheral neuropathy and neutropenia. These results show that STX140 has a greater anti-cancer efficacy, TIX, and reduced neurotoxicity compared to paclitaxel in C3(1/SV40 T-Ag mice and therefore may be of significant benefit to patients with breast cancer.

In Vivo Tumor Targeting by the B-Subunit of Shiga Toxin

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Thomas Viel

2008-11-01

Full Text Available Delivery of drugs to the appropriate target cells would improve efficacy and reduce potential side effects. The nontoxic B-subunit of the intestinal pathogen-produced Shiga toxin (STxB binds specifically to the glycosphingolipid Gb3, overex-pressed in membranes of certain tumor cells, and enters these cells through the retrograde pathway. Therefore, STxB binding to Gb3 receptors may be useful for cell-specific vectorization or imaging purposes. Here we labeled STxB with a fluorophore to evaluate its potential as an in vivo cell-specific targeting reagent in two different models of human colorectal carcinoma. Fluorescent STxB was administered systemically to xenografted nude mice, and its biodistribution was studied by optical imaging. The use of fluorescent STxB allowed the combination of the macroscopic observations with analyses at the cellular level using confocal microscopy. After administration, the fluorescent STxB was slowly eliminated by renal excretion. However, it accumulated in the tumor area. Furthermore, STxB was demonstrated to enter the Gb3-expressing tumoral cells, as well as the epithelial cells of the neovascularization and the monocytes and macrophages surrounding the xenografts.

Essential role of STX6 in esophageal squamous cell carcinoma growth and migration

Energy Technology Data Exchange (ETDEWEB)

Du, Jin [Department of Cardiothoracic Surgery, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210029 (China); Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028 (China); Liu, Xiang [Department of Cardiothoracic Surgery, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210029 (China); Wu, Yanhu, E-mail: wuyanhu@njmu.edu.cn [Department of Cardiothoracic Surgery, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210029 (China); Zhu, Jinfu; Tang, Yihu [Department of Cardiothoracic Surgery, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210029 (China)

2016-03-25

Abnormalities in endosomes, or dysregulation in their trafficking, play an important role directly in many diseases including oncogenesis. Syntaxin-6 (STX6) is involved in diverse cellular functions in a variety of cell types and has been shown to regulate many intracellular membrane trafficking events such as endocytosis, recycling and anterograde and retrograde trafficking. However, its expression pattern and biological functions in esophageal squamous cell carcinoma (ESCC) remained unknown. Here, we have found that the expression of STX6 was up-regulated in ESCC samples, its expression was significantly correlated with tumor size, histological differentiation, lymph node metastasis and depth. On one hand, STX6 silencing inhibited ESCC cells viability and proliferation in a p53-dependent manner. On the other hand, STX6 effect integrin trafficking and regulate ESCC cells migration. Taken together, our study revealed the oncogenic roles of STX6 in the progression of ESCC, and it might be a valuable target for ESCC therapy.

Rare autism-associated variants implicate syntaxin 1 (STX1 R26Q) phosphorylation and the dopamine transporter (hDAT R51W) in dopamine neurotransmission and behaviors.

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Cartier, Etienne; Hamilton, Peter J; Belovich, Andrea N; Shekar, Aparna; Campbell, Nicholas G; Saunders, Christine; Andreassen, Thorvald F; Gether, Ulrik; Veenstra-Vanderweele, Jeremy; Sutcliffe, James S; Ulery-Reynolds, Paula G; Erreger, Kevin; Matthies, Heinrich J G; Galli, Aurelio

2015-02-01

Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA. We adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD). Here, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms involve decreased phosphorylation of STX1 at Ser14 mediated by casein kinase 2 as well as a reduction in STX1/DAT interaction. These findings point to STX1/DAT interactions and STX1 phosphorylation as key regulators of DA homeostasis. We determine the molecular identity and the impact of these variants with the intent of defining DA dysfunction and associated behaviors as possible complications of ASD.

Influence of RNase E deficiency on the production of stx2-bearing phages and Shiga toxin in an RNase E-inducible strain of enterohaemorrhagic Escherichia coli (EHEC) O157:H7.

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Thuraisamy, Thujitha; Lodato, Patricia B

2018-05-01

In enterohaemorrhagic Escherichia coli (EHEC), stx1 or stx2 genes encode Shiga toxin (Stx1 or Stx2, respectively) and are carried by prophages. The production and release of both stx phages and toxin occur upon initiation of the phage lytic cycle. Phages can further disseminate stx genes by infecting naïve bacteria in the intestine. Here, the effect of RNase E deficiency on these two virulence traits was investigated. Cultures of the EHEC strains TEA028-rne containing low versus normal RNase E levels or the parental strain (TEA028) were treated with mitomycin C (MMC) to induce the phage lytic cycle. Phages and Stx2 titres were quantified by the double-agar assay and the receptor ELISA technique, respectively. RNase E deficiency in MMC-treated cells significantly reduced the yield of infectious stx2 phages. Delayed cell lysis and the appearance of encapsidated phage DNA copies suggest a slow onset of the lytic cycle. However, these observations do not entirely explain the decrease of phage yields. stx1 phages were not detected under normal or deficient RNase E levels. After an initial delay, high levels of toxin were finally produced in MMC-treated cultures. RNase E scarcity reduces stx2 phage production but not toxin. Normal concentrations of RNase E are likely required for correct phage morphogenesis. Our future work will address the mechanism of RNase E action on phage morphogenesis.

Commensal E. coli Stx2 lysogens produce high levels of phages after spontaneous prophage induction

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Hildegunn eIversen

2015-02-01

Full Text Available Enterohemorrhagic E. coli (EHEC is a food-borne pathogen that causes disease ranging from uncomplicated diarrhea to life-threatening hemolytic uremic syndrome (HUS and nervous system complications. Shiga toxin 2 (Stx2 is the major virulence factor of EHEC and is critical for development of HUS. The genes encoding Stx2 are carried by lambdoid bacteriophages and the toxin production is tightly linked to the production of phages during lytic cycle. It has previously been suggested that commensal E. coli could amplify the production of Stx2-phages and contribute to the severity of disease. In this study we examined the susceptibility of commensal E. coli strains to the Stx2-converting phage ϕ734, isolated from a highly virulent EHEC O103:H25 (NIPH-11060424. Among 38 commensal E. coli strains from healthy children below five years, 15 were lysogenized by the ϕ734 phage, whereas lytic infection was not observed. Three of the commensal E. coli ϕ734 lysogens were tested for stability, and appeared stable and retained the phage for at least 10 cultural passages. When induced to enter lytic cycle by H2O2 treatment, 8 out of 13 commensal lysogens produced more ϕ734 phages than NIPH-11060424. Strikingly, five of them even spontaneously (non-induced produced higher levels of phage than the H2O2 induced NIPH-11060424. An especially high frequency of HUS (60% was seen among children infected by NIPH-11060424 during the outbreak in 2006. Based on our findings, a high Stx2 production by commensal E. coli lysogens cannot be ruled out as a contributor to the high frequency of HUS during this outbreak.

Phylogenetic diversity and similarity of active sites of Shiga toxin (stx) in Shiga toxin-producing Escherichia coli (STEC) isolates from humans and animals.

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Asakura, H; Makino, S; Kobori, H; Watarai, M; Shirahata, T; Ikeda, T; Takeshi, K

2001-08-01

Nucleotide sequences of Shiga toxin (Stx) genes in STEC from various origins were determined and characterized by phylogenetic analysis based on Shiga toxin (Stx) with those deposited in GenBank. The phylogenetic trees placed Stx1 and Stx2 into two and five groups respectively, and indicated that Stx1 in sheep-origin STEC were placed into a different group from those in other STEC, and that Stx2 of deer-origin STEC also belonged to the unique group and appeared to be distantly related to human-origin STEC. On the other hand, Stx of STEC isolated from cattle, seagulls and flies were closely related to those of human-origin STEC. Such a diversity of Stx suggested that STEC might be widely disseminated in many animal species, and be dependent on their host species or their habitat. In addition, the active sites in both toxins were compared; the active sites in both subunits of Stx in all the animal-origin STEC were identical to those in human-origin STEC, suggesting that all the toxin of STEC from animals might be also cytotoxic, and therefore, such animal-origin STEC might have potential pathogenicity for humans.

Heterogeneity in Induction Level, Infection Ability, and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

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Bonanno, Ludivine; Petit, Marie-Agnès; Loukiadis, Estelle; Michel, Valérie

2016-01-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event. PMID:26826235

Characterization of stx2 and its variants in Escherichia coli O157:H7 ...

African Journals Online (AJOL)

Yomi

2011-04-11

Apr 11, 2011 ... from Chile and Chinese Mainland and both suggested that, pig might be ... De Baets et al. (2004) stx2-R .... the presence of 5% CO2. The tissue ... the cell concentration was adjusted to 1 x 109 CFU/ml, bacterial cultures were ...

Comparative genomics and stx phage characterization of LEE-negative Shiga toxin-producing Escherichia coli

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Susan Renee Steyert

2012-11-01

Full Text Available Infection by Escherichia coli and Shigella species are among the leading causes of death due to diarrheal disease in the world. Shiga toxin producing Escherichia coli (STEC that do not encode the locus of enterocyte effacement (LEE-negative STEC often possess Shiga toxin gene variants and have been isolated from humans and a variety of animal sources. In this study, we compare the genomes of nine LEE-negative STEC harboring various stx alleles with four complete reference LEE-positive STEC isolates. Compared to a representative collection of prototype E. coli and Shigella isolates representing each of the pathotypes, the whole genome phylogeny demonstrated that these isolates are diverse. Whole genome comparative analysis of the 13 genomes revealed that in addition to the absence of the LEE pathogenicity island, phage encoded genes including non-LEE encoded effectors, were absent from all nine LEE-negative STEC genomes. Several plasmid-encoded virulence factors reportedly identified in LEE-negative STEC isolates were identified in only a subset of the nine LEE-negative isolates further confirming the diversity of this group. In combination with whole genome analysis, we characterized the lambdoid phages harboring the various stx alleles and determined their genomic insertion sites. Although the integrase gene sequence corresponded with genomic location, it was not correlated with stx variant, further highlighting the mosaic nature of these phages. The transcription of these phages in different genomic backgrounds was examined. Expression of the Shiga toxin genes, stx1 and/or stx2, as well as the Q genes, were examined with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR assays. A wide range of basal and induced toxin induction was observed. Overall, this is a first significant foray into the genome space of this unexplored group of emerging and divergent pathogens.

High temperature in combination with UV irradiation enhances horizontal transfer of stx2 gene from E. coli O157:H7 to non-pathogenic E. coli.

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Wan-Fu Yue

Full Text Available Shiga toxin (stx genes have been transferred to numerous bacteria, one of which is E. coli O157:H7. It is a common belief that stx gene is transferred by bacteriophages, because stx genes are located on lambdoid prophages in the E. coli O157:H7 genome. Both E. coli O157:H7 and non-pathogenic E. coli are highly enriched in cattle feedlots. We hypothesized that strong UV radiation in combination with high temperature accelerates stx gene transfer into non-pathogenic E. coli in feedlots.E. coli O157:H7 EDL933 strain were subjected to different UV irradiation (0 or 0.5 kJ/m(2 combination with different temperature (22, 28, 30, 32, and 37 °C treatments, and the activation of lambdoid prophages was analyzed by plaque forming unit while induction of Stx2 prophages was quantified by quantitative real-time PCR. Data showed that lambdoid prophages in E. coli O157:H7, including phages carrying stx2, were activated under UV radiation, a process enhanced by elevated temperature. Consistently, western blotting analysis indicated that the production of Shiga toxin 2 was also dramatically increased by UV irradiation and high temperature. In situ colony hybridization screening indicated that these activated Stx2 prophages were capable of converting laboratory strain of E. coli K12 into new Shiga toxigenic E. coli, which were further confirmed by PCR and ELISA analysis.These data implicate that high environmental temperature in combination with UV irradiation accelerates the spread of stx genes through enhancing Stx prophage induction and Stx phage mediated gene transfer. Cattle feedlot sludge are teemed with E. coli O157:H7 and non-pathogenic E. coli, and is frequently exposed to UV radiation via sunlight, which may contribute to the rapid spread of stx gene to non-pathogenic E. coli and diversity of shiga toxin producing E. coli.

Inhibition of Shiga Toxin 2 (Stx2) in Apple Juices and its Resistance to Pasteurization

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In the present study, we evaluated Shiga toxin (Stx2) activity in apple juices by measuring a decrease in dehydrogenase activity of Vero cells with the microculture tetrazolium (MTT) assay. Freshly prepared juice from Red Delicious apples and Golden Delicious apples inhibited the biological activity...

Prevalence of Stx-producing Shigella species isolated from French Travelers Returning from the Caribbean: An Emerging Pathogen with International Implications

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Gray, Miranda D.; Lacher, David W.; Leonard, Susan R.; Abbott, Jason; Zhao, Shaohua; Lampel, Keith A.; Prothery, Estelle; Gouali, Malika; Weill, François-Xavier; Maurelli, Anthony T.

2015-01-01

Shiga toxins are potent cytotoxins that inhibit host cell protein synthesis, leading to cell death. Classically, these toxins are associated with intestinal infections due to Shiga toxin-producing Escherichia coli or Shigella dysenteriae serotype 1 and infections with these strains can lead to hemolytic uremic syndrome. Over the past decade there is increasing recognition that Shiga toxin is produced by additional Shigella species. We recently reported the presence and expression of stx genes in Shigella flexneri 2a clinical isolates. The toxin genes were carried by a new stx-encoding bacteriophage and infection with these strains correlated with recent travel to Haiti or the Dominican Republic. In this study we further explored the epidemiological link to this region by utilizing the French National Reference Center for Escherichia coli, Shigella and Salmonella collection to survey the frequency of Stx-producing Shigella species isolated from French travelers returning from the Caribbean. About 21% of the isolates tested were found to encode and produce Stx. These isolates included strains of S. flexneri 2a, S. flexneri Y, and S. dysenteriae 4. All of the travelers whom were infected with Stx-producing Shigella had recently traveled to Haiti, the Dominican Republic, or French Guiana. Furthermore, whole genome sequencing found that the toxin genes were encoded by a prophage that was highly identical to the phage we identified in our previous study. These findings demonstrate that this new stx-encoding prophage is circulating within that geographical area, has spread to other continents, and is capable of spreading to multiple Shigella serogroups. PMID:25980352

Occurrence of Escherichia coli harbouring stx genes in popiah, a Malaysian street food

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Elexson, N.

2017-01-01

Full Text Available Irrespective of its health effects, street foods are very popular with the consumers. The main purpose of this research was to study the biosafety of Escherichia coli in popiah, a Malaysian street food sold at a roadside food stall and a restaurant in Sri Serdang, Selangor, Malaysia, using the combination of the most probable number (MPN-Polymerase Chain Reaction (PCR assay-plating on Eosin Methylene Blue (EMB agar methods. Using these biomolecular methods, E. coli was detected in 12/15 (80% and 11/15 (73% of the collected samples from the roadside food stall and the restaurant respectively. The incidence of stx virulence-associated genes was detected in 1/15 (7% among the E. coli isolated from samples taken from the roadside food stall while the E. coli isolated from the restaurant was 3/15 (20%. The density of E. coli ranged from 1100 MPN/g and the density of E. coli positive with stx genes was <3 to 53 MPN /g in samples from both the roadside food stall and the restaurant. The presence of the stx-positive E. coli in popiah are significant to risk assessments of food and epidemiological studies. Therefore, from the information obtained in this study, it is obvious that the screening for STEC markers in food samples would be useful for food safety survey.

Immunization of pregnant cows with Shiga toxin-2 induces high levels of specific colostral antibodies and lactoferrin able to neutralize E. coli O157:H7 pathogenicity.

Science.gov (United States)

Albanese, Adriana; Sacerdoti, Flavia; Seyahian, E Abril; Amaral, Maria Marta; Fiorentino, Gabriela; Fernandez Brando, Romina; Vilte, Daniel A; Mercado, Elsa C; Palermo, Marina S; Cataldi, Angel; Zotta, Elsa; Ibarra, Cristina

2018-03-20

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Specific egg yolk immunoglobulin as a new preventive approach for Shiga-toxin-mediated diseases.

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Paola Neri

Full Text Available Shiga toxins (Stxs are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection.

Metabolic and Pharmacokinetic Differentiation of STX209 and Racemic Baclofen in Humans

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Raymundo Sanchez-Ponce

2012-09-01

Full Text Available STX209 is an exploratory drug comprising the single, active R-enantiomer of baclofen which is in later stage clinical trials for the treatment of fragile x syndrome (FXS and autism spectrum disorders (ASD. New clinical data in this article on the metabolism and pharmacokinetics of the R- and S-enantiomers of baclofen presents scientific evidence for stereoselective metabolism of only S-baclofen to an abundant oxidative deamination metabolite that is sterically resolved as the S-enantiomeric configuration. This metabolite undergoes some further metabolism by glucuronide conjugation. Consequences of this metabolic difference are a lower Cmax and lower early plasma exposure of S-baclofen compared to R-baclofen and marginally lower urinary excretion of S-baclofen after racemic baclofen administration. These differences introduce compound-related exposure variances in humans in which subjects dosed with racemic baclofen are exposed to a prominent metabolite of baclofen whilst subjects dosed with STX209 are not. For potential clinical use, our findings suggest that STX209 has the advantage of being a biologically defined and active enantiomer.

Rare autism-associated variants implicate syntaxin 1 (STX1 R26Q) phosphorylation and the dopamine transporter (hDAT R51W) in dopamine neurotransmission and behaviors

DEFF Research Database (Denmark)

Cartier, Etienne; Hamilton, Peter J; Belovich, Andrea N

2015-01-01

BACKGROUND: Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through...... the high-affinity re-uptake of synaptically released DA. METHODS: We adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD). OUTCOMES......: Here, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms...

The Design of New Adjuvants for Mucosal Immunity to Neisseria meningitidis B in Nasally Primed Neonatal Mice for Adult Immune Response

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Tatiane Ferreira

2012-01-01

Full Text Available The aim of this study was to determine the value of detoxified Shiga toxins Stx1 and Stx2 (toxoids of Escherichia coli as mucosal adjuvants in neonatal mice for immunogenicity against the outer membrane proteins (OMPs of Neisseria meningitidis B. Mucosal immunization has been shown to be effective for the induction of antigen-specific immune responses in both the systemic and mucosal compartments. Systemic antibody levels (IgG, IgG1, IgG2a, IgG2b, IgM, and IgA and mucosal IgM and IgA were measured by ELISA using an N. meningitidis as an antigen. In addition, IFN-γ and IL-6 production were measured after stimulated proliferation of immune cells. Intranasal administration elicited a higher anti-OMP IgA response in both saliva and vaginal fluids. Our results suggest that both Stx1 and Stx2 toxoids are effective mucosal adjuvants for the induction of Ag-specific IgG, IgM, and IgA antibodies. The toxoids significantly enhanced the IgG and IgM response against OMPs with a potency equivalent to CT, with the response being characterized by both IgG1 and IgG2a isotypes, and increased IFN-gamma production. Additionally, bactericidal activity was induced with IgG and IgM antibodies of high avidity. These results support the use of the new toxoids as potent inducing adjuvants that are particularly suitable for mucosal immunization.

Detection of Enterohemorrhagic Escherichia coli Related Genes in E. coli Strains Belonging to B2 Phylogroup Isolated from Urinary Tract Infections in Combination with Antimicrobial Resistance Phenotypes

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Hamid Staji

2017-07-01

Full Text Available Background:  This study was conducted to detect the prevalence of EHEC virulence genes and antimicrobial resistance profile of Escherichia coli strains belonging to B2 phylogroup implicated in Urinary tract infections in Semnan, Iran.Methods:   From 240 urine samples 160 E. coli strains were isolated, biochemically. Then, E. coli isolates were examined by Multiplex-PCR for phylogenetic typing and detection of virulence genes (hly, stx1, stx2, eae associated with Enterohemorrhagic E. coli. Finally, Antimicrobial resistance of E. coli isolates were characterized using Disk Diffusion method.  Results:  From 160 E. coli isolates, 75 strains (47% were assigned to B2 phylogenetic group and prevalence of virulence genes were as follow: hly (21.3%, stx1 (16%, stx2 (10.6% and eae (6.7%, subsequently.  Phenotypic antimicrobial resistance of B2 isolates showed that all isolates were sensitive to Meropenem and Furazolidone and then highest frequency of resistance was observed to Streptomycin, Oxytetracycline, Neomycin, Nalidixic acid and Ampicillin (98.7% to 49.3%. Also low resistance prevalence was observed in case of Ceftizoxime, Lincospectin, Imipenem, Chloramphenicol and flurefenicole (16% to 1.3%.Conclusion:   The data suggest a high prevalence of antibiotic resistance in UPEC strains belonging to B2 phylogroup even for the antimicrobials using in pet and farm animals and their potential to cause EHEC specific clinical symptoms which may represent a serious health risk since these strains can be transmitted to GI tract and act as a reservoir for other uropathogenic E. coli and commensal strains.

Vaccination of pregnant cows with EspA, EspB, γ-intimin, and Shiga toxin 2 proteins from Escherichia coli O157:H7 induces high levels of specific colostral antibodies that are transferred to newborn calves.

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Rabinovitz, B C; Gerhardt, E; Tironi Farinati, C; Abdala, A; Galarza, R; Vilte, D A; Ibarra, C; Cataldi, A; Mercado, E C

2012-06-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of intestinal disease and hemolytic uremic syndrome, a serious systemic complication that particularly affects children. Cattle are primary reservoirs for EHEC O157:H7 and the main source of infection for humans. Vaccination of cattle with different combinations of bacterial virulence factors has shown efficacy in decreasing EHEC O157:H7 shedding. It is, therefore, important to demonstrate whether vaccination of pregnant cows with EHEC O157:H7 induces high titers of transferable antibodies to avoid early colonization of calves by the bacteria. In this study we evaluated the ability of EspA, EspB, the C-terminal fragment of 280 amino acids of γ-intimin (γ-intimin C₂₈₀) and inactivated Shiga toxin (Stx) 2 proteins to induce specific antibodies in colostrum and their passive transference to colostrum-fed calves. Friesian pregnant cows immunized by the intramuscular route mounted significantly high serum and colostrum IgG responses against EspB and γ-intimin C₂₈₀ that were efficiently transferred to their calves. Antibodies to EspB and γ-intimin C₂₈₀ were detected in milk samples of vaccinated cows at d 40 postparturition. Significant Stx2-neutralizing titers were also observed in colostrum from Stx2-vaccinated cows and sera from colostrum-fed calves. The results presented showed that bovine colostrum with increased levels of antibodies against EHEC O157:H7 may be obtained by systemic immunization of pregnant cows, and that these specific antibodies are efficiently transferred to newborn calves by feeding colostrum. Hyperimmune colostrum and milk may be an alternative to protect calves from early colonization by EHEC O157:H7 and a possible key source of antibodies to block colonization and toxic activity of this bacterium. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Rare Autism-Associated Variants Implicate Syntaxin 1 (STX1 R26Q) Phosphorylation and the Dopamine Transporter (hDAT R51W) in Dopamine Neurotransmission and Behaviors

OpenAIRE

Cartier, Etienne; Hamilton, Peter J.; Belovich, Andrea N.; Shekar, Aparna; Campbell, Nicholas G.; Saunders, Christine; Andreassen, Thorvald F.; Gether, Ulrik; Veenstra-Vanderweele, Jeremy; Sutcliffe, James S.; Ulery-Reynolds, Paula G.; Erreger, Kevin; Matthies, Heinrich J.G.; Galli, Aurelio

2015-01-01

Background: Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA. Methods: We adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gen...

Genetic characterization of Shiga toxin-producing Escherichia coli O26:H11 strains isolated from animal, food, and clinical samples

Science.gov (United States)

Krüger, Alejandra; Lucchesi, Paula M. A.; Sanso, A. Mariel; Etcheverría, Analía I.; Bustamante, Ana V.; Burgán, Julia; Fernández, Luciana; Fernández, Daniel; Leotta, Gerardo; Friedrich, Alexander W.; Padola, Nora L.; Rossen, John W. A.

2015-01-01

The Shiga-toxin producing Escherichia coli (STEC) may cause serious illness in human. Here we analyze O26:H11 strains known to be among the most reported STEC strains causing human infections. Genetic characterization of strains isolated from animal, food, and clinical specimens in Argentina showed that most carried either stx1a or stx2a subtypes. Interestingly, stx2a-positive O26:H11 rarely isolated from cattle in other countries showed to be an important proportion of O26:H11 strains circulating in cattle and food in our region. Seventeen percent of the isolates harbored more than one gene associated with antimicrobial resistance. In addition to stx, all strains contained the virulence genes eae-β, tir, efa, iha, espB, cif, espA, espF, espJ, nleA, nleB, nleC, and iss; and all except one contained ehxA, espP, and cba genes. On the other hand, toxB and espI genes were exclusively observed in stx2-positive isolates, whereas katP was only found in stx1a-positive isolates. Our results show that O26:H11 STEC strains circulating in Argentina, including those isolated from humans, cattle, and meat products, present a high pathogenic potential, and evidence that cattle can be a reservoir of O26:H11 strains harboring stx2a. PMID:26539413

Protection of Human Podocytes from Shiga Toxin 2-Induced Phosphorylation of Mitogen-Activated Protein Kinases and Apoptosis by Human Serum Amyloid P Component

Science.gov (United States)

Dettmar, Anne K.; Binder, Elisabeth; Greiner, Friederike R.; Liebau, Max C.; Kurschat, Christine E.; Jungraithmayr, Therese C.; Saleem, Moin A.; Schmitt, Claus-Peter; Feifel, Elisabeth; Orth-Höller, Dorothea; Kemper, Markus J.; Pepys, Mark; Würzner, Reinhard

2014-01-01

Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38α MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option. PMID:24566618

Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

Science.gov (United States)

Li, Wenna; Ke, Yuehua; Wang, Yufei; Yang, Mingjuan; Gao, Junguang; Zhan, Shaoxia; Xinying, Du; Huang, Liuyu; Li, Wenfeng; Chen, Zeliang; Li, Juan

2016-08-26

Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp. Copyright © 2016 Elsevier Inc. All rights reserved.

ArgO145, a Stx2a prophage of a bovine O145 : H-STEC strain, is closely related to phages of virulent human strains

NARCIS (Netherlands)

Krüger, A; Burgán, J; Friedrich, A W; Jwa, Rossen; Pma, Lucchesi

Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both

Genomic comparison of Escherichia coli O104:H4 isolates from 2009 and 2011 reveals plasmid, and prophage heterogeneity, including shiga toxin encoding phage stx2.

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Sanaa A Ahmed

Full Text Available In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493 and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071. Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

[On the risk of spread of E. coli/EHEC O104:H4 stx 2 positive bacteria via sewerage treatment plants during the 2011 EHEC outbreak in north Germany].

Science.gov (United States)

Heinemeyer, E-A; Luden, K; Monazahian, M

2013-08-01

During an EHEC outbreak with E. coli O104:H4 stx2-pos in northern Germany 2 sewage treatment plants (Cuxhaven and Stade) of highly affected areas were monitored for the presence of the outbreak strain. 7 efflux water samples were collected at 1 h and 6 h intervals. The overall E. coli content of the treated sewage water was approximately 35 000 CFU/100 mL in both treatment plants. Among these about 500 were ESBL-E. coli (1.4%). ESBL-Agar was used as selective medium as the outbreak strain is highly resistant to 3rd generation cephalosporins. From the ESBL-isolates 208 strains have been typed by molecular methods for markers specific to the outbreak strain (O104rfb -351 base pairs (bp), flic H4-201 bp, stx2-584 bp, Tellur D - 434 bp). No outbreak strain was detected. The number of E. coli O104:H4 stx2-pos was calculated to be less than 3 per 100 mL in the treated sewage at the time of the study. Therefore it can be concluded that there was no threat for bathers to fall sick with this highly pathogenic strain from possibly sewage-contaminated bathing waters during the outbreak. The national limit value of 1 800 E. coli in 100 mL offers a high safety margin. © Georg Thieme Verlag KG Stuttgart · New York.

Subtype-specific suppression of Shiga toxin 2 released from Escherichia coli upon exposure to protein synthesis inhibitors

DEFF Research Database (Denmark)

Pedersen, Malene Gantzhorn; Hansen, Claus; Riise, Erik

2008-01-01

Shiga toxins (Stx) are important virulence factors in the pathogenesis of severe disease including hemolytic-uremic syndrome, caused by Stx-producing Escherichia coli (STEC). STEC strains increase the release of Stx in vitro following the addition of fluoroquinolones, whereas protein synthesis...... inhibitors previously have been reported to suppress the release of Stx. The amount of Stx released from wild-type STEC strains incubated with protein synthesis inhibitors was examined by a Vero cell cytotoxicity assay. The amounts released were compared to the Stx type (Stx1 or Stx2) and additionally...... to the individual subtypes and toxin variants of Stx2. In general, Stx2 release was suppressed significantly upon exposure to protein synthesis inhibitors at MICs, which was not observed in the case of Stx1. Also, the average amount of different Stx2 toxin variants released was suppressed to various levels ranging...

An essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1α, IL-1β, IL-12b, and CCL4 from differentiated HL-60 cells.

Science.gov (United States)

Naegelen, Isabelle; Plançon, Sébastien; Nicot, Nathalie; Kaoma, Tony; Muller, Arnaud; Vallar, Laurent; Tschirhart, Eric J; Bréchard, Sabrina

2015-03-01

Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the rapid secretion of cytokines orchestrates the action of other immune cells at the infection site and thus, can contribute to the development and chronicity of inflammatory diseases, we aimed to determine the intracellular SNARE machinery responsible for the regulation of cytokine secretion and degranulation. From a constructed gene-expression network, we first selected relevant cytokines for functional validation by the CBA approach. We established a cytokine-secretion profile for human neutrophils and dHL-60 cells, underlining their similar ability to secrete a broad variety of cytokines within proinflammatory conditions mimicked by LPS stimulation. Secondly, after screening of SNARE genes by microarray experiments, we selected STX3 for further functional studies. With the use of a siRNA strategy, we show that STX3 is clearly required for the maximal release of IL-1α, IL-1β, IL-12b, and CCL4 without alteration of other cytokine secretion in dHL-60 cells. In addition, we demonstrate that STX3 is involved in MMP-9 exocytosis from gelatinase granules, where STX3 is partly localized. Our results suggest that the secretion of IL-1α, IL-1β, IL-12b, and CCL4 occurs during gelatinase degranulation, a process controlled by STX3. In summary, these findings provide first evidence that STX3 has an essential role in trafficking pathways of cytokines in neutrophil granulocytes. © Society for Leukocyte Biology.

Genetic makeup of Shiga toxin-producing Escherichia coli in relation to clinical symptoms and duration of shedding: a microarray analysis of isolates from Swedish children.

Science.gov (United States)

Matussek, A; Jernberg, C; Einemo, I-M; Monecke, S; Ehricht, R; Engelmann, I; Löfgren, S; Mernelius, S

2017-08-01

Shiga toxin (Stx)-producing Escherichia coli (STECs) cause non-bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome, and are the primary cause of acute renal failure in children worldwide. This study investigated the correlation of genetic makeup of STEC strains as revealed by DNA microarray to clinical symptoms and the duration of STEC shedding. All STEC isolated (n = 96) from patients <10 years of age in Jönköping County, Sweden from 2003 to 2015 were included. Isolates were characterized by DNA microarray, including almost 280 genes. Clinical data were collected through a questionnaire and by reviewing medical records. Of the 96 virulence genes (including stx) in the microarray, 62 genes were present in at least one isolate. Statistically significant differences in prevalence were observed for 21 genes when comparing patients with bloody diarrhea (BD) and with non-bloody stool (18 of 21 associated with BD). Most genes encode toxins (e.g., stx2 alleles, astA, toxB), adhesion factors (i.e. espB_O157, tir, eae), or secretion factors (e.g., espA, espF, espJ, etpD, nleA, nleB, nleC, tccP). Seven genes were associated with prolonged stx shedding; the presence of three genes (lpfA, senB, and stx1) and the absence of four genes (espB_O157, espF, astA, and intI1). We found STEC genes that might predict severe disease outcome already at diagnosis. This can be used to develop diagnostic tools for risk assessment of disease outcome. Furthermore, genes associated with the duration of stx shedding were detected, enabling a possible better prediction of length of STEC carriage after infection.

Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

Science.gov (United States)

Parma, Y. R.; Chacana, P. A.; Lucchesi, P. M. A.; Rogé, A.; Granobles Velandia, C. V.; Krüger, A.; Parma, A. E.; Fernández-Miyakawa, M. E.

2012-01-01

Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933, and stx1d were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli. PMID:22919675

Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

Directory of Open Access Journals (Sweden)

Yanil R Parma

2012-06-01

Full Text Available Enterohemorrhagic Escherichia coli (EHEC, a subset of Shiga toxin producing E. coli (STEC is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic uremic syndrome (HUS. Regardless of serotype, Shiga toxins (Stx1 and/or Stx2 are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx was developed using anti-Stx2 B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933 and stx1d were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 400 ng /ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for 2 strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli.

Simple method for Shiga toxin 2e purification by affinity chromatography via binding to the divinyl sulfone group.

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Hideyuki Arimitsu

Full Text Available Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e, a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

A Staphylococcus aureus TIR domain protein virulence factor blocks TLR2-mediated NF-κB signaling.

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Askarian, Fatemeh; van Sorge, Nina M; Sangvik, Maria; Beasley, Federico C; Henriksen, Jørn R; Sollid, Johanna U E; van Strijp, Jos A G; Nizet, Victor; Johannessen, Mona

2014-01-01

Signaling through Toll-like receptors (TLRs), crucial molecules in the induction of host defense responses, requires adaptor proteins that contain a Toll/interleukin-1 receptor (TIR) domain. The pathogen Staphylococcus aureus produces several innate immune-evasion molecules that interfere with the host's innate immune response. A database search analysis suggested the presence of a gene encoding a homologue of the human TIR domain in S. aureus MSSA476 which was named staphylococcal TIR domain protein (TirS). Ectopic expression of TirS in human embryonic kidney, macrophage and keratinocyte cell lines interfered with signaling through TLR2, including MyD88 and TIRAP, NF-κB and/or mitogen-activated protein kinase pathways. Moreover, the presence of TirS reduced the levels of cytokines MCP-1 and G-CSF secreted in response to S. aureus. The effects on NF-κB pathway were confirmed using S. aureus MSSA476 wild type, an isogenic mutant MSSA476ΔtirS, and complemented MSSA476ΔtirS +pTirS in a Transwell system where bacteria and host cells were physically separated. Finally, in a systematic mouse infection model, TirS promoted bacterial accumulation in several organs 4 days postinfection. The results of this study reveal a new S. aureus virulence factor that can interfere with PAMP-induced innate immune signaling in vitro and bacterial survival in vivo. © 2014 S. Karger AG, Basel.

Evaluation of ELISA tests specific for Shiga toxin 1 and 2 in food and water samples

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Two enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their effectiveness in detecting and differentiating between Shiga toxin 1 and 2 (Stx1 and Stx2) produced by Shiga toxin-producing E. coli (STEC) inoculated into food and water samples. Each kit incorporated monoclonal antibodies ...

A part of patients with autism spectrum disorder has haploidy of HPC-1/syntaxin1A gene that possibly causes behavioral disturbance as in experimentally gene ablated mice.

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Kofuji, Takefumi; Hayashi, Yuko; Fujiwara, Tomonori; Sanada, Masumi; Tamaru, Masao; Akagawa, Kimio

2017-03-22

Autism spectrum disorder (ASD) is highly heritable and encompasses a various set of neuropsychiatric disorders with a wide-ranging presentation. HPC-1/syntaxin1A (STX1A) encodes a neuronal plasma membrane protein that regulates the secretion of neurotransmitters and neuromodulators. STX1A gene ablated mice (null and heterozygote mutant) exhibit abnormal behavioral profiles similar to human autistic symptoms, accompanied by reduction of monoamine secretion. To determine whether copy number variation of STX1A gene and the change of its expression correlate with ASD as in STX1A gene ablated mice, we performed copy number assay and real-time quantitative RT-PCR using blood or saliva samples from ASD patients. We found that some ASD patients were haploid for the STX1A gene similar to STX1A heterozygote mutant mice. However, copy number of STX1A gene was normal in the parents and siblings of ASD patients with STX1A gene haploidy. In ASD patients with gene haploidy, STX1A mRNA expression was reduced to about half of their parents. Thus, a part of ASD patients had haploidy of STX1A gene and lower STX1A gene expression. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of highly sensitive detection method for toxins and other pathogenic factors by phage-displayed monoclonal antibody using radioisotopes

International Nuclear Information System (INIS)

Izumiya, Hidemasa; Watanabe, Haruo

2000-01-01

To prepare anti-Shiga toxin (Stx) antibody, a recombinant strain of E coli that can produce the subunit B of Stx was constructed. DNA fragment coding the Stx subunit B, about 0.2 kb in length was amplified using a plasmid containing Stx gene as the template by PCR. After digesting with a restriction enzyme, the DNA fragment was inserted into pmal-c2 vector (New England Biolabs) to produce a fusion protein with maltose binding protein (MBP). E.coli K12 (DH5α) including the pmal-stx plasmid was cultured in the presence of isopropylthiogalactoside (IPTG) and thus, MBP-stx fusion protein was obtained. After purification by Millipore membrane filter, this fusion protein was used as the antigen. Then, mice BALB/c were immunized by intraperitoneal injection of the suspension of MBP-stx and adjuvant. The antibody purified from the spleen was submitted to phage display system. The phage specifically binding to the antigen was proliferated through repeated infection to E coli and the anti-Stx antibody was obtained from the culture of its colony grown on IPTG plate. Three different colonies specifically responding to the recombinant Stx antigen were obtained. In near future, labeled antibody would be produced by addition of 35 S compound in to the culture medium. (M.N.)

Kjemi 1

DEFF Research Database (Denmark)

Brandt, Harald; Hushovd, Odd T.

Kemi 1 er en lærebog til den gymnasiale kemiundervisning i Norge (VG2, studieforberedende) tilsvarende det danske kemi B-niveau i det almene gymnasium (stx). Lærebogen er et del af af et bogsystem med tilhørende netsted til elever og online-lærervejledning (lokus.no).......Kemi 1 er en lærebog til den gymnasiale kemiundervisning i Norge (VG2, studieforberedende) tilsvarende det danske kemi B-niveau i det almene gymnasium (stx). Lærebogen er et del af af et bogsystem med tilhørende netsted til elever og online-lærervejledning (lokus.no)....

Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

Energy Technology Data Exchange (ETDEWEB)

Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D.; Varnum, Susan M.

2014-10-21

Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

UV-Sensitivity of Shiga Toxin-Converting Bacteriophage Virions Φ24B, 933W, P22, P27 and P32

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Sylwia Bloch

2015-09-01

Full Text Available Shiga toxin-converting bacteriophages (Stx phages are present as prophages in Shiga toxin-producing Escherichia coli (STEC strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m2, their infectivity dropped by 1–3 log10, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells.

Cotransport of clay colloids and viruses through water-saturated vertically oriented columns packed with glass beads: Gravity effects.

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Syngouna, Vasiliki I; Chrysikopoulos, Constantinos V

2016-03-01

The cotransport of clay colloids and viruses in vertically oriented laboratory columns packed with glass beads was investigated. Bacteriophages MS2 and ΦX174 were used as model viruses, and kaolinite (ΚGa-1b) and montmorillonite (STx-1b) as model clay colloids. A steady flow rate of Q=1.5 mL/min was applied in both vertical up (VU) and vertical down (VD) flow directions. In the presence of KGa-1b, estimated mass recovery values for both viruses were higher for VD than VU flow direction, while in the presence of STx-1b the opposite was observed. However, for all cases examined, the produced mass of viruses attached onto suspended clay particles were higher for VD than VU flow direction, suggesting that the flow direction significantly influences virus attachment onto clays, as well as packed column retention of viruses attached onto suspended clays. KGa-1b hindered the transport of ΦX174 under VD flow, while STx-1b facilitated the transport of ΦX174 under both VU and VD flow directions. Moreover, KGa-1b and STx-1b facilitated the transport of MS2 in most of the cases examined except of the case where KGa-1b was present under VD flow. Also, the experimental data were used for the estimation of virus surface-coverages and virus surface concentrations generated by virus diffusion-limited attachment, as well as virus attachment due to sedimentation. Both sedimentation and diffusion limited virus attachment were higher for VD than VU flow, except the case of MS2 and STx-1b cotransport. The diffusion-limited attachment was higher for MS2 than ΦΧ174 for all cases examined. Copyright © 2015 Elsevier B.V. All rights reserved.

STX140, but Not Paclitaxel, Inhibits Mammary Tumour Initiation and Progression in C3(1)/SV40 T/t-Antigen Transgenic Mice

OpenAIRE

Meyer-Losic, Florence; Newman, Simon P.; Day, Joanna M.; Reed, Michael J.; Kasprzyk, Philip G.; Purohit, Atul; Foster, Paul A.

2013-01-01

Despite paclitxael's clinical success, treating hormone-refractory breast cancer remains challenging. Paclitaxel has a poor pharmacological profile, characterized by a low therapeutic index (TIX) caused by severe dose limiting toxicities, such as neutropenia and peripheral neuropathy. Consequently, new drugs are urgently required. STX140, a compound previously shown to have excellent efficacy against many tumors, is here compared to paclitaxel in three translational in vivo breast cancer mode...

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources.

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Beutin, L; Steinrück, H; Krause, G; Steege, K; Haby, S; Hultsch, G; Appel, B

2007-03-01

To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.

Antibody Response to Shiga Toxins in Argentinean Children with Enteropathic Hemolytic Uremic Syndrome at Acute and Long-Term Follow-Up Periods

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Fernández-Brando, Romina J.; Bentancor, Leticia V.; Mejías, María Pilar; Ramos, María Victoria; Exeni, Andrea; Exeni, Claudia; Laso, María del Carmen; Exeni, Ramón; Isturiz, Martín A.; Palermo, Marina S.

2011-01-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools. PMID:21559455

Detecting Protein-Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS.

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Han, Ling; Kitova, Elena N; Klassen, John S

2016-11-01

This study reports on the use of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay, combined with glycomicelles, as a method for detecting specific interactions between water-soluble proteins and glycolipids (GLs) in aqueous solution. The B subunit homopentamers of cholera toxin (CTB 5 ) and Shiga toxin type 1 B (Stx1B 5 ) and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2 served as model systems for this study. The CTB 5 exhibits broad specificity for gangliosides and binds to GM1, GM2, GM3, GD1a, GD1b, and GT1b; Stx1B 5 does not recognize gangliosides. The CaR-ESI-MS assay was used to analyze solutions of CTB 5 or Stx1B 5 and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. The high affinity interaction of CTB 5 with GM1 was successfully detected. However, the apparent affinity, as determined from the mass spectra, is significantly lower than that of the corresponding pentasaccharide or when GM1 is presented in model membranes such as nanodiscs. Interactions between CTB 5 and the low affinity gangliosides GD1a, GD1b, and GT1b, as well as GD2, which served as a negative control, were detected; no binding of CTB 5 to GM2 or GM3 was observed. The CaR-ESI-MS results obtained for Stx1B 5 reveal that nonspecific protein-ganglioside binding can occur during the ESI process, although the extent of binding varies between gangliosides. Consequently, interactions detected for CTB 5 with GD1a, GD1b, and GT1b are likely nonspecific in origin. Taken together, these results reveal that the CaR-ESI-MS/glycomicelle approach for detecting protein-GL interactions is prone to false positives and false negatives and must be used with caution. Graphical Abstract GRAPHICAL ABSTRACT TEXT HERE] -->.

A High-Throughput, Precipitating Colorimetric Sandwich ELISA Microarray for Shiga Toxins

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Andrew Gehring

2014-06-01

Full Text Available Shiga toxins 1 and 2 (Stx1 and Stx2 from Shiga toxin-producing E. coli (STEC bacteria were simultaneously detected with a newly developed, high-throughput antibody microarray platform. The proteinaceous toxins were immobilized and sandwiched between biorecognition elements (monoclonal antibodies and pooled horseradish peroxidase (HRP-conjugated monoclonal antibodies. Following the reaction of HRP with the precipitating chromogenic substrate (metal enhanced 3,3-diaminobenzidine tetrahydrochloride or DAB, the formation of a colored product was quantitatively measured with an inexpensive flatbed page scanner. The colorimetric ELISA microarray was demonstrated to detect Stx1 and Stx2 at levels as low as ~4.5 ng/mL within ~2 h of total assay time with a narrow linear dynamic range of ~1–2 orders of magnitude and saturation levels well above background. Stx1 and/or Stx2 produced by various strains of STEC were also detected following the treatment of cultured cells with mitomycin C (a toxin-inducing antibiotic and/or B-PER (a cell-disrupting, protein extraction reagent. Semi-quantitative detection of Shiga toxins was demonstrated to be sporadic among various STEC strains following incubation with mitomycin C; however, further reaction with B-PER generally resulted in the detection of or increased detection of Stx1, relative to Stx2, produced by STECs inoculated into either axenic broth culture or culture broth containing ground beef.

Salt at concentrations relevant to meat processing enhances Shiga toxin 2 production in Escherichia coli O157:H7.

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Harris, Shaun M; Yue, Wan-Fu; Olsen, Sarena A; Hu, Jia; Means, Warrie J; McCormick, Richard J; Du, Min; Zhu, Mei-Jun

2012-10-15

Escherichia coli (E. coli) O157:H7 remains a major food safety concern associated with meat, especially beef products. Shiga toxins (Stx) are key virulence factors produced by E. coli O157:H7 that are responsible for hemorrhagic colitis and Hemolytic Uremic Syndrome. Stx are heat stable and can be absorbed after oral ingestion. Despite the extensive study of E. coli O157:H7 survival during meat processing, little attention is paid to the production of Stx during meat processing. The objective of this study was to elucidate the effect of salt, an essential additive to processed meat, at concentrations relevant to meat processing (0%, 1%, 2%, 3%, W/V) on Stx2 production and Stx2 prophage induction by E. coli O157:H7 strains. For both E. coli O157:H7 86-24 and EDL933 strains, including 2% salt in LB broth decreased (Pmeat processing enhances Stx production, a process linked to bacterial stress response and lambdoid prophage induction. Published by Elsevier B.V.

Clinical Significances of Serum Vitamin B12, Folate and Ferritin Levels in Patients with Malignant Tumors

International Nuclear Information System (INIS)

Monn, Youn Sung; Soung, In Whan; Kim, Sam Yong; Ro, Heung Kyu; Lee, Bok Hee

1987-01-01

In order to evaluate the clinical significances of the serum vitamin B 12 , folate and ferritin levels in patients with malignant tumors, the levels were measured in 10 normal control subjects, 70 patients with malignant tumors, 7 patients with liver cirrhosis and 25 patients with other benign diseases. The results are as follows: 1) In normal control subjects, mean serum values for vitamin B 12 , folate and ferritin level were 588.80±131.58 pg/ml, 5.59±1.52 ng/ml and 89.22±42.78 ng/ml retrospectively. 2) There was no significant difference in serum levels between patients with benign diseases and normal control subjects. 3) The serum vitamin B 12 and ferritin levels in patients with liver cirrhosis were significantly higher than in normal control, and the serum folate levels in these patients were lower than in normal control subjects. 4) The serum vitamin B 12 and ferritin levels in patients with malignant tumors were significantly higher than in normal control subjects, and the serum folate levels in these patients were significantly lower than in normal control subjects. The above results suggest that the serum vitamin B 12 and ferritin may be useful as tumor markers in patients with malignant tumors.

Lipid reorganization induced by Shiga toxin clustering on planar membranes.

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Barbara Windschiegl

Full Text Available The homopentameric B-subunit of bacterial protein Shiga toxin (STxB binds to the glycolipid Gb(3 in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb(3 clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb(3 lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC/cholesterol/Gb(3 (65:30:5 bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb(3 (40:35:20:5 phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb(3, STxB-Gb(3 complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.

Characteristics of Shiga toxin-producing Escherichia coli from meat and milk products of different origins and association with food producing animals as main contamination sources.

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Martin, Annett; Beutin, Lothar

2011-03-15

Shiga toxin-producing strains of Escherichia coli (STEC) cause diarrhoea and haemorrhagic colitis in humans. Most human infections are attributed to consumption of STEC contaminated foodstuff. Food producing animals constitute important reservoirs of STEC and serve as source of food contamination. In this study, we have analyzed 593 foodborne STEC strains for their serotypes and for nine virulence genes (stx1, stx1c, stx1d, stx2, stx2b, stx2e, stx2g, E-hly and eae). The 593 STEC strains grouped into 215 serotypes, and 123 serotypes (57.2%) were represented each by only one STEC isolate. Fifteen serotypes (7.0%) were attributed to 198 (33.3%) of the 593 STEC strains. The foodborne STEC were grouped into different categories in relation to the species of the food producing animal (cattle, pigs, sheep, goats, red deer, wild-boar and hare). Univariate and multivariate statistical analyses revealed significant similarities between the animal origin of the food and the virulence markers of foodborne STEC. Significant associations (pfood producing animals. Virulence profiles and serotypes of STEC from food showed remarkable similarities to those of faecal STEC that were from the same animal species. The findings from our study clearly indicate that the food producing animals represent the most important source for the entry of STEC in the food chain. Sound hygiene measures implemented at critical stages of food production (milking, slaughtering, and evisceration) should be most effective in reducing the frequency of STEC contamination of food derived from domestic and wildlife animals. Copyright © 2011 Elsevier B.V. All rights reserved.

Shiga toxin-producing Escherichia coli in yaks (Bos grunniens from the Qinghai-Tibetan Plateau, China.

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Xiangning Bai

Full Text Available Shiga toxin (Stx-producing Escherichia coli (STEC are recognized as important human pathogens of public health concern. Many animals are the sources of STEC. In this study we determined the occurrence and characteristics of the STEC in yaks (Bos grunniens from the Qinghai-Tibetan plateau, China. A total of 728 yak fecal samples was collected from June to August, 2012 and was screened for the presence of the stx 1 and stx 2 genes by TaqMan real-time PCR after the sample was enriched in modified Tryptone Soya Broth. Of the 138 (18.96% stx 1 and/or stx 2-positive samples, 85 (61.59% were confirmed to have at least 1 STEC isolate present by culture isolation, from which 128 STEC isolates were recovered. All STEC isolates were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE and characterized for the presence of 16 known virulence factors. Fifteen different O serogroups and 36 different O:H serotypes were identified in the 128 STEC isolates with 21 and 4 untypable for the O and H antigens respectively. One stx 1 subtype (stx 1a and 5 stx 2 subtypes (stx 2a, stx 2b, stx 2c, stx 2d and stx 2g were present in these STEC isolates. Apart from lpfA O157/OI-141, lpfA O157/OI-154, lpfA O113, katP and toxB which were all absent, other virulence factors screened (eaeA, iha, efa1, saa, paa, cnf1, cnf2, astA, subA, exhA and espP were variably present in the 128 STEC isolates. PFGE were successful for all except 5 isolates and separated them into 67 different PFGE patterns. For the 18 serotypes with 2 or more isolates, isolates of the same serotypes had the same or closely related PFGE patterns, demonstrating clonality of these serotypes. This study was the first report on occurrence and characteristics of STEC isolated from yaks (Bos grunniens from the Qinghai-Tibetan plateau, China, and extended the genetic diversity and reservoir host range of STEC.

Immunization of Mice with a Live Transconjugant Shigella Hybrid Strain Induced Th1 and Th17 Cell-Mediated Immune Responses and Confirmed Passive Protection Against Heterologous Shigellae.

Science.gov (United States)

Nag, D; Koley, H; Sinha, R; Mukherjee, P; Sarkar, C; Withey, J H; Gachhui, R

2016-02-01

An avirulent, live transconjugant Shigella hybrid (LTSHΔstx) strain was constructed in our earlier study by introducing a plasmid vector, pPR1347, into a Shiga toxin gene deleted Shigella dysenteriae 1. Three successive oral administrations of LTSHΔstx to female adult mice produced comprehensive passive heterologous protection in their offspring against challenge with wild-type shigellae. Production of NO and different cytokines such asIL-12p70, IL-1β and IL-23 in peritoneal mice macrophages indicated that LTSHΔstx induced innate and adaptive immunity in mice. Furthermore, production of IFN-γ, IL-10 and IL-17 in LTSH-primed splenic CD4+ T cell suggested that LTSHΔstx may induce Th1 and Th17 cell-mediated immune responses. Exponential increase of the serum IgG and IgA titre against whole shigellae was observed in immunized adult mice during and after the immunization with the highest peak on day 35. Antigen-specific sIgA was also determined from intestinal lavage of immunized mice. The stomach extracts of neonates from immunized mice, mainly containing mother's milk, contained significant levels of anti-LTSHΔstx immunoglobulin. These studies suggest that the LTSHΔstx could be a new live oral vaccine candidate against shigellosis in the near future. © 2015 The Foundation for the Scandinavian Journal of Immunology.

Genome-wide DNA methylation analysis of pseudohypoparathyroidism patients with GNAS imprinting defects.

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Rochtus, Anne; Martin-Trujillo, Alejandro; Izzi, Benedetta; Elli, Francesca; Garin, Intza; Linglart, Agnes; Mantovani, Giovanna; Perez de Nanclares, Guiomar; Thiele, Suzanne; Decallonne, Brigitte; Van Geet, Chris; Monk, David; Freson, Kathleen

2016-01-01

Pseudohypoparathyroidism (PHP) is caused by (epi)genetic defects in the imprinted GNAS cluster. Current classification of PHP patients is hampered by clinical and molecular diagnostic overlaps. The European Consortium for the study of PHP designed a genome-wide methylation study to improve molecular diagnosis. The HumanMethylation 450K BeadChip was used to analyze genome-wide methylation in 24 PHP patients with parathyroid hormone resistance and 20 age- and gender-matched controls. Patients were previously diagnosed with GNAS-specific differentially methylated regions (DMRs) and include 6 patients with known STX16 deletion (PHP(Δstx16)) and 18 without deletion (PHP(neg)). The array demonstrated that PHP patients do not show DNA methylation differences at the whole-genome level. Unsupervised clustering of GNAS-specific DMRs divides PHP(Δstx16) versus PHP(neg) patients. Interestingly, in contrast to the notion that all PHP patients share methylation defects in the A/B DMR while only PHP(Δstx16) patients have normal NESP, GNAS-AS1 and XL methylation, we found a novel DMR (named GNAS-AS2) in the GNAS-AS1 region that is significantly different in both PHP(Δstx16) and PHP(neg), as validated by Sequenom EpiTYPER in a larger PHP cohort. The analysis of 58 DMRs revealed that 8/18 PHP(neg) and 1/6 PHP(Δstx16) patients have multi-locus methylation defects. Validation was performed for FANCC and SVOPL DMRs. This is the first genome-wide methylation study for PHP patients that confirmed that GNAS is the most significant DMR, and the presence of STX16 deletion divides PHP patients in two groups. Moreover, a novel GNAS-AS2 DMR affects all PHP patients, and PHP patients seem sensitive to multi-locus methylation defects.

Detection of E.Coli Strains Containing Shiga Toxin (Stx1/2 Gene in Diarrheal Specimens from Children Less than 5 Years Old by PCR Technique and Study of the Patterns of Antibiotic Resistance

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MR Pourmand

2009-10-01

Full Text Available Introduction: Shiga toxin- producing Escherichia coli (STEC is an emerging bacterial pathogen in developing countries that causes several diseases such as diarrhea, hemorrhagic colitis (HC and hemolytic uremic syndrome (HUS, particularly in children. Aim of the research was detection of STEC in diarrheal specimens from under 5 year olds and study of the patterns of antibiotic resistance of these strains. Methods: In the study,300 fecal samples were collected from children with diarrhea referring to Ali Asghar Hospital. E.coli species were isolated by standard bacteriological and biochemical tests. Presence of shiga toxin genes (stx1/2 was investigated by PCR technique (Qiagen. Antibiogram test for strains containing the toxin gene was performed using 16 different antibiotic discs (MAST by disc diffusion agar (Kirby-Bauer method. Results: From 39 E.coli isolates, 9(23.1% strains were detected by PCR to contain stx1/2 gene. One strain was resistant to all 16 antibiotics. All the STEC strains were sensitive to meropenem (MRP, imipenem (IMI, gentamycin (GEN and nitrofurantoin (NI. 4(44.44% strains showed multi-drug resistant pattern. All these 4strains were resistant to cotrimoxazole(SxT. Also, 6(66.66% strains were resistant to at least one antibiotic. Conclusion: In Iran, shiga toxin- producing Escherichia coli (STEC may be a commonly bacterial pathogen causing diarrhea, particularly in children. Therefore, we should use new techniques for investigation of these strains. Increase in number of emerging and new strains that could be resistant to classic antibiotics such as cotrimoxazole may be foreseen. It is suggested that antibiotics prescription programs in treatment of diarrhea causing E.coli strains be updated.

Quiescent complement in nonhuman primates during E coli Shiga toxin-induced hemolytic uremic syndrome and thrombotic microangiopathy.

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Lee, Benjamin C; Mayer, Chad L; Leibowitz, Caitlin S; Stearns-Kurosawa, D J; Kurosawa, Shinichiro

2013-08-01

Enterohemorrhagic Escherichia coli (EHEC) produce ribosome-inactivating Shiga toxins (Stx1, Stx2) responsible for development of hemolytic uremic syndrome (HUS) and acute kidney injury (AKI). Some patients show complement activation during EHEC infection, raising the possibility of therapeutic targeting of complement for relief. Our juvenile nonhuman primate (Papio baboons) models of endotoxin-free Stx challenge exhibit full spectrum HUS, including thrombocytopenia, hemolytic anemia, and AKI with glomerular thrombotic microangiopathy. There were no significant increases in soluble terminal complement complex (C5b-9) levels after challenge with lethal Stx1 (n = 6) or Stx2 (n = 5) in plasma samples from T0 to euthanasia at 49.5 to 128 hours post-challenge. d-dimer and cell injury markers (HMGB1, histones) confirmed coagulopathy and cell injury. Thus, complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these preclinical models, and benefits or risks of complement inhibition should be studied further for this infection.

Detection and characterization of Shiga toxin-producing Escherichia coli in game meat and ready-to-eat meat products.

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Díaz-Sánchez, S; Sánchez, S; Sánchez, M; Herrera-León, S; Hanning, I; Vidal, D

2012-11-15

A total of 142 samples of game meat and ready-to-eat meat products from red deer and wild boar were analysed in order to assess the presence of Shiga toxin-producing Escherichia coli (STEC). Shiga-toxin encoding genes (stx genes) were detected by PCR in 36 (25.4%) of the samples and STEC was isolated from 8 (5.6%) of the same samples. None of the samples tested positive for E. coli O157:H7. Four different serotypes were found among the 8 STEC isolates, with serotype O27:H30 being predominant (62.5%, 5/8). The PCR assay indicated the presence of the stx2 gene in all of the STEC isolates and further subtyping resulted in detection of three different subtypes: stx2a, stx2b and stx2g. The only stx1-positive isolate was further subtyped as stx1c. The ehxA gene was detected in 3 (37.5%) of the isolates and none of them contained the eae gene. All STEC isolates were sensitive to the 13 antibiotics tested. Some isolates possessed serotypes and virulence gene profiles previously associated with STEC infections in humans. The isolation of a STEC strain carrying the stx2a subtype from a ready-to-eat meat product from deer suggests the role of these products as a potential source of STEC infections in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

An immunoradiometric assay for Helicobacter pylori urease B in serum

International Nuclear Information System (INIS)

Bai Yanyan; Wang Zhaoyue; Bai Xia; Ruan Changgen; Miao Jingcheng; Gu Guanbing

2008-01-01

Objective: The aim of this study was to establish and evaluate a novel immunoradiometric assay (IRMA) for detecting helicobacter pylori (HP) urease B (ureB) in serum. Methods: HP ureB was prepared by the DNA recombinant technique. Monoclonal antibodies against HP ureB were produced by hybridoma technique and were used to establish IRMA for detecting HP ureB. Fifty patients with chronic gastric diseases were recruited in this study, including 36 with gastric ulcer disease, 9 with chronic gastritis and 5 with gastric cancer. The IRMA was compared with rapid urease test (RUT), polymerase chain reaction (PCR) test and direct-ELISA test of serum anti-ureB antibody, using HP culture from gastric biopsy samples as a 'golden standard'. Results: The sensitivity and specificity of IRMA for HP ureB were 94.6% and 76.9%, respectively, with positive and negative predictive values being 92.1% and 83.3%. The sensitivity and specificity of RUT, PCR and serum anti-ureB antibody test were 94.6% and 23.1% , 66.8% and 84.6%, 86.5% and 38.5%, respectively. IRMA and the test of serum anti-ureB antibody was not significantly different from the 'golden standard' (X 2 =1.165, P>0.05), but RUT and PCR showed statistically significant difference with the 'gold standard' (X 2 =5.333, 9.436; all P<0.05). Conclusion: IRMA for HP ureB antigen is a sensitive and specific method for detection of HP infection, and can be used in the study of etiology and pathogenesis of gastric and some other related diseases. (authors)

Investigation of an Escherichia coli O145 outbreak in a child day-care centre - extensive sampling and characterization of eae- and stx1-positive E. coli yields epidemiological and socioeconomic insight

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Bruheim Torkjel

2011-09-01

Full Text Available Abstract Background On October 29th 2009 the health authorities in the city of Trondheim, Norway were alerted about a case of Shiga toxin-positive E. coli (STEC O145 in a child with bloody diarrhoea attending a day-care centre. Symptomatic children in this day-care centre were sampled, thereby identifying three more cases. This initiated an outbreak investigation. Methods A case was defined as a child attending the day-care centre, in whom eae- and stx1- but not stx2-positive E. coli O145:H28 was diagnosed from a faecal sample, with multilocus variable number of tandem repeat analysis (MLVA profile identical to the index isolate. All 61 children, a staff of 14 in the day-care centre, and 74 close contacts submitted faecal samples. Staff and parents were interviewed about cases' exposure to foods and animals. Faecal samples from 31 ewes from a sheep herd to which the children were exposed were analyzed for E. coli O145. Results Sixteen cases were identified, from which nine presented diarrhoea but not haemolytic uremic syndrome (HUS. The attack rate was 0.26, and varied between age groups (0.13-0.40 and between the three day-care centre departments (0.20-0.50, and was significantly higher amongst the youngest children. Median duration of shedding was 20 days (0-71 days. Children were excluded from the day-care centre during shedding, requiring parents to take compassionate leave, estimated to be a minimum total of 406 days for all cases. Atypical enteropathogenic E. coli (aEPEC were detected among 14 children other than cases. These isolates were genotypically different from the outbreak strain. Children in the day-care centre were exposed to faecal pollution from a sheep herd, but E. coli O145 was not detected in the sheep. Conclusions We report an outbreak of stx1- and eae-positive STEC O145:H28 infection with mild symptoms among children in a day-care centre. Extensive sampling showed occurrence of the outbreak strain as well as other STEC and

The new allelic variant of the subtilase cytotoxin (subAB2) is common among Shiga toxin-producing Escherichia coli strains from large game animals and their meat and meat products.

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Sánchez, Sergio; Díaz-Sánchez, Sandra; Martínez, Remigio; Llorente, María Teresa; Herrera-León, Silvia; Vidal, Dolors

2013-10-25

Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Two allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, and subAB2, located on a pathogenicity island (PAI) together with tia gene. While subAB1 has been reported to be more frequent among bovine strains, subAB2 has been mainly associated with strains from small ruminants. We investigated the presence of the two variants of subAB among 59 eae-negative STEC from large game animals (deer and wild boar) and their meat and meat products in order to assess the role of other species in the epidemiology of subAB-positive, eae-negative STEC. For this approach, the strains were PCR-screened for the presence of subAB, including the specific detection of both allelic variants, for the presence of saa, tia and sab, and for stx subtyping. Overall, subAB genes were detected in 71.2% of the strains: 84.1% of the strains from deer and 33.3% of the strains from wild boar. Most of them (97.6%) possessed subAB2 and most of these subAB2-positive strains (92.7%) were also positive for tia and negative for saa, suggesting the presence of the subAB2-harbouring PAI. Subtype stx2b was present in most of the strains (67.8%) and a statistically significant association could be established between subAB2 and stx2b. Our results suggest that large game animals, mainly deer, may represent an important animal reservoir of subAB2-positive, eae-negative STEC, and also highlight the risk of human infection posed by the consumption of large game meat and meat products. Copyright © 2013 Elsevier B.V. All rights reserved.

Unusual social behavior in HPC-1/syntaxin1A knockout mice is caused by disruption of the oxytocinergic neural system.

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Fujiwara, Tomonori; Sanada, Masumi; Kofuji, Takefumi; Akagawa, Kimio

2016-07-01

HPC-1/syntaxin1A (STX1A), a neuronal soluble N-ethylmaleimide-sensitive fusion attachment protein receptor, contributes to neural function in the CNS by regulating transmitter release. Recent studies reported that STX1A is associated with human neuropsychological disorders, such as autism spectrum disorder and attention deficit hyperactivity disorder. Previously, we showed that STX1A null mutant mice (STX1A KO) exhibit neuropsychological abnormalities, such as fear memory deficits, attenuation of latent inhibition, and unusual social behavior. These observations suggested that STX1A may be involved in the neuropsychological basis of these abnormalities. Here, to study the neural basis of social behavior, we analyzed the profile of unusual social behavior in STX1A KO with a social novelty preference test, which is a useful method for quantification of social behavior. Interestingly, the unusual social behavior in STX1A KO was partially rescued by intracerebroventricular administration of oxytocin (OXT). In vivo microdialysis studies revealed that the extracellular OXT concentration in the CNS of STX1A KO was significantly lower compared with wild-type mice. Furthermore, dopamine-induced OXT release was reduced in STX1A KO. These results suggested that STX1A plays an important role in social behavior through regulation of the OXTergic neural system. Dopamine (DA) release is reduced in CNS of syntaxin1A null mutant mice (STX1A KO). Unusual social behavior was observed in STX1A KO. We found that oxytocin (OXT) release, which was stimulated by DA, was reduced and was rescued the unusual social behavior in STX1A KO was rescued by OXT. These results indicated that STX1A plays an important role in promoting social behavior through regulation of DA-induced OXT release in amygdala. © 2016 International Society for Neurochemistry.

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation.

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Lai, Jeffrey K F; Sam, I-Ching; Verlhac, Pauline; Baguet, Joël; Eskelinen, Eeva-Liisa; Faure, Mathias; Chan, Yoke Fun

2017-07-04

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N -ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

Impact of urbanization and agriculture on the occurrence of bacterial pathogens and stx genes in coastal waterbodies of central California.

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Walters, Sarah P; Thebo, Anne L; Boehm, Alexandria B

2011-02-01

Fecal pollution enters coastal waters through multiple routes, many of which originate from land-based activities. Runoff from pervious and impervious land surfaces transports pollutants from land to sea and can cause impairment of coastal ocean waters. To understand how land use practices and water characteristics influence concentrations of fecal indicator bacteria (FIB) and pathogens in natural waters, fourteen coastal streams, rivers, and tidal lagoons, surrounded by variable land use and animal densities, were sampled every six weeks over two years (2008 & 2009). Fecal indicator bacteria (FIB; Escherichia coli and Enterococci) and Salmonella concentrations, the occurrence of Bacteroidales human, ruminant, and pig-specific fecal markers, E. coli O157:H7, and Shiga toxin (stx) genes present in E. coli, were measured. In addition, environmental and climatic variables (e.g., temperature, salinity, rainfall), as well as human and livestock population densities and land cover were quantified. Concentrations of FIB and Salmonella were correlated with each other, but the occurrence of host-specific Bacteroidales markers did not correlate with FIB or pathogens. FIB and Salmonella concentrations, as well as the occurrence of E. coli harboring stx genes, were positively associated with the fraction of the surrounding subwatershed that was urban, while the occurrence of E. coli O157:H7 was positively associated with the agricultural fraction. FIB and Salmonella concentrations were negatively correlated to salinity and temperature, and positively correlated to rainfall. Areal loading rates of FIB, Salmonella and E. coli O157:H7 to the coastal ocean were calculated for stream and river sites and varied with land cover, salinity, temperature, and rainfall. Results suggest that FIB and pathogen concentrations are influenced, in part, by their flux from the land, which is exacerbated during rainfall; once waterborne, bacterial persistence is affected by water temperature and

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

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Xu, Xuefang; McAteer, Sean P.; Tree, Jai J.; Shaw, Darren J.; Wolfson, Eliza B. K.; Beatson, Scott A.; Roe, Andrew J.; Allison, Lesley J.; Chase-Topping, Margo E.; Mahajan, Arvind; Tozzoli, Rosangela; Woolhouse, Mark E. J.; Morabito, Stefano; Gally, David L.

2012-01-01

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins. PMID:22615557

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes.

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Beutin, Lothar; Miko, Angelika; Krause, Gladys; Pries, Karin; Haby, Sabine; Steege, Katja; Albrecht, Nadine

2007-08-01

We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.

Emerging types of Shiga toxin-producing E. coli (STEC O178 present in cattle, deer and humans from Argentina and Germany

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Angelika eMiko

2014-06-01

Full Text Available More than 400 serotypes of Shiga toxin-producing Escherichia coli (STEC have been implicated in outbreaks and sporadic human diseases. In recent years STEC strains belonging to serogroup O178 have been commonly isolated from cattle and food of bovine origin in South America and Europe. In order to explore the significance of these STEC strains as potential human pathogens, 74 German and Argentinean E. coli O178 strains from animals, food and humans were characterized phenotypically and investigated for their serotypes, stx-genotypes and forty-three virulence-associated markers by a real-time PCR-microarray. The majority (n=66 of the O178 strains belonged to serotype O178:H19. The remaining strains divided into O178:H7 (n=6, O178:H10 (n=1 and O178:H16 (n=1. STEC O178:H19 strains were mainly isolated from cattle and food of bovine origin, but one strain was from a patient with hemolytic uremic syndrome (HUS. Genotyping of the STEC O178:H19 strains by pulsed-field gel electrophoresis revealed two major clusters of genetically highly related strains which differ in their stx-genotypes and non-Stx putative virulence traits, including adhesins, toxins and serine-proteases. Cluster A-strains including the HUS-strain (n=35 carried genes associated with severe disease in humans (stx2a, stx2d, ehxA, saa, subAB1, lpfAO113, terE combined with stx1a, espP, iha. Cluster B-strains (n=26 showed a limited repertoire of virulence genes (stx2c, pagC, lpfAO113, espP, iha. Among O178:H7 strains isolated from deer meat and patients with uncomplicated disease a new STEC variant was detected that is associated with the genotype stx1c/stx2b/ehxA/subAB2/espI/[terE]/espP/iha. None of the STEC O178 strains was positive for locus of enterocyte effacement (LEE- and nle-genes. Results indicate that STEC O178:H19 strains belong to the growing group of LEE-negative STEC that should be considered with respect to their potential to cause diseases in humans.

[Stx2a-producing enteroaggregative Escherichia coli O104:H4-ST678. Microbiological diagnostic already, for this and other STEC/VTEC serotypes!].

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Blanco, Jorge

2012-02-01

A Stx2a-producing Escherichia coli (STEC) strain belonging to serotype O104:H4, with virulence features common to the enteroaggregative E. coli pathotype, was reported as the cause of the recent 2011 outbreak in Germany. In addition, the German outbreak strain was found to possess several virulence factors of extra-intestinal pathogenic E. coli and to have acquired resistance to numerous antibiotics, including third-generation cephalosporins, owing to several plasmid-borne genes encoding TEM-1 and CTX-M-15 β-lactamases. There are only a few reports of serotype O104:H4, which is very rare in humans, and has never been detected in animals or food. Once the serotype of the German outbreak strain became known, specific molecular methods were developed for its detection based on conventional and real-time PCR. Data from Galicia suggest that, per year in Spain, STEC O157:H7 is responsible for more than 500 cases of infection, and non-O157 for more than 2,000. A microbiological diagnosis for O104:H4, O157:H7 and other STEC serotypes is required in Spanish hospitals. Copyright © 2011 Elsevier España, S.L. All rights reserved.

The clinical significance of determination of serum CTGF, PDGF and TGF-β1 levels in patients with post-hepatitis B liver cirrhosis

International Nuclear Information System (INIS)

Li Qingru; Sang Shibiao; Zhao Zhenhua; Jiang Jiwei

2010-01-01

Objective: To explore the clinical significance of changes of serum connective tissue growth factor (CTGF), Platelet-derived growth factor (PDGF) and transforming growth factor-β 1 (TGF-β 1 )levels in patients with post-hepatitis B liver cirrhosis. Methods: Serum TGF-β 1 (with RIA), CTGF and PDGF (with ELISA) levels were determined in 50 patients with liver cirrhosis (mild, n=15 moderate n=16 severe, n=19) and 45 controls. Results: The serum level of CTGF in patients with mild post-hepatitis B liver cirrhosis were in significantly higher than those in the controls (P>0.05). The serum level of CTGF were significantly higher in patients with moderate (P 1 were also significantly higher in all the patients than those in the controls (P<0.05, P<0.01, P<0.01). Conclusion: The changes of those 3 markers serum levels were related to the progress of post-hepatitis B liver cirrhosis and the determination was helpful for outcome prediction. (authors)

Subject-specific increases in serum S-100B distinguish sports-related concussion from sports-related exertion.

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Kiechle, Karin; Bazarian, Jeffrey J; Merchant-Borna, Kian; Stoecklein, Veit; Rozen, Eric; Blyth, Brian; Huang, Jason H; Dayawansa, Samantha; Kanz, Karl; Biberthaler, Peter

2014-01-01

The on-field diagnosis of sports-related concussion (SRC) is complicated by the lack of an accurate and objective marker of brain injury. To compare subject-specific changes in the astroglial protein, S100B, before and after SRC among collegiate and semi-professional contact sport athletes, and compare these changes to differences in S100B before and after non-contact exertion. Longitudinal cohort study. From 2009-2011, we performed a prospective study of athletes from Munich, Germany, and Rochester, New York, USA. Serum S100B was measured in all SRC athletes at pre-season baseline, within 3 hours of injury, and at days 2, 3 and 7 post-SRC. Among a subset of athletes, S100B was measured after non-contact exertion but before injury. All samples were collected identically and analyzed using an automated electrochemiluminescent assay to quantify serum S100B levels. Forty-six athletes (30 Munich, 16 Rochester) underwent baseline testing. Thirty underwent additional post-exertion S100B testing. Twenty-two athletes (16 Rochester, 6 Munich) sustained a SRC, and 17 had S100B testing within 3 hours post-injury. The mean 3-hour post-SRC S100B was significantly higher than pre-season baseline (0.099±0.008 µg/L vs. 0.058±0.006 µg/L, p = 0.0002). Mean post-exertion S100B was not significantly different than the preseason baseline. S100B levels at post-injury days 2, 3 and 7 were significantly lower than the 3-hour level, and not different than baseline. Both the absolute change and proportional increase in S100B 3-hour post-injury were accurate discriminators of SRC from non-contact exertion without SRC (AUC 0.772 and 0.904, respectively). A 3-hour post-concussion S100B >0.122 µg/L and a proportional S100B increase of >45.9% over baseline were both 96.7% specific for SRC. Relative and absolute increases in serum S100B can accurately distinguish SRC from sports-related exertion, and may be a useful adjunct to the diagnosis of SRC.

Subject-specific increases in serum S-100B distinguish sports-related concussion from sports-related exertion.

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Karin Kiechle

Full Text Available The on-field diagnosis of sports-related concussion (SRC is complicated by the lack of an accurate and objective marker of brain injury.To compare subject-specific changes in the astroglial protein, S100B, before and after SRC among collegiate and semi-professional contact sport athletes, and compare these changes to differences in S100B before and after non-contact exertion.Longitudinal cohort study.From 2009-2011, we performed a prospective study of athletes from Munich, Germany, and Rochester, New York, USA. Serum S100B was measured in all SRC athletes at pre-season baseline, within 3 hours of injury, and at days 2, 3 and 7 post-SRC. Among a subset of athletes, S100B was measured after non-contact exertion but before injury. All samples were collected identically and analyzed using an automated electrochemiluminescent assay to quantify serum S100B levels.Forty-six athletes (30 Munich, 16 Rochester underwent baseline testing. Thirty underwent additional post-exertion S100B testing. Twenty-two athletes (16 Rochester, 6 Munich sustained a SRC, and 17 had S100B testing within 3 hours post-injury. The mean 3-hour post-SRC S100B was significantly higher than pre-season baseline (0.099±0.008 µg/L vs. 0.058±0.006 µg/L, p = 0.0002. Mean post-exertion S100B was not significantly different than the preseason baseline. S100B levels at post-injury days 2, 3 and 7 were significantly lower than the 3-hour level, and not different than baseline. Both the absolute change and proportional increase in S100B 3-hour post-injury were accurate discriminators of SRC from non-contact exertion without SRC (AUC 0.772 and 0.904, respectively. A 3-hour post-concussion S100B >0.122 µg/L and a proportional S100B increase of >45.9% over baseline were both 96.7% specific for SRC.Relative and absolute increases in serum S100B can accurately distinguish SRC from sports-related exertion, and may be a useful adjunct to the diagnosis of SRC.

Role of hyaluronic acid and laminin as serum markers for predicting significant fibrosis in patients with chronic hepatitis B

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Feng Li

Full Text Available OBJECTIVES: The aim of this study was to evaluate the diagnostic performance of serum HA and LN as serum markers for predicting significant fibrosis in CHB patients. METHODS: Serum HA and LN levels of 87 patients with chronic hepatitis B and 19 blood donors were assayed by RIA. Liver fibrosis stages were determined according to the Metavir scoring-system. The diagnostic performances of all indexes were evaluated by the receiver operating characteristic (ROC curves. RESULTS: Serum HA and LN concentrations increased significantly with the stage of hepatic fibrosis, which showed positive correlation with the stages of liver fibrosis (HA: r = 0.875, p < 0.001; LN: r = 0.610, p < 0.001. There were significant differences of serum HA and LN levels between F2-4 group in comparison with those in F0-F1 group (p < 0.001 and controls (p < 0.001, respectively. From ROC curves, 185.3 ng/mL as the optimal cut-off value of serum HA for diagnosis of significant fibrosis, giving its sensitivity, specificity, PPV, NPV, LR+, LR- and AC of 84.2%, 83.3%, 90.6%, 73.5%, 5.04, 0.19 and 83.9, respectively. While 132.7 ng/mL was the optimal cut-off value of serum LN, the sensitivity, specificity, PPV, NPV, LR+, LR- and AC were 71.9%, 80.0%, 87.2%, 60.0%, 3.59%, 0.35% and 74.7, respectively. Combinations of HA and LN by serial tests showed a perfect specificity and PPV of 100%, at the same time sensitivity declined to 63.2% and LR+ increased to 18.9, while parallel tests revealed a good sensitivity of 94.7%, NPV to 86.4%, and LR- declined to 0.08. CONCLUSIONS: Serum HA and LN concentrations showed positive correlation with the stages of liver fibrosis. Detection of serum HA and LN in predicting significant fibrosis showed good diagnostic performance, which would be further optimized by combination of the two indices. HA and LN would be clinically useful serum markers for predicting significant fibrosis in patients with chronic hepatitis B, when liver biopsy is

Prevalence and characteristics of Shiga toxin-producing Escherichia coli in finishing pigs: Implications on public health.

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Cha, Wonhee; Fratamico, Pina M; Ruth, Leah E; Bowman, Andrew S; Nolting, Jacqueline M; Manning, Shannon D; Funk, Julie A

2018-01-02

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens, which can cause serious illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. To study the epidemiology of STEC in finishing pigs and examine the potential risks they pose for human STEC infections, we conducted a longitudinal cohort study in three finishing sites. Six cohorts of pigs (2 cohorts/site, 20 pigs/cohort) were randomly selected, and fecal samples (n=898) were collected every two weeks through their finishing period. Eighty-two pigs (68.3%) shed STEC at least once, and the proportion of STEC-positive pigs varied across sites (50-97.5%) and cohorts (15-100%). Clinically important serotypes, O157:H7 (stx 2c , eae) and O26:H11 (stx 1a , eae), were recovered from two pigs at sites C and A, respectively. The most common serotype isolated was O59:H21 (stx 2e ), which was particularly prevalent in site B as it was recovered from all STEC positive pigs (n=39). Each cohort showed different patterns of STEC shedding, which were associated with the prevalent serotype. The median shedding duration of STEC in pigs was 28days, consistent with our prior study. However, among pigs shedding O59:H21 at least once, pigs in cohort B2 had a significantly longer shedding duration of 42days (P<0.05) compared to other cohorts. Stx2e was the most commonly observed stx variant in finishing pigs (93.9%), in accordance with the previous studies. Stx2e has been reported to be significantly associated with edema disease in pigs, however, the pathogenicity in humans warrants further investigations. Nonetheless, our findings affirm that pigs are an important reservoir for human STEC infections, and that the circulating serotypes in a cohort and site management factors may significantly affect the prevalence of STEC. Molecular characterization of STEC isolates and epidemiological studies to identify risk factors for shedding in pigs are strongly warranted to further address the

Plasma Homocysteine, Serum Folic Acid, Serum Vitamin B12, Serum Vitamin B6, MTHFR, and Risk of Normal-Tension Glaucoma.

Science.gov (United States)

Li, Jinmiao; Xu, Fan; Zeng, Rui; Gong, Haijun; Lan, Yuqing

2016-02-01

This meta-analysis aims to comprehensively evaluate the association between total homocysteine (tHcy) levels, serum folic acid, vitamin B12, vitamin B6 levels, methylenetetrahydrofolate reductase (MTHFR) C677T genotype, and risk of normal-tension glaucoma (NTG). A systematic search of the EMBASE and PubMed databases was performed to evaluate plasma tHcy levels, serum folic acid, B vitamins' mean difference, and odds ratios of MTHFR C677T genotype between cases and controls. A total of 7 studies including 458 cases and 555 controls meeting the inclusion criteria were involved in this meta-analysis. There were 4 studies for tHcy (149 cases and 148 controls), 2 studies for vitamin B6, vitamin B12, and folate (90 cases and 82 controls), and 4 studies for MTHFR (343 cases and 449 controls). Overall, the mean plasma tHcy levels, serum folic acids, vitamin B12, and vitamin B6 levels were 1.16 μmol/L [95% confidence interval (CI), -0.13, 2.45], -0.62 μmol/L (95% CI, -1.98, 0.74), 5.81 μmol/L (95% CI, -3.53, 15.14), and -16.79 μmol/L (95% CI, -86.09, 52.51). MTHFR TT genotype was found to be unrelated to NTG risk (odds ratio=1.08; 95% CI, 0.69, 1.69). NTG is not associated with elevated plasma tHcy, serum folic acid, serum vitamin B12, serum vitamin B6, and MTHFR C677T genotype.

Human aldolase B subunit-specific radioimmunoassay

International Nuclear Information System (INIS)

Asaka, M.; Alpert, E.

1983-01-01

A radioimmunoassay was developed for the direct quantification of aldolase B in human serum and tissues. The method is a double-antibody radioimmunoassay technique using radioiodinated aldolase B homopolymer as ligand, chicken antibodies to aldolase B and rabbit antibodies to chicken IgG. This radioimmunoassay was shown to be specific for the aldolase B subunit, with no cross-reactivity with either human aldolase A subunit or homopolymeric human aldolase C (C 4 ). The lowest measurable amount by this method was 2 ng/ml. Aldolase B is predominantly found in normal liver tissue, with relatively-high aldolase B levels also observed in kidney. Aldolase B levels in the serum obtained from 11 normal subjects ranged from 23 to 38 ng/ml, with a mean of 28.5 +- 9.2 (S.D.) ng/ml. Almost all of patients with hepatitis had serum aldolase B levels greater than 30 ng/ml. In cancer patients, serum aldolase B was slightly elevated in patients with metastatic liver cancer and primary lever cell carcinoma, whereas no elevation of serum aldolase B was shown in patients without liver metastasis. (Auth.)

Evolution of saxitoxin synthesis in cyanobacteria and dinoflagellates.

Science.gov (United States)

Hackett, Jeremiah D; Wisecaver, Jennifer H; Brosnahan, Michael L; Kulis, David M; Anderson, Donald M; Bhattacharya, Debashish; Plumley, F Gerald; Erdner, Deana L

2013-01-01

Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some

Characterizing RecA-independent induction of Shiga toxin2-encoding phages by EDTA treatment.

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Lejla Imamovic

Full Text Available BACKGROUND: The bacteriophage life cycle has an important role in Shiga toxin (Stx expression. The induction of Shiga toxin-encoding phages (Stx phages increases toxin production as a result of replication of the phage genome, and phage lysis of the host cell also provides a means of Stx toxin to exit the cell. Previous studies suggested that prophage induction might also occur in the absence of SOS response, independently of RecA. METHODOLOGY/PRINCIPAL FINDINGS: The influence of EDTA on RecA-independent Stx2 phage induction was assessed, in laboratory lysogens and in EHEC strains carrying Stx2 phages in their genome, by Real-Time PCR. RecA-independent mechanisms described for phage λ induction (RcsA and DsrA were not involved in Stx2 phage induction. In addition, mutations in the pathway for the stress response of the bacterial envelope to EDTA did not contribute to Stx2 phage induction. The effect of EDTA on Stx phage induction is due to its chelating properties, which was also confirmed by the use of citrate, another chelating agent. Our results indicate that EDTA affects Stx2 phage induction by disruption of the bacterial outer membrane due to chelation of Mg(2+. In all the conditions evaluated, the pH value had a decisive role in Stx2 phage induction. CONCLUSIONS/SIGNIFICANCE: Chelating agents, such as EDTA and citrate, induce Stx phages, which raises concerns due to their frequent use in food and pharmaceutical products. This study contributes to our understanding of the phenomenon of induction and release of Stx phages as an important factor in the pathogenicity of Shiga toxin-producing Escherichia coli (STEC and in the emergence of new pathogenic strains.

[Clinical significance of determination of serum B7-H4 in patients with malignant hematologic diseases].

Science.gov (United States)

Wang, Xiao-Mei; Hu, Guo-Yan; Liu, Wei; Zheng, Shu-Hua; Lv, Jing; Wang, Hong-Mei; Xu, Jun-Fa

2010-09-01

To study the clinical significance of determination of serum B7-H4 in patients with malignant hematologic diseases. Serum B7-H4 levels were determined in 65 patients with leucemia, 34 patients with lymphoma, 12 patients with multiple myeloma as well as in 50 healthy controls. The serum B7-H4 levels in patients with lymphoma [(38.81+/-10.34) kappag/L] were significantly higher than healthy controls [(31.62+/-9.850) kappag/L] (Pleucemia, patients with multiple myeloma and healthy controls. These results suggest that the B7-H4 may correlated with lymphoma, but uncorrelated with leucemia and multiple myeloma. Measurement of serum B7-H4 level provide useful information for distinctive diagnosis of different kinds of malignant hematologic diseases.

[Virulence markers of Escherichia coli O1 strains].

Science.gov (United States)

Makarova, M A; Kaftyreva, L A; Grigor'eva, N S; Kicha, E V; Lipatova, L A

2011-01-01

To detect virulence genes in clinical isolates of Escherichia coli O1 using polymerase chain reaction (PCR). One hundred and twenty strains of E.coli O1 strains isolated from faeces of patients with acute diarrhea (n = 45) and healthy persons (n = 75) were studied. PCR with primers for rfb and fliC genes, which control synthesis of O- and H- antigens respectively, was used. Fourteen virulence genes (pap, aaf, sfa, afa, eaeA, bfpA, ial, hly, cnf, stx1, stx2, lt, st, and aer) were detected by PCR primers. K1-antigen was determined by Pastorex Meningo B/E. coli O1 kit (Bio-Rad). rfb gene controlling O-antigen synthesis in serogroup O1 as well as fliC gene controlling synthesis of H7 and K1 antigens were detected in all strains. Thus all E. coli strains had antigenic structure O1:K1 :H-:F7. Virulence genes aafl, sfa, afa, eaeA, bfpA, ial, hly, cnf, stx1, stx2, lt, and st were not detected. All strains owned pap and aer genes regardless of the presence of acute diarrhea symptoms. It was shown that E. coli O1:KI:H-:F7 strains do not have virulence genes which are characteristic for diarrhea-causing Escherichia. In accordance with the presence of pap and aer genes they could be attributed to uropathogenic Escherichia (UPEC) or avian-pathogenic Escherichia (APEC). It is necessary to detect virulence factors in order to determine E. coli as a cause of intestinal infection.

Serum inhibin B in healthy pubertal and adolescent boys

DEFF Research Database (Denmark)

Andersson, A M; Juul, A; Petersen, J H

1997-01-01

correlated strongly with age, and when the effect of age was taken into account, only the partial correlation between inhibin B and LH/testosterone remained statistically significant. At stage II of puberty, the positive partial correlation between inhibin B and LH/testosterone was still present. At stage......Inhibin B levels were measured in serum from 400 healthy Danish prepubertal, pubertal, and adolescent males, aged 6-20 yr, in a cross-sectional study using a recently developed immunoassay that is specific for inhibin B, the physiologically important inhibin form in men. In addition, serum levels...... of FSH, LH, testosterone, and estradiol levels were measured. Serum levels of inhibin B, FSH, LH, testosterone, and estradiol all increased significantly between stages I and II of puberty. From stage II of puberty the inhibin B level was relatively constant, whereas the FSH level continued to increase...

Synanthropic rodents as possible reservoirs of shigatoxigenic Escherichia coli strains

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Ximena eBlanco Crivelli

2012-11-01

Full Text Available Shigatoxigenic Escherichia coli (STEC strains are worldwide zoonotic pathogen responsible for different cases of human disease including hemolytic uremic syndrome (HUS. Transmission of STEC to humans occurs through the consumption of food and water contaminated by faeces of carriers and by person-to-person contact.The objective of this study was twofold: (a to investigate whether synanthropic rodents are possible reservoirs of STEC in the urban area and (b whether a particular genus out of synanthropic rodent is the principal carrier of STEC.One hundred forty-five rodents were captured in Buenos Aires City. Screening for stx1/stx2 and rfbO157 was done by PCR from the confluence zone. STEC isolates were further characterized with biochemical tests by standard methods. Additional virulence factors (eae, ehxA and saa were also determined by PCR. Forty-one of the rodents were necropsied and sample of kidney and small and large intestine were taken for histopathological diagnosis. The samples sections were stained with hematoxylin-eosin, and observed by light microscopy to evaluate the systemic involvement of these species in natural infections. STEC was isolated from seven out of twenty seven suspect animals at screening. The following genotypes were found in the STEC strains: stx1/stx2/ehxA (1, stx2 (4, stx2/ehxA (1, stx2/ehxA/eae (1. Neither gross nor microscopic lesions compatible with those produced by Shiga toxin were observed in the studied organs of necropsied rodents.The bivariate analysis including the hundred forty-five rodents data showed that the isolation of STEC is associated positively to Rattus genus. This synanthropic species may play a role in the transmissibility of the agent thus being a risk to the susceptible population. Their control should be included specifically in actions to dismiss the contamination of food and water by STEC in the urban area, as additional strategies for epidemiological control.

Investigation of Non-Covalent Interactions of Aflatoxins (B1, B2, G1, G2, and M1 with Serum Albumin

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Miklós Poór

2017-10-01

Full Text Available Aflatoxins are widely spread mycotoxins produced mainly by Aspergillus species. Consumption of aflatoxin-contaminated foods and drinks causes serious health risks for people worldwide. It is well-known that the reactive epoxide metabolite of aflatoxin B1 (AFB1 forms covalent adducts with serum albumin. However, non-covalent interactions of aflatoxins with human serum albumin (HSA are poorly characterized. Thus, in this study the complex formation of aflatoxins was examined with HSA applying spectroscopic and molecular modelling studies. Our results demonstrate that aflatoxins form stable complexes with HSA as reflected by binding constants between 2.1 × 104 and 4.5 × 104 dm3/mol. A binding free energy value of −26.90 kJ mol−1 suggests a spontaneous binding process between AFB1 and HSA at room-temperature, while the positive entropy change of 55.1 JK−1 mol−1 indicates a partial decomposition of the solvation shells of the interacting molecules. Modeling studies and investigations with site markers suggest that Sudlow’s Site I of subdomain IIA is the high affinity binding site of aflatoxins on HSA. Interaction of AFB1 with bovine, porcine, and rat serum albumins was also investigated. Similar stabilities of the examined AFB1-albumin complexes were observed suggesting the low species differences of the albumin-binding of aflatoxins.

Cloning and Expression of Fusion Genes of Domain A-1 Protective Antigen of Bacillus Anthracis and Shigella Enterotoxin B Subunit (Stxb In E. Coil

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AH ahmadi

2015-02-01

Conclusion: The findings of the current study revealed that this antigen can be raised as an anti-cancer and recombinant vaccine candidate against types of Shigella, Escherichia coli and Bacillus anthracis which can be due to such factors as identification of antigen(PA by antibody PA20, its apoptosis induction properties, property of immunogenicity, adjuvant and delivery of STxB protein and high expression levels of Gb3 in human cancer cells.

Clinical significance of measurement of changes of serum IL-2, SIL-2R levels, B lymphocyte number and T-cell subsets after chemotherapy in patients with malignant hydatidiform mole

International Nuclear Information System (INIS)

Zhang Guangcai

2007-01-01

Objective: To study the clinical significance of changes of serum IL-2, SIL-2R level, peripheral blood B lymphocyte number and T-cell subsets after chemotherapy in patients with malignant hydatidiform mole. Methods: Serum IL-2 ( with RIA), SIL-2R level (with ELISA) and peripheral blood B lymphocytes number as well as T subsets (with monoclonal antibody technique) were measured both before and after chemotherapy in 32 patients with malignant hydatidiform mole as well as in 35 controls. Results: Before chemotherapy serum SIL-2R level and B lymphocyte were significantly higher in the patients than those in controls (P<0.01), while the serum IL-2 level, CD3, CD4, CD4/CD8 were significantly lower (P<0.01). Six months after chemotherapy the levels changed markedly toward normal, but remained significantly different from those in controls (P<0.05). Conclusion: Abnormal immuno-regulation were present in patients with malignant mole. (authors)

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains.

Science.gov (United States)

Rivera, I G; Chowdhury, M A; Sanchez, P S; Sato, M I; Huq, A; Colwell, R R; Martins, M T

1995-09-01

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx (-)/zot (-) whereas strains isolated during the epidemic were ctx (+)/zot (+). All V. cholerae non-O1 strains tested in the study were ctx (-)/zot (-), whereas all V. cholerae O139 strains were ctx (+)/zot (+). Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.

In vitro study of vitamins B1, B2 and B6 adsorption on zeolite

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Basić Zorica

2011-01-01

Full Text Available Background/Aim. Zeolites are the hydratised alumosilicates of alcali and earthalcali cations, which have a long three-dimensional crystal structure. Preparations on the basis of zeolites are used for adsorption of organic and nonorganic toxic substances and they, also, find more and more use in veterinary and human medicine and pharmacy. The aim of this study was to evaluate the possibilities of zeolite to adsorb vitamins B1, B2 and B6 in acid and neutral solutions, as well as the characteristics of the process (saturability, reversibility and competitivness. Methods. The specific and sensitive HPLC method with fluorescent detector was used for determination of vitamins B1, B2 and B6. Analyte separation and detection were carried out by applying the reverse-phase method on column C18. An in vitro experiment was done by testing the influence of pH value (2 and 7, concentration of vitamin solution (1, 2 and 5 mg/L, the lenght of contact with zeolite (10-180 min and cation competitiveness on the exchange capacity, which is achieved by media and zeolite contact, as well as a possible vitamins desorption through changing pH value of the solution at 37°C. Jon competitiveness was examined by adding commercial feed mixture (grower with a defined content of the examined vitamines in zeolite solutions the pH = 2 and pH = 7. Results. Vitamins B1, B2 and B6 were stable in both pH=2 and pH = 7 solutions at 37°C, in the defined time intervals. In acid solution concentrations of vitamins significantly declined in the first 10 min, with no significant decline in further 30 min for all the three concentrations testch. In neutral solution, after the addition of 1% zeolite, decrease in vitamins concentrations was slightly lower than in acid solution, but also significant in the first 10 min of the contact with zeolite. It was found that zeolite, which adsorbed vitamins in acid solution, transferred in the neutral one released a significant quantity of adsorbed

Molecular screening of bovine raw milk for the presence of Shiga toxin-producing Escherichia coli (STEC on dairy farms

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Tatiane Vendramin

2014-09-01

Full Text Available Milkborne transmission of Shiga toxin- producing Escherichia coli (STEC has raised considerable concern due to recent outbreaks worldwide and poses a threat to public health. The aim of this study was to develop a sensitive and specific multiplex PCR assay to detect the presence of STEC in bovine raw milk. To identify E. coli (ATCC 25922 contamination, the gene uspA was used, and PCR sensitivity and specificity were accessed by testing diluted samples ranging from 2 to 2.0 × 10(6 CFU/mL. To detect STEC, the stx1 and stx2 genes were selected as targets. After reaction standardization, the multiplex assay was tested in raw milk collected from 101 cows on dairy farms. PCR assay for E. coli detection had a specificity of 100% and sensitivity of 79% (P<0.0001, with a lower detection limit of 2 CFU/mL. Multiplex PCR assay had 100% sensitivity for E. coli positive raw milk samples, and 31.1% were contaminated with STEC, 28.3% of stx2, and 1.9% of stx1. The multiplex PCR assay described in the present study can be employed to identify and screen E. coli harboring stx1 and stx2 genes in raw milk on dairy farms and in industries.

Characteristics of Escherichia coli from raw vegetables at a retail market in the Czech Republic.

Science.gov (United States)

Skočková, Alena; Karpíšková, Renáta; Koláčková, Ivana; Cupáková, Šárka

2013-10-15

A large epidemic caused by shigatoxigenic Escherichia coli (E. coli) in spring 2011 in Germany resulted in reduction of trust in the health safety of raw vegetables and sprouted seeds. This study focused on the detection and characterization of E. coli in raw vegetables and sprouted seeds sold in the Czech Republic. Out of 91 samples, 24 (26.4%) were positive for the presence of E. coli. Resistance to antimicrobial agents was determined by the disk diffusion method and E-test. Polymerase chain reaction was used for the detection of selected genes encoding virulence--eaeA, hly, stx1, and stx2 and genes encoding resistance to tetracycline--tet(A), tet(B), tet(C), and tet(G) and to β-lactams--blaTEM, blaSHV, and blaCTX. The blaTEM gene was detected in two isolates, the tet(B) gene in three and tet(A) in one isolate. No hly, stx1, or stx2 genes were present, but the eaeA gene was found in three (11.1%) isolates from imported vegetables. These isolates can be considered as potentially enteropathogenic. Results of this study show that raw vegetables and sprouted seeds sold in the retail market can represent a potential risk for consumers. © 2013.

From farm to table: follow-up of Shiga toxin-producing Escherichia coli throughout the pork production chain in Argentina

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Rocío eColello

2016-02-01

Full Text Available Pigs are important reservoirs of Shiga toxin-producing Escherichia coli (STEC. The entrance of these strains into the food chain implies a risk to consumers because of the severity of hemolytic uremic syndrome. This study reports the prevalence and characterization of Shiga toxin-producing Escherichia coli (STEC throughout the pork production chain. From 764 samples, 31 (4.05% were stx positive by PCR screening. At farms, 2.86% of samples were stx positive; at slaughter, 4.08% of carcasses were stx positive and at boning rooms, 6% of samples were stx positive. These percentages decreased in pork meat ready for sale at sales markets (4.59%. From positive samples, 50 isolates could be characterized. At farms 37.5% of the isolates carried stx1/stx2 genes, 37.5% possessed stx2e and 25%, carried only stx2. At slaughter we detected 50% of isolates positive for stx2, 33% for stx2e and 16% for stx1/stx2. At boning rooms 59% of the isolates carried stx1/stx2, 14% stx2e and 5% stx1/stx2/stx2e. At retail markets 66% of isolates were positive for stx2, 17% stx2e and 17% stx1/stx2. For the other virulence factors, ehxA and saa were not detected and eae gene was detected in 12% of the isolates. Concerning putative adhesins, agn43 was detected in 72%, ehaA in 26%, aida in 8% and iha in 6% of isolates. The strains were typed into 14 E. coli O groups (O1, O2, O8, O15, O20, O35, O69, O78, O91, O121, O138, O142, O157, O180 and ten H groups (H9, H10, H16, H21, H26, H29, H30, H32, H45, H46. This study reports the prevalence and characterization of STEC strains through the chain pork suggesting the vertical transmission. STEC contamination originates in the farms and is transferred from pigs to carcasses in the slaughter process and increase in meat pork at boning rooms and sales markets. These results highlight the need to implement an integrated STEC control system based on good management practices on the farm and critical control point systems in the food chain.

Respiratory syncytial virus infection in sheep bronchial explants is associated with enhanced ETB receptor-mediate contractile functional and autoradiographic studies

International Nuclear Information System (INIS)

Fernandes, L.B.; D'Aprile, A.C.; Betts, R.J.; Goldie, R.G.

2001-01-01

Full text: Respiratory syncytial virus (RSV) is an important precipitant of asthma in children. The impact of RSV infection on endothelin (ET) receptor density and function in airways is unknown. In the present study, sheep bronchial rings were maintained as explants in culture for up to 48 h. During this time, both the structural integrity of the epithelium and carbachol responsiveness were preserved. Bronchial rings in culture were exposed to non-infected culture medium or to RSV (1/50 TCID 50 ) for 0, 24 and 48 h which caused marked damage to and loss of the epithelium. RSV infection did not significantly alter responsiveness to ET-1 at either 24 (Control EC 40 = 102 nM, 95% confidence limits, 76-138 nM vs RSV EC 40 = 66 nM, 95% confidence limits, 48-91 nM, n=5-6, P>0.05) or 48 h (Control EC 40 35 nM, 95% confidence limits, 19-66 nM vs RSV EC 40 = 55 nM, 95% confidence limits, 32-93 nM, n=8, P>0.05). As seen previously (Goldie et al., 1994), sarafotoxin S6c (StxS6c, ET B -selective) did not cause contraction in non-infected sheep bronchial explants. In contrast, StxS6c (300 nM) increased tone by 8±3% carbachol Emax (n=6-8) in explants exposed to RSV for 24 or 48 h. Light microscopic autoradiography was used to determine the relative distribution of ET A and ET B receptors using [ 125 I]-ET-1, BQ-123 (ET A -selective) and StxS6c. Sheep airway smooth muscle contains a homogeneous population of ET A receptors (Goldie et al., 1994). Since StxS6c caused significant contraction in RSV-infected bronchial explants, it was surprising that autoradiographic techniques failed to detect airway smooth muscle ET B receptors in these preparations. It is likely that ET B receptors fell below the level of detection of autoradiography. The significant StxS6c-induced contraction of sheep bronchi suggests the novel expression of ET B receptors triggered by RSV which might be relevant to RSV-associated asthma. Copyright (2001) Australasian Society of Clinical and Experimental

Sekuen Nukleotida Gene Shiga like toxin-2 dari Isolat Lokal Escherichia coli O157:H7 asal Hewan dan Manusia (NUCLEOTIDES SQUENCES OF SHIGA-LIKE TOXIN 2 GENES OF ESCHERICHIA COLI O157:H7 LOCAL ISOLATES ORIGINATED FROM ANIMALS AND HUMAN

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I Wayan Suardana

2017-04-01

Full Text Available Animals/livestock, especially cattle, are known as the main reservoir of Escherichia coli O157: H7. As the only one of zoonotic E. coli, the pathogenicity of these bacteria is determined by its ability to produce one or more very potent cytotoxin known as Shiga-like toxin (Stx or verocytotoxin, particularly of the Stx2 type that is closely related to the incidence of hemolytic uremic syndrome (HUS in humans. This study analyzed the nucleotide sequences of stx2 gene between isolates from animals and humans in an effort to assess the potential zoonoses of the agent. The research activity was initiated by cultivating 20 isolates of E. coli O157:H7 collection based on result in the previous study i.e. 2 isolates originated from cattle feces, 2 isolates originated from beef, 2 isolates originated from chicken feces, 2 isolates originated from human feces, and 12 non-clinical isolates originated from human fecal who were suffering with renal failure. All isolates were confirmed on selective medium Sorbitol MacConkey Agar (SMAC followed by testing on aglutination O157 latex test, and H7 antisera. Molecular analysis of stx2 gene covering open reading frame (ORF of the stx2 gene was performed using the primer which was designed by researcher i.e. Stx2 (F/Stx2 (R. The results showed, there were 2 isolates i.e. KL-48 (2 originated from human feces and SM-25 (1 originated from cattle feces were positive for carrying a stx2 gene, which was marked by the 1587 bp PCR product. Analysis of sequencing showed both isolates had identical to stx2 nucleotide squences with E. phaga 933 as well as E. coli ATCC 933. These results indicate the both local isolates are potential as zoonotic agents with clinical effects similar to E. phaga 933 and E. coli ATCC 43894. ABSTRAK Hewan ternak khususnya sapi, dikenal sebagai reservoir utama Escherichia coli O157:H7. Sebagai satu-satunya serotipe E. coli yang bersifat zoonosis, patogenitas bakteri ini ditentukan oleh kemampuannya

Prevalence and Characterization of Shiga Toxin-Producing Escherichia coli in Swine Feces Recovered in the National Animal Health Monitoring System's Swine 2000 Study

Science.gov (United States)

Fratamico, Pina M.; Bagi, Lori K.; Bush, Eric J.; Solow, Barbara T.

2004-01-01

A study was conducted to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in swine feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from 13 of the top 17 swine-producing states were tested for the presence of STEC. After enrichment of swine fecal samples in tryptic soy broth, the samples were tested for the presence of stx1 and stx2 by use of the TaqMan E. coli STX1 and STX2 PCR assays. Enrichments of samples positive for stx1 and/or stx2 were plated, and colony hybridization was performed using digoxigenin-labeled probes complementary to the stx1 and stx2 genes. Positive colonies were picked and confirmed by PCR for the presence of the stx1, stx2, or stx2e genes, and the isolates were serotyped. Out of 687 fecal samples tested using the TaqMan assays, 70% (484 of 687) were positive for Shiga toxin genes, and 54% (370 of 687), 64% (436 of 687), and 38% (261 of 687) were positive for stx1, stx2, and both toxin genes, respectively. Out of 219 isolates that were characterized, 29 (13%) produced stx1, 14 (6%) produced stx2, and 176 (80%) produced stx2e. Twenty-three fecal samples contained at least two STEC strains that had different serotypes but that had the same toxin genes or included a strain that possessed stx1 in addition to a strain that possessed stx2 or stx2e. The STEC isolates belonged to various serogroups, including O2, O5, O7, O8, O9, OX10, O11, O15, OX18, O20, O57, O65, O68, O69, O78, O91, O96, O100, O101, O120, O121, O152, O159, O160, O163, and O untypeable. It is noteworthy that no isolates of serogroup O157 were recovered. Results of this study indicate that swine in the United States harbor STEC that can potentially cause human illness. PMID:15574914

Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E-coli (STEC) infections in the Netherlands, January 2008 to December 2011

NARCIS (Netherlands)

Friesema, I.; van der Zwaluw, K.; Schuurman, T.; Kooistra-Smid, M.; Franz, E.; van Duynhoven, Y.; van Pelt, W.

2014-01-01

The Shiga toxins of Shiga toxin-producing Escherichia coli (STEC) can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) with several sub-variants. Variant Stx(2f) is one of the latest described, but has been rarely associated with symptomatic human infections. In the enhanced STEC

Clinical significance of measurement of changes of serum IL-2, SIL-2R, TNF-α levels, B lymphocyte count and T subsets distribution type after treatment in patients with vitiligo

International Nuclear Information System (INIS)

Xie Chuntao

2007-01-01

Objective: To study the changes of serum IL-2, SIL-2R, TNF-α levels B lymphocytes count and T lyonphocyte subsets distribation type after treatment in patients with vitiligo. Methods: Serum IL-2, TNF-α (with RIA), SIL-2R (with ELISA) levels, B lymphocytes count and T subsets (with monoclonal antibody technique) were examined in 40 patients with vitiligo both before and after treatment as well as in 35 controls. Results: Before treatment, serum SIL-2R, TNF-α levels and B lymphocytes count were significantly higher than those in controls (P<0.01), while the serum IL-2, CD3, CD4 levels CD4/CD8 ratio were significantly lower(P<0.01). After treatment for 6 months, the data were greatly corrected but remanied significantly different from those in controls (P<0.05). Conclusion: Vitiligo is a kind of auto-immune diseases with abnormal immuno-regulation. (authors)

Preconception serum 1,1,1-trichloro-2,2,bis(p-chlorophenyl)ethane and B-vitamin status: independent and joint effects on women's reproductive outcomes.

Science.gov (United States)

Ouyang, Fengxiu; Longnecker, Matthew P; Venners, Scott A; Johnson, Sara; Korrick, Susan; Zhang, Jun; Xu, Xiping; Christian, Parul; Wang, Mei-Cheng; Wang, Xiaobin

2014-12-01

Although preconception 1,1,1-trichloro-2,2,bis(p-chlorophenyl)ethane (DDT) exposure and B-vitamin deficiencies have each been shown to negatively affect human reproductive outcomes, little is known about their joint effect. We sought to examine whether B-vitamin sufficiency protects against adverse effects of DDT on clinical pregnancy (CP) and subclinical early pregnancy loss (EPL). We measured preconception concentrations of plasma B vitamins (vitamin B-6, vitamin B-12, and folate) and serum total DDT [sum of p,p' and o,p' isomers of DDT and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] in 291 nulligravid women from Anhui, China, who were studied in 1996-1998. The women were followed prospectively from the time they stopped contraception until CP (gestational age ≥42 d) or 12 mo (whichever occurred first). EPL was identified by using daily urinary human chorionic gonadotropin. The women were categorized according to B-vitamin status (deficiency compared with sufficiency) and DDT concentration (high compared with low). Of 291 study women, a total of 385 conceptions (31% of which ended in EPL) and 265 CPs occurred. Compared with women with adequate B-vitamins and low DDT, incidence rates of CP were reduced in women with B-vitamin deficiency and a high DDT concentration (P vitamin B-12, DDT was not associated with the incidence of CP; in contrast, in women with vitamin B-12 deficiency, high DDT was associated with a lower incidence of CP (HR: 0.44; 95% CI: 0.23, 0.84); and the test for interaction was significant (P vitamin B-12 and folate sufficiency may help protect against adverse reproductive effects of DDT exposure. Additional studies are needed to confirm our findings. © 2014 American Society for Nutrition.

SU-F-T-601: Equivalence of CyberKnife and TrueBeam STx for Spine Irradiation

Energy Technology Data Exchange (ETDEWEB)

Herrington, J [University of Rhode Island, Kingston, RI (United States); Price, M; Brindle, J [University of Rhode Island, Kingston, RI (United States); Rhode Island Hospital, Providence, RI (United States); Alpert Medical School of Brown University (United States); Schmidt, M; Knutson, N [University of Rhode Island, Kingston, RI (United States); Rhode Island Hospital, Providence, RI (United States)

2016-06-15

Purpose: To evaluate the equivalence of spine SBRT treatment plans created in Eclipse for the TrueBeam STx (Varian Medical System, Palo Alto, CA) compared to plans using CyberKnife and MultiPlan (Accuray, Sunnyvale, CA). Methods: CT data and contours for 23 spine SBRT patients previously treated using CyberKnife (CK) were exported from MultiPlan treatment planning system into Eclipse where they were planned using static IMRT 6MV coplanar beams. Plans were created according to the original prescription dose and fractionation schedule while limiting spinal dose according to the RTOG 0631 protocol and maintaining target coverage comparable to the original CK plans. Plans were evaluated using new conformity index (nCI), homogeneity index (HI), dose-volume histogram data, number of MU, and estimated treatment time. To ensure all Eclipse plans were deliverable, standard clinical IMRT QA was performed. The plan results were matched with their complimentary CK plans for paired statistical analysis. Results: Plans generated in Eclipse demonstrated statistically significant (p<0.01) improvements compared to complimentary CK plans in median values of maximum spinal cord dose (17.39 vs. 18.12 Gy), RTOG spinal cord constraint dose (14.50 vs. 16.93 Gy), nCI (1.28 vs. 1.54), HI (1.13 vs. 1.27), MU (3918 vs. 36416), and estimated treatment time (8 vs. 48 min). All Eclipse generated plans passed our clinically used protocols for IMRT QA. Conclusion: CK spine SBRT replanned utilizing Eclipse for LINAC delivery demonstrated dosimetric advantages. We propose improvements in plan quality metrics reviewed in this study may be attributed to dynamic MLCs that facilitate treatment of complicated geometries as well as posterior beams ideal for centrally located and/or posterior targets afforded by gantry-based RT delivery.

Both serum apolipoprotein B and the apolipoprotein B/apolipoprotein A-I ratio are associated with carotid intima-media thickness.

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Fei Huang

Full Text Available BACKGROUND: Previous studies indicated that apolipoprotein measurements predicted cardiovascular disease (CVD risk; however, associations between apolipoproteins and carotid intima-media thickness (CIMT were less explored. METHODOLOGY AND PRINCIPAL FINDINGS: The cross-sectional study included 6069 participants aged 40 years or older with NGT from Shanghai, China. Serum fasting traditional lipids (total cholesterol [TC], low-density lipoprotein cholesterol [LDL-C], high-density lipoprotein cholesterol [HDL-C] and triglycerides [TG], apoA-I and apoB were assessed. A high-resolution B-mode ultrasonography was performed to measure CIMT. We found CIMT increased progressively across the quartiles of serum apoB (p for trend <0.0001. In logistic regression, concentrations of apoB (odds ratio [OR] 1.27, 95% confidence interval [CI] 1.18-1.36, TC (OR 1.23, 95% CI 1.14-1.32, LDL-C (OR 1.25, 95% CI 1.16-1.34 and TG (OR 1.11, 95% CI 1.04-1.20 were significantly related to elevated CIMT after adjusted for age and sex. Meanwhile, the apoB/apoA-I ratio (OR 1.25, 95% CI 1.17-1.34 related to elevated CIMT. ApoB (OR 1.23, 95% CI 1.00-1.51 and the apoB/apoA-I ratio (OR 1.19, 95% CI 1.04-1.36 remained significantly associated with elevated CIMT, after adjusted for the traditional CVD risk factors including traditional lipids. CONCLUSIONS AND SIGNIFICANCE: There were significant associations between serum apoB, the apoB/apoA-I ratio and elevated CIMT. Serum apoB and the apoB/apoA-I ratio might be independent predictors of early atherosclerosis in NGT.

Changes of MCP-1, FKN, and related cytokines in the serum and cerebrospinal fluid in children with epidemic encephalitis B

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Qian Wang

2017-09-01

Full Text Available Objective: To explore the changes of MCP-1, FKN, and related cytokines in the serum and cerebrospinal fluid (CSF in children with epidemic encephalitis B. Methods: A total of 40 children with epidemic encephalitis B who were admitted in our hospital from June, 2014 to June, 2017 were included in the study and divided into the severe group (n=15 and general group (n=25 according to the severity group. Moreover, 20 children who were suffered from oblique inguinal hernia, perineal adhesion, and cryptorchidism were served as the control group. The serum and CSF specimens were collected 24 h after admission and during the recovery period in children with epidemic encephalitis B. The serum specimen was collected 24 h after admission in the control group, and CSF specimen was collected during the lumbar puncture. ELISA was used to detect CMP-1, FKN, IL-1β, IL-18, and TNF-α levels in the serum and CSF. CMP-1, FKN, IL-1β, IL-18, and TNF-α levels in children with epidemic encephalitis B on the day after admission and 2-3 weeks after admission and in the control group were compared. The changes of CMP-1, FKN, IL-1β, IL-18, and TNF-α in children with severe and general epidemic encephalitis B were observed. Results: CMP-1 and FKN levels in the serum and CSF in children with epidemic encephalitis B in the critical stage were significantly higher than those in the recovery stage and in the control group. The serum CMP-1 and FKN levels in children with epidemic encephalitis B during the recovery stage were not significantly different from those in the control group, while CMP-1 and FKN levels in CSF were significantly higher than those in the control group. CMP-1 and FKN levels in the serum and CSF in children with severe epidemic encephalitis B were significantly higher than those in the general group. IL-1β, TNF-α, and IL-18 levels in the serum and CSF in children with epidemic encephalitis B during the critical stage were significantly higher than

Clinical significance of determination of changes of serum SF, folic acid and vitamin B{sub 12} levels in pregnant women of different gestational stages

Energy Technology Data Exchange (ETDEWEB)

Hua, Jiang [Taizhou City Women and Children Health Station, Jiangsu, Taizhou (China)

2007-10-01

Objective: To explore the changes of serum SF, FA and Vitamin Bn levels in pregnant women of different gestational stages. Methods: Levels of serum SF, fore acid and Vitamin B{sub 12} were detected with RIA in 132 pregnant women of different gestational stages and 35 controls (non-pregant women). Results: Serum SF, folic acid and VitB{sub 12} levels were significantly lower in pregnant women of 2nd and 3rd trimester than those in controls (P<0.01), but expect in pregnant women of 1st trimester. Conclusion: Serum SF, folio acid and Vitamin B{sub l2} levels were low in late pregnancy predisposing to development anemia. (authors)

Serum thromboxane B2 (TXB2 Determination is Influenced by Sample Incubation Temperature in Healthy Beagle Dogs

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Aleš Jerin

2009-01-01

Full Text Available The measurement of serum thromboxane B2 (TXB2 production by platelets is a specific test for assessment of platelet cyclooxygenase (COX-1 activity following administration of non-steroidal anti-inflammatory drugs (NSAIDs. The aim of this study was to investigate the influence of sample incubation at 37 °C for one hour on serum TXB2 concentration in comparison with incubation at room temperature. A total of 54 blood samples for serum TXB2 measurements were collected from six healthy beagle dogs into two separate serum tubes. While one group of tubes was incubated in a 37 °C water bath, the second group of tubes was left to coagulate at room temperature, both for one hour. Serum TXB2 concentrations were measured by ELISA. The mean concentration (± SD of serum TXB2 in the group of samples that were incubated at 37 °C was significantly (P 2 concentration in healthy beagle dogs and demonstrate that validated methods for assessment of COX-1 activity by measurement of serum TXB2 should be used in order to make results more reliable and comparable between different studies. The results of this study might be of great help in planning NSAID studies in dogs by providing the information that TXB2 generation by platelets is influenced profoundly by incubation temperature.

Shiga toxin-producing Escherichia coli distribution and characterization in a pasture-based cow-calf production system.

Science.gov (United States)

Baltasar, Patrícia; Milton, Stewart; Swecker, William; Elvinger, François; Ponder, Monica

2014-05-01

Shiga toxin-producing Escherichia coli (STEC) strains are commonly found in cattle gastrointestinal tracts. In this study, prevalence and distribution of E. coli virulence genes (stx1, stx2, hlyA, and eaeA) were assessed in a cow-calf pasture-based production system. Angus cows (n = 90) and their calves (n = 90) were kept in three on-farm locations, and fecal samples were collected at three consecutive times (July, August, and September 2011). After enrichment of samples, stx1, stx2, eaeA, and hlyA were amplified and detected with a multiplex PCR (mPCR) assay. Fecal samples positive for stx genes were obtained from 93.3% (84 of 90) of dams and 95.6% (86 of 90) of calves at one or more sampling times. Age class (dam or calf), spatial distribution of cattle (farm locations B, H, K), and sampling time influenced prevalence and distribution of virulence genes in the herd. From 293 stx-positive fecal samples, 744 E. coli colonies were isolated. Virulence patterns of isolates were determined through mPCR assay: stx1 was present in 41.9% (312 of 744) of the isolates, stx2 in 6.5% (48 of 744), eaeA in 4.2% (31 of 744), and hlyA in 2.4% (18 of 744). Prevalence of non-O157 STEC was high among the isolates: 33.8% (112 of 331) were STEC O121, 3.6% (12 of 331) were STEC O103, and 1.8% (6 of 331) were STEC O113. One isolate (0.3%) was identified as STEC O157. Repetitive element sequence-based PCR (rep-PCR) fingerprinting was used to study genetic diversity of stx-positive E. coli isolates. Overall, rep-PCR fingerprints were highly similar, supporting the hypothesis that strains are transmitted between animals but not necessarily from a dam to its calf. Highly similar STEC isolates were obtained at each sampling time, but isolates obtained from dams were more diverse than those from calves, suggesting that strain differences in transference may exist. Understanding the transfer of E. coli from environmental and animal sources to calves may aid in developing intervention

Prevalence, Variability and Bioconcentration of Saxitoxin-Group in Different Marine Species Present in the Food Chain.

Science.gov (United States)

Oyaneder Terrazas, Javiera; Contreras, Héctor R; García, Carlos

2017-06-12

The saxitoxin-group (STX-group) corresponds to toxic metabolites produced by cyanobacteria and dinoflagellates of the genera Alexandrium, Gymnodinium , and Pyrodinium . Over the last decade, it has been possible to extrapolate the areas contaminated with the STX-group worldwide, including Chile, a phenomenon that has affected ≈35% of the Southern Pacific coast territory, generating a high economic impact. The objective of this research was to study the toxicity of the STX-group in all aquatic organisms (bivalves, algae, echinoderms, crustaceans, tunicates, cephalopods, gastropods, and fish) present in areas with a variable presence of harmful algal blooms (HABs). Then, the toxic profiles of each species and dose of STX equivalents ingested by a 60 kg person from 400 g of shellfish were determined to establish the health risk assessment. The toxins with the highest prevalence detected were gonyautoxin-4/1 (GTX4/GTX1), gonyautoxin-3/2 (GTX3/GTX2), neosaxitoxin (neoSTX), decarbamoylsaxitoxin (dcSTX), and saxitoxin (STX), with average concentrations of 400, 2800, 280, 200, and 2000 µg kg -1 respectively, a species-specific variability, dependent on the evaluated tissue, which demonstrates the biotransformation of the analogues in the trophic transfer with a predominance of α-epimers in all toxic profiles. The identification in multiple vectors, as well as in unregulated species, suggests that a risk assessment and risk management update are required; also, chemical and specific analyses for the detection of all analogues associated with the STX-group need to be established.

Radioimmunoassay of serum antibodies with B-streptococcus specificity in pregnant women and infants

International Nuclear Information System (INIS)

Frey, C.W.

1980-01-01

In a specific competitive radioimmunoassay of purified rabbit antibodies, labeled with iodine 125 against group- and type-antigens of streptococcus agalactiae (streptococci type B), we investigated the amount of serum anti-bodies providing specificity of streptococci type B in not preselected pregnant women, newborn and babies with colonies of streptococci type B or with diseases due to streptococci type B and in some of their mothers. These antibodies could be detected in 26 of 45 pregnant women and in 3 of 7 children with colonies of streptococci type B. 5 of 18 newborn with the ''early-onset'' type of infection and 6 of 7 of their mothers provided antibodies with specificity of streptococci type B as did one of two newborn with the ''late onset'' type of infection. Contrary to the supposition of Baker and Kasper and in accordance with the findings of Wilkinson, the ''risk group'' cannot be determined only by detecting the antibodies against streptococci type B. The risk group comprises those persons in whom the colonisation of streptococci agalactiae leads to the frequently life-threatening infecton of neonatals with streptococci type B. (orig.) [de

Hybrids of Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Among Human and Animal Isolates in Finland.

Science.gov (United States)

Nyholm, O; Heinikainen, S; Pelkonen, S; Hallanvuo, S; Haukka, K; Siitonen, A

2015-11-01

Diarrhoeagenic Escherichia coli (DEC) cause serious foodborne infections in humans. Total of 450 Shigatoxigenic E. coli (STEC) strains isolated from humans, animals and environment in Finland were examined by multiplex PCR targeting the virulence genes of various DEC pathogroups simultaneously. One per cent (3/291) of the human STEC and 14% (22/159) of the animal and environmental STEC had genes typically present in enterotoxigenic E. coli (ETEC). The strains possessed genes encoding both Shiga toxin 1 and/or 2 (stx1 and/or stx2 ) and ETEC-specific heat-stable (ST) enterotoxin Ia (estIa). The identified stx subtypes were stx1a, stx1c, stx2a, stx2d and stx2g. The three human STEC/ETEC strains were isolated from the patients with haemolytic uraemic syndrome and diarrhoea and from an asymptomatic carrier. The animal STEC/ETEC strains were isolated from cattle and moose. The human and animal STEC/ETEC strains belonged to 11 serotypes, of which O2:H27, O15:H16, O101:H-, O128:H8 and O141:H8 have previously been described to be associated with human disease. Identification of multiple virulence genes offers further information for assessing the virulence potential of STEC and other DEC. The emergence of novel hybrid pathogens should be taken into account in the patient care and epidemiological surveillance. © 2015 Blackwell Verlag GmbH.

A Topographical Atlas of Shiga Toxin 2e Receptor Distribution in the Tissues of Weaned Piglets

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Daniel Steil

2016-11-01

Full Text Available Shiga toxin (Stx 2e of Stx-producing Escherichia coli (STEC is the primary virulence factor in the development of pig edema disease shortly after weaning. Stx2e binds to the globo-series glycosphingolipids (GSLs globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer and globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer, the latter acting as the preferential Stx2e receptor. We determined Stx receptor profiles of 25 different tissues of a male and a female weaned piglet using immunochemical solid phase binding assays combined with mass spectrometry. All probed tissues harbored GSL receptors, ranging from high (category I over moderate (category II to low content (category III. Examples of Gb4Cer expression in category I tissues are small intestinal ileum, kidney pelvis and whole blood, followed by colon, small intestinal duodenum and jejunum belonging to category II, and kidney cortex, cerebrum and cerebellum as members of category III organs holding true for both genders. Dominant Gb3Cer and Gb4Cer lipoforms were those with ceramides carrying constant sphingosine (d18:1 and a variable C16:0, C22:0 or C24:1/C24:0 fatty acid. From the mapping data, we created a topographical atlas for Stx2e receptors in piglet tissues and organs, which might be helpful to further investigations on the molecular and cellular mechanisms that underlie infections of Stx2e-producing STEC in pigs and their zoonotic potential for humans.

Uptake and release of paralytic shellfish toxins by the clam Ruditapes decussatus exposed to Gymnodinium catenatum and subsequent depuration.

Science.gov (United States)

Botelho, Maria João; Vale, Carlos; Grilo, Rita Velez; Ferreira, João Gomes

2012-06-01

A laboratory experiment was performed with the clam Ruditapes decussatus, fed with the toxic dinoflagellate Gymnodinium catenatum and the non-toxic algae Isochrysis galbana (14 days) and subsequently only with I. galbana (15 days). Individual paralytic shellfish toxins were determined by LC-FLD in G. catenatum cells, whole clam tissues, and particulate organic matter (POM) produced by clams. The toxins dcSTX and dcGTX2 + 3 in the algae were less abundant than C1 + 2 and B1, but were predominant in clams during both the exposure and depuration phases. The toxin dcNEO was only detected in clams during a short period, indicating conversion from other compounds. The toxin composition of the POM indicated the export of dcSTX as faeces or pseudo-faeces along the entire experiment (2.5-14 nmol mg(-1)), B1 was present in a short period of the exposure and C1 + 2 and dcGTX2 + 3 absent. A mass balance calculation indicated that approximately 95% of C1 + 2 and 85% of B1 supplied to the clams were converted into other toxins or lost in solution. Conversely, the net gain of 512, 61 and 31 nmol for dcSTX, dcGTX2 + 3 and dcNEO, respectively, suggests the conversion from other assimilated compounds by clams during exposure and depuration phases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Flanking sequence determination and specific PCR identification of transgenic wheat B102-1-2.

Science.gov (United States)

Cao, Jijuan; Xu, Junyi; Zhao, Tongtong; Cao, Dongmei; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

2014-01-01

The exogenous fragment sequence and flanking sequence between the exogenous fragment and recombinant chromosome of transgenic wheat B102-1-2 were successfully acquired using genome walking technology. The newly acquired exogenous fragment encoded the full-length sequence of transformed genes with transformed plasmid and corresponding functional genes including ubi, vector pBANF-bar, vector pUbiGUSPlus, vector HSP, reporter vector pUbiGUSPlus, promoter ubiquitin, and coli DH1. A specific polymerase chain reaction (PCR) identification method for transgenic wheat B102-1-2 was established on the basis of designed primers according to flanking sequence. This established specific PCR strategy was validated by using transgenic wheat, transgenic corn, transgenic soybean, transgenic rice, and non-transgenic wheat. A specifically amplified target band was observed only in transgenic wheat B102-1-2. Therefore, this method is characterized by high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B102-1-2.

A study of serum lipid profile and serum apolipoproteins A1 and B in Indian male violent criminal offenders.

Science.gov (United States)

Chakrabarti, Nandini; Sinha, V K

2006-01-01

High cholesterol has been advanced as the most important factor in the development of coronary artery disease. Most panels have recommended population-wide dietary restrictions, yet a body of evolving data yields evidence of the hazards of low cholesterol, including links to aggression and hostility. The aim of this study was to compare the serum lipid profile and serum apolipoproteins A1 and B of men with a violent criminal record and men with no criminal history. Fasting blood samples were collected from 30 men with a known history of violent crime and 30 men with no criminal record. Serum lipid profile and serum apolipoproteins A1 and B were measured in each sample, and compared between the two groups. The group with the violent criminal record showed significantly lower total cholesterol, lower LDL cholesterol, higher apolipoprotein A1 and lower apolipoprotein B compared with the control group. Lower total cholesterol, lower LDL cholesterol, higher apolipoprotein A1 and lower apolipoprotein B could predispose to violence. Future research might explore the possibility that diets offered in prison could affect relevant pathways in lipid metabolism. Copyright (c) 2006 John Wiley & Sons, Ltd.

Mouse in Vivo Neutralization of Escherichia coli Shiga Toxin 2 with Monoclonal Antibodies

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Larry H. Stanker

2013-10-01

Full Text Available Shiga toxin-producing Escherichia coli (STEC food contaminations pose serious health concerns, and have been the subject of massive food recalls. STEC has been identified as the major cause of the life-threatening complication of hemolytic uremic syndrome (HUS. Besides supportive care, there currently are no therapeutics available. The use of antibiotics for combating pathogenic E. coli is not recommended because they have been shown to stimulate toxin production. Clearing Stx2 from the circulation could potentially lessen disease severity. In this study, we tested the in vivo neutralization of Stx2 in mice using monoclonal antibodies (mAbs. We measured the biologic half-life of Stx2 in mice and determined the distribution phase or t1/2 α to be 3 min and the clearance phase or t1/2 β to be 40 min. Neutralizing mAbs were capable of clearing Stx2 completely from intoxicated mouse blood within minutes. We also examined the persistence of these mAbs over time and showed that complete protection could be passively conferred to mice 4 weeks before exposure to Stx2. The advent of better diagnositic methods and the availability of a greater arsenal of therapeutic mAbs against Stx2 would greatly enhance treatment outcomes of life threatening E. coli infections.

Depuration kinetics of paralytic shellfish toxins in Mytilus galloprovincialis exposed to Gymnodinium catenatum: laboratory and field experiments.

Science.gov (United States)

Botelho, Maria João; Vale, Carlos; Mota, Ana M; S Simões Gonçalves, Maria de Lurdes

2010-12-01

The kinetics of paralytic shellfish toxins in Mytilus galloprovincialis, previously exposed to Gymnodinium catenatum, was studied under depuration laboratory conditions and over a declining bloom of the dinoflagellate in the field. The variation of the levels observed throughout the laboratory experiment was characterized by a fast depuration of B1, C1 + 2, dcSTX and dcGTX2 + 3, possibly due to the gut evacuation of unassimilated toxins or microalgae cells, or loss during digestive mechanisms. Subsequent enhancements were observed for all compounds with emphasis to dcSTX and dcGTX2 + 3, pointing to biotransformation of the assimilated toxins. Then levels decreased gradually. A first-order depuration kinetic model fitted well to the decrease of B1, C1 + 2 and dcGTX2 + 3 concentrations, but not for dcSTX. Mussels exposed to a declining bloom of Gymnodinium catenatum exhibited a loss of toxins following the same pattern. Despite the low abundance of this dinoflagellate, a similar kinetic model was applied to the field data. The depuration rate of dcGTX2 + 3 in the field experiment (0.153 ± 0.03 day(-1)) significantly exceeded the value calculated in the laboratory (0.053 ± 0.01 day(-1)), while smaller differences were obtained for B1 (0.071 ± 0.02 and 0.048 ± 0.01 day(-1)) and similar values for C1 + 2 (0.082 ± 0.03 and 0.080 ± 0.03 day(-1)). The slower depuration rate of dcGTX2 + 3 in the heavily contaminated mussels at the laboratory may be related to a more effective contribution of C1 + 2 biotransformation.

Prevalence, Variability and Bioconcentration of Saxitoxin-Group in Different Marine Species Present in the Food Chain

Science.gov (United States)

Oyaneder Terrazas, Javiera; Contreras, Héctor R.; García, Carlos

2017-01-01

The saxitoxin-group (STX-group) corresponds to toxic metabolites produced by cyanobacteria and dinoflagellates of the genera Alexandrium, Gymnodinium, and Pyrodinium. Over the last decade, it has been possible to extrapolate the areas contaminated with the STX-group worldwide, including Chile, a phenomenon that has affected ≈35% of the Southern Pacific coast territory, generating a high economic impact. The objective of this research was to study the toxicity of the STX-group in all aquatic organisms (bivalves, algae, echinoderms, crustaceans, tunicates, cephalopods, gastropods, and fish) present in areas with a variable presence of harmful algal blooms (HABs). Then, the toxic profiles of each species and dose of STX equivalents ingested by a 60 kg person from 400 g of shellfish were determined to establish the health risk assessment. The toxins with the highest prevalence detected were gonyautoxin-4/1 (GTX4/GTX1), gonyautoxin-3/2 (GTX3/GTX2), neosaxitoxin (neoSTX), decarbamoylsaxitoxin (dcSTX), and saxitoxin (STX), with average concentrations of 400, 2800, 280, 200, and 2000 µg kg−1 respectively, a species-specific variability, dependent on the evaluated tissue, which demonstrates the biotransformation of the analogues in the trophic transfer with a predominance of α-epimers in all toxic profiles. The identification in multiple vectors, as well as in unregulated species, suggests that a risk assessment and risk management update are required; also, chemical and specific analyses for the detection of all analogues associated with the STX-group need to be established. PMID:28604648

Prevalence, Variability and Bioconcentration of Saxitoxin-Group in Different Marine Species Present in the Food Chain

Directory of Open Access Journals (Sweden)

Javiera Oyaneder Terrazas

2017-06-01

Full Text Available The saxitoxin-group (STX-group corresponds to toxic metabolites produced by cyanobacteria and dinoflagellates of the genera Alexandrium, Gymnodinium, and Pyrodinium. Over the last decade, it has been possible to extrapolate the areas contaminated with the STX-group worldwide, including Chile, a phenomenon that has affected ≈35% of the Southern Pacific coast territory, generating a high economic impact. The objective of this research was to study the toxicity of the STX-group in all aquatic organisms (bivalves, algae, echinoderms, crustaceans, tunicates, cephalopods, gastropods, and fish present in areas with a variable presence of harmful algal blooms (HABs. Then, the toxic profiles of each species and dose of STX equivalents ingested by a 60 kg person from 400 g of shellfish were determined to establish the health risk assessment. The toxins with the highest prevalence detected were gonyautoxin-4/1 (GTX4/GTX1, gonyautoxin-3/2 (GTX3/GTX2, neosaxitoxin (neoSTX, decarbamoylsaxitoxin (dcSTX, and saxitoxin (STX, with average concentrations of 400, 2800, 280, 200, and 2000 µg kg−1 respectively, a species-specific variability, dependent on the evaluated tissue, which demonstrates the biotransformation of the analogues in the trophic transfer with a predominance of α-epimers in all toxic profiles. The identification in multiple vectors, as well as in unregulated species, suggests that a risk assessment and risk management update are required; also, chemical and specific analyses for the detection of all analogues associated with the STX-group need to be established.

Comparison of Escherichia coli Isolates from humans, food, and farm and companion animals for presence of Shiga toxin-producing E. coli virulence markers.

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Murinda, Shelton E; Nguyen, Lien T; Landers, Tippi L; Draughon, F Ann; Mathew, Alan G; Hogan, Joseph S; Smith, K Larry; Hancock, Dale D; Oliver, Stephen P

2004-01-01

The objective of this study was to characterize Escherichia coli isolates from dairy cows/feedlots, calves, mastitis, pigs, dogs, parrot, iguana, human disease, and food products for prevalence of Shiga toxin-producing E. coli (STEC) virulence markers. The rationale of the study was that, isolates of the same serotypes that were obtained from different sources and possessed the same marker profiles, could be cross-species transmissible. Multiplex polymerase chain reaction (PCR) was used to detect presence of genes encoding Shiga toxin 1 and 2 (stx1 and stx2), H7 flagella (flicC), enterohemolysin (hly) and intimin (eaeA) in E. coli isolates (n = 400). Shiga toxin-producing isolates were tested for production of Shiga toxins (Stx1 and Stx2 and enterohemolysin. Of the E. coli O157:H7/H- strains, 150 of 164 (mostly human, cattle, and food) isolates were stx+. Sixty-five percent of O157 STEC produced both Stx1 and Stx2; 32% and 0.7% produced Stx2 or Stx1, respectively. Ninety-eight percent of O157 STEC had sequences for genes encoding intimin and enterohemolysin. Five of 20 E. coli O111, 4 of 14 O128 and 4 of 10 O26 were stx+ . Five of 6 stx+ O26 and O111 produced Stx1, however, stx+ O128 were Stx-negative. Acid resistance (93.3%) and tellurite resistance (87.3%) were common attributes of O157 STEC, whereas, non-O157 stx+ strains exhibited 38.5% and 30.8% of the respective resistances. stx-positive isolates were mostly associated with humans and cattle, whereas, all isolates from mastitis (n = 105), and pigs, dogs, parrot and iguanas (n = 48) were stx-negative. Multiplex PCR was an effective tool for characterizing STEC pathogenic profiles and distinguished STEC O157:H7 from other STEC. Isolates from cattle and human disease shared similar toxigenic profiles, whereas isolates from other disease sources had few characteristics in common with the former isolates. These data suggest interspecies transmissibility of certain serotypes, in particular, STEC O157:H7, between

Identification of genes expressed in cultures of E. coli lysogens carrying the Shiga toxin-encoding prophage Φ24B

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Riley Laura M

2012-03-01

Full Text Available Abstract Background Shigatoxigenic E. coli are a global and emerging health concern. Shiga toxin, Stx, is encoded on the genome of temperate, lambdoid Stx phages. Genes essential for phage maintenance and replication are encoded on approximately 50% of the genome, while most of the remaining genes are of unknown function nor is it known if these annotated hypothetical genes are even expressed. It is hypothesized that many of the latter have been maintained due to positive selection pressure, and that some, expressed in the lysogen host, have a role in pathogenicity. This study used Change Mediated Antigen Technology (CMAT™ and 2D-PAGE, in combination with RT-qPCR, to identify Stx phage genes that are expressed in E. coli during the lysogenic cycle. Results Lysogen cultures propagated for 5-6 hours produced a high cell density with a low proportion of spontaneous prophage induction events. The expression of 26 phage genes was detected in these cultures by differential 2D-PAGE of expressed proteins and CMAT. Detailed analyses of 10 of these genes revealed that three were unequivocally expressed in the lysogen, two expressed from a known lysogenic cycle promoter and one uncoupled from the phage regulatory network. Conclusion Propagation of a lysogen culture in which no cells at all are undergoing spontaneous lysis is impossible. To overcome this, RT-qPCR was used to determine gene expression profiles associated with the growth phase of lysogens. This enabled the definitive identification of three lambdoid Stx phage genes that are expressed in the lysogen and seven that are expressed during lysis. Conservation of these genes in this phage genome, and other Stx phages where they have been identified as present, indicates their importance in the phage/lysogen life cycle, with possible implications for the biology and pathogenicity of the bacterial host.

Shiga Toxin-Producing Escherichia coli in Plateau Pika (Ochotona curzoniae) on the Qinghai-Tibetan Plateau, China.

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Bai, Xiangning; Zhang, Wang; Tang, Xinyuan; Xin, Youquan; Xu, Yanmei; Sun, Hui; Luo, Xuelian; Pu, Ji; Xu, Jianguo; Xiong, Yanwen; Lu, Shan

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) are an emerging group of zoonotic pathogens. Ruminants are the natural reservoir of STEC. In this study we determined the prevalence and characteristics of the STEC in plateau pika (Ochotona curzoniae) on the Qinghai-Tibetan Plateau, China. A total of 1116 pika samples, including 294 intestinal contents samples, 317 fecal samples, and 505 intestinal contents samples, were collected from May to August in the years 2012, 2013, and 2015, respectively. Twenty-one samples (1.88%) yielded at least one STEC isolate; in total, 22 STEC isolates were recovered. Thirteen different O serogroups and 14 serotypes were identified. One stx 1 subtype (stx 1a) and three stx 2 subtypes (stx 2a, stx 2b, and stx 2d) were present in the STEC isolates. Fifteen, fourteen, and three STEC isolates harbored the virulence genes ehxA, subA, and astA, respectively. Adherence-associated genes iha and saa were, respectively, present in 72.73 and 68.18% of the STEC isolates. Twenty antibiotics were active against all the STEC isolates; all strains were resistant to penicillin G, and some to cephalothin or streptomycin. The 22 STEC isolates were divided into 16 pulsed-field gel electrophoresis patterns and 12 sequence types. Plateau pikas may play a role in the ongoing circulation of STEC in the Qinghai-Tibetan plateau. This study provides the first report on STEC in plateau pikas and new information about STEC reservoirs in wildlife. Based on the serotypes, virulence gene profiles and multi-locus sequence typing (MLST) analysis, the majority of these pika STECs may pose a low public health risk.

Isolation and Characterization of Plantaricin Produced by Lactobacillus plantarum Strains (IIA-1A5, IIA-1B1, IIA-2B2

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I. I. Arief

2013-08-01

Full Text Available Bacteriocins produced by Indonesian lactic acid bacteria Lactobacillus plantarum IIA-1A5, IIA-1B1, IIA-2B2 were purified and characterized. Plantaricin W gene had been successfully amplified from all strains. This amplicon showed the expected 200 bp size of plantaricin W gene. This bacteriocins purified from L. plantarum IIA-1A5, IIA-1B1, and IIA-2B2 were named plantaricin IIA-1A5, IIA-1B1, and IIA-2B2. Purification by cation exchange chromatography increased the purity (fold and activity of plantaricins. Purity of plantaricin IIA-1A5 was increased by 3.13 fold with specific activity 13.40 AU/mg. Plantaricin IIA-1B1 had 2.98 fold purity with specific activity 5.12 AU/mg, while purity of plantaricin IIA-2B2 was 1.37 fold with specific activity 7.70 AU/mg. All plantaricins could inhibit the growth of pathogenic bacteria, such as Escherichia coli, Salmonella typhimurium, Bacillus cereus, and Staphylococcus aureus. Plantaricins could be digested by trypsin. Stability of plantaricins at 80 oC for 30 min and at 121 oC for 15 min were affected by type of plantaricin and species of pathogenic bacteria. Generally, plantaricin IIA-1A5 was better as antimicrobial agent than plantaricin IIA-1B1 and plantaricin IIA-2B2.

Failure of manganese to protect from Shiga toxin.

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Marsha A Gaston

Full Text Available Shiga toxin (Stx, the main virulence factor of Shiga toxin producing Escherichia coli, is a major public health threat, causing hemorrhagic colitis and hemolytic uremic syndrome. Currently, there are no approved therapeutics for these infections; however manganese has been reported to provide protection from the Stx1 variant isolated from Shigella dysenteriae (Stx1-S both in vitro and in vivo. We investigated the efficacy of manganese protection from Stx1-S and the more potent Stx2a isoform, using experimental systems well-established for studying Stx: in vitro responses of Vero monkey kidney cells, and in vivo toxicity to CD-1 outbred mice. Manganese treatment at the reported therapeutic concentration was toxic to Vero cells in culture and to CD-1 mice. At lower manganese concentrations that were better tolerated, we observed no protection from Stx1-S or Stx2a toxicity. The ability of manganese to prevent the effects of Stx may be particular to certain cell lines, mouse strains, or may only be manifested at high, potentially toxic manganese concentrations.

Host cell interactions of outer membrane vesicle-associated virulence factors of enterohemorrhagic Escherichia coli O157: Intracellular delivery, trafficking and mechanisms of cell injury

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Greune, Lilo; Jarosch, Kevin-André; Steil, Daniel; Zhang, Wenlan; He, Xiaohua; Lloubes, Roland; Fruth, Angelika; Kim, Kwang Sik; Schmidt, M. Alexander; Dobrindt, Ulrich; Mellmann, Alexander; Karch, Helge

2017-01-01

Outer membrane vesicles (OMVs) are important tools in bacterial virulence but their role in the pathogenesis of infections caused by enterohemorrhagic Escherichia coli (EHEC) O157, the leading cause of life-threatening hemolytic uremic syndrome, is poorly understood. Using proteomics, electron and confocal laser scanning microscopy, immunoblotting, and bioassays, we investigated OMVs secreted by EHEC O157 clinical isolates for virulence factors cargoes, interactions with pathogenetically relevant human cells, and mechanisms of cell injury. We demonstrate that O157 OMVs carry a cocktail of key virulence factors of EHEC O157 including Shiga toxin 2a (Stx2a), cytolethal distending toxin V (CdtV), EHEC hemolysin, and flagellin. The toxins are internalized by cells via dynamin-dependent endocytosis of OMVs and differentially separate from vesicles during intracellular trafficking. Stx2a and CdtV-B, the DNase-like CdtV subunit, separate from OMVs in early endosomes. Stx2a is trafficked, in association with its receptor globotriaosylceramide within detergent-resistant membranes, to the Golgi complex and the endoplasmic reticulum from where the catalytic Stx2a A1 fragment is translocated to the cytosol. CdtV-B is, after its retrograde transport to the endoplasmic reticulum, translocated to the nucleus to reach DNA. CdtV-A and CdtV-C subunits remain OMV-associated and are sorted with OMVs to lysosomes. EHEC hemolysin separates from OMVs in lysosomes and targets mitochondria. The OMV-delivered CdtV-B causes cellular DNA damage, which activates DNA damage responses leading to G2 cell cycle arrest. The arrested cells ultimately die of apoptosis induced by Stx2a and CdtV via caspase-9 activation. By demonstrating that naturally secreted EHEC O157 OMVs carry and deliver into cells a cocktail of biologically active virulence factors, thereby causing cell death, and by performing first comprehensive analysis of intracellular trafficking of OMVs and OMV-delivered virulence factors

Immunogenicity, reactogenicity, and safety of a P1.7b,4 strain-specific serogroup B meningococcal vaccine given to preteens.

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Hosking, Jamie; Rasanathan, Kumanan; Mow, Florina Chan; Jackson, Catherine; Martin, Diana; O'Hallahan, Jane; Oster, Philipp; Ypma, Ellen; Reid, Stewart; Aaberge, Ingeborg; Crengle, Sue; Stewart, Joanna; Lennon, Diana

2007-11-01

New Zealand (NZ) has experienced a Neisseria meningitidis serogroup B epidemic since 1991. MeNZB, a strain-specific outer membrane vesicle vaccine made using an NZ epidemic strain isolate, NZ98/254 (B:4:P1.7b,4), from two manufacturing sites, the Norwegian Institute of Public Health (NIPH) and Chiron Vaccines (CV; now Novartis), was evaluated for safety, immunogenicity, and reactogenicity in this observer-blind trial with 8- to 12-year-old children. In year 1, cohort A (n = 302) was randomized 4:1 for receipt of NIPH-MeNZB or MenBvac (Norwegian parent vaccine strain 44/76; B:15:P1.7,16). In year 2, cohort B (n = 313) was randomized 4:1 for receipt of CV-MeNZB or NIPH-MeNZB. Participants all received three vaccinations 6 weeks apart. Local and systemic reactions were monitored for 7 days. Seroresponse was defined as a fourfold or greater rise in the serum bactericidal antibody titer from the baseline titer as measured by a serum bactericidal assay. Those with baseline titers of /=1:8 to serorespond. Intention-to-treat (ITT) and per protocol (PP) analyses are presented. In cohort A, 74% (ITT) and 73% (PP) of NIPH-MeNZB recipients demonstrated seroresponses against NZ98/254 after three doses, versus 32% (ITT and PP) of MenBvac recipients. In cohort B, seroresponses against NZ98/254 after three doses occurred in 79% (ITT and PP) of CV-MeNZB versus 75% (ITT) and 76% (PP) of NIPH-MeNZB recipients. Vaccines were tolerable, with no vaccine-related serious adverse events. In conclusion, the NZ strain meningococcal B vaccine (MeNZB) from either manufacturing site was immunogenic against New Zealand epidemic vaccine strain meningococci with no safety concerns when given in three doses to these 8- to 12-year-old children.

Clinical significance of combined determination of the changes of the serum ADA, HA and PIIIP levels in patients with hepatitis B

International Nuclear Information System (INIS)

Wang Xiaoyan

2009-01-01

Objective: To explore the clinical significance of the changes of serum ADA, HA and PIIIP levels in patients with hepatitis B. Methods: Serum adenosine deaminase (ADA) (with biochemistry) and HA PIIIP (with RIA) levels were determined in 102 patients with hepatitis B of various stages and 35 controls. Results: Serum ADA, HA and PIIIP levels in patients with hepatitis B were significantly higher than those in controls (P<0.01). Particularly in patients with cirrhosis (n=13) (P<0.001). Conclusion: The serum ADA, HA and PIIIP levels were closely related to the diseases process of hepatitis B and were of great clinical values. (authors)

Racial difference in serum Vitamin B12 levels

International Nuclear Information System (INIS)

Kwee, H.G.; Bowman, H.S.; Wells, L.W.

1985-01-01

Measurements of the serum Vitamin B 12 concentrations of 49 black and 49 white healthy adults demonstrate a significantly higher mean serum Vitamin B 12 level in blacks when compared to whites. The reason for this difference appears to be genetic, although environmental factors may also be involved. It is suggested that clinical laboratories should establish their own separate reference values of serum Vitamin B 12 for blacks and whites in order to prevent misinterpretation of test results

Serum Vitamin B12 Levels in Type 2 Diabetes Patients on Metformin Compared to those Never on Metformin: A Cross-sectional Study.

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Raizada, Nishant; Jyotsna, Viveka P; Sreenivas, Vishnubhatla; Tandon, Nikhil

2017-01-01

There are limited data about the effect of metformin use on serum Vitamin B12 levels in type 2 diabetes patients from India. We studied serum Vitamin B12 levels in patients with type 2 diabetes mellitus who were receiving metformin and compared them to those never treated with metformin. A total of 183 patients ("metformin" group 121, "no metformin" group 63) of type 2 diabetes from the endocrinology clinic of a tertiary care center in North India were studied. Serum Vitamin B12 levels were measured in all patients. Diabetic neuropathy symptom score (DNS) and diabetic neuropathy examination score (DNE) were used to assess peripheral neuropathy while hemoglobin and mean corpuscular volume (MCV) were used to assess anemia. The serum Vitamin B12 levels were 267.7 ± 194.4 pmol/l in metformin group and 275.1 ± 197.2 pmol/l in the no metformin group ( P = 0.78). When adjusted for duration of diabetes, metformin use was associated with a 87.7 ± 37.7 pmol/l (95% confidence interval [CI], -162.1--3.3, P = 0.02) lower serum Vitamin B12 levels. No significant increase in the prevalence of neuropathy (DNS and DNE scores), anemia, or MCV was found in the Vitamin B12 deficient patients (levels <150 pmol/l) as compared to patients with normal Vitamin B12. However, serum Vitamin B12 levels for the entire cohort were higher by 12.2 ± 3.0 pmol/l (95% CI 6.4-18.0, P < 0.001) for every 1 year increase in the duration of diabetes. Metformin use was associated with a lower serum Vitamin B12 levels when adjusted for duration of diabetes. Increasing duration of diabetes was associated with higher serum Vitamin B12 levels.

Impact of line parameter database, continuum absorption, full grind configuration, and L1B update on GOSAT TIR methane retrieval

Science.gov (United States)

Yamada, A.; Saitoh, N.; Nonogaki, R.; Imasu, R.; Shiomi, K.; Kuze, A.

2016-12-01

The thermal infrared (TIR) band of Thermal and Near-infrared Sensor for Carbon Observation Fourier Transform Spectrometer (TANSO-FTS) onboard Greenhouse Gases Observing Satellite (GOSAT) observes CH4 profile at wavenumber range from 1210 cm-1 to 1360 cm-1 including CH4 ν4 band. The current retrieval algorithm (V1.0) uses LBLRTM V12.1 with AER V3.1 line database to calculate optical depth. LBLRTM V12.1 include MT_CKD 2.5.2 model to calculate continuum absorption. The continuum absorption has large uncertainty, especially temperature dependent coefficient, between BPS model and MT_CKD model in the wavenumber region of 1210-1250 cm-1(Paynter and Ramaswamy, 2014). The purpose of this study is to assess the impact on CH4 retrieval from the line parameter databases and the uncertainty of continuum absorption. We used AER v1.0 database, HITRAN2004 database, HITRAN2008 database, AER V3.2 database, and HITRAN2012 database (Rothman et al. 2005, 2009, and 2013. Clough et al., 2005). AER V1.0 database is based on HITRAN2000. The CH4 line parameters of AER V3.1 and V3.2 databases are developed from HITRAN2008 including updates until May 2009 with line mixing parameters. We compared the retrieved CH4 with the HIPPO CH4 observation (Wofsy et al., 2012). The difference of AER V3.2 was the smallest and 24.1 ± 45.9 ppbv. The differences of AER V1.0, HITRAN2004, HITRAN2008, and HITRAN2012 were 35.6 ± 46.5 ppbv, 37.6 ± 46.3 ppbv, 32.1 ± 46.1 ppbv, and 35.2 ± 46.0 ppbv, respectively. Compare AER V3.2 case to HITRAN2008 case, the line coupling effect reduced difference by 8.0 ppbv. Median values of Residual difference from HITRAN2008 to AER V1.0, HITRAN2004, AER V3.2, and HITRAN2012 were 0.6 K, 0.1 K, -0.08 K, and 0.08 K, respectively, while median values of transmittance difference were less than 0.0003 and transmittance differences have small wavenumber dependence. We also discuss the retrieval error from the uncertainty of the continuum absorption, the test of full grid

The clinical significance of detection of serum Pre-S1 antigen

International Nuclear Information System (INIS)

Jiang Xuehua; Huang Zhuqing; Han Yi; Gong Shoujun

2003-01-01

To study the clinical significance of detection of serum Pre-S 1 Ag, the serum Pre-S 1 Ag, HBV-marks and HBV-DNA were detected in 338 patients with hepatitis B. The positive rate and the relationship between them were analyzed and compared. In 338 patients, the positive rate of serum Pre-S 1 Ag, HBeAg, HBV-DNA was 63.02%, 48.52%, 68.05% respectively, and the co-positive rate of Pre-S 1 Ag with HBV-DNA, HBeAg was 78.56%, 81.17% respectively. There was a significant correlation between Pre-S 1 Ag, HBeAg and HBV-DNA (P 1 Ag could well reflect the reproductive status of hepatitis B virus, and so it could be used as the clinical marker of the reproductive status of hepatitis B virus

Stronger relationship of serum apolipoprotein A-1 and B with diabetic retinopathy than traditional lipids

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B S Ankit

2017-01-01

Full Text Available Aim: Diabetic retinopathy (DR is the most common preventable cause of blindness where early detection and treatment can be sight-saving. Search for biomarkers of the disease has been relentless. We aimed to determine whether lipoproteins apolipoproteins A1 and B1 (Apo-A1 and Apo-B1 have stronger associations with DR in contrast to conventionally measured low-density lipoprotein (LDL and high-density lipoprotein cholesterol levels. Materials and Methods: We performed a cross-sectional study and studied 117 patients. Serum lipid profile was assessed by autoanalyzer. Serum Apo-A1 and Apo-B were measured using immunoturbidimetric kit on an autoanalyzer. Apo-B/A1 ratio was calculated. Retinopathy was graded from the digital retinal photographs, taken with nonmydriatic auto fundus camera and classified according to International Clinical DR Disease Severity Scale. Results: Mean Apo-A1 for mild, moderate, severe retinopathy, and proliferative DR (PDR shows a significant negative correlation (P = 0.001 with severity of retinopathy. Mean Apo-B for mild, moderate, severe, PDR displayed a significant positive correlation with severity of retinopathy (P = 0.001. Mean Apo-B/A1 for mild, moderate, severe, PDR showed highly significant positive correlation with severity of retinopathy (P < 0.001. In contrast, mean LDL for mild, moderate, severe, PDR showed insignificant association with severity of DR (P = 0.081. Conclusion: Apo-A1 and Apo-B have a stronger association with the development of DR than traditional lipids and can thus facilitate early detection and treatment of the disease.

Detection of Shiga toxins genes by Multiplex PCR in clinical samples

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2013-09-01

Full Text Available Background: Different methods have been used for detection of shiga toxins; such as,  cell culture, ELISA, and RFPLA. However, all of these methods suffer from high cost, time-consumption and relatively low sensitivity. In this study we used Multiplex PCR method for detection of genes encoding shiga toxins. Material and Methods: In this study, 63 clinical samples were obtained from positive cultures of Shigella and E. coli O157, from Bahman 1391 until Ordibehesht 1392 in Mazandaran province. Initial confirmation of shiga toxins producing bacteria was performed by biochemical and serological methods. After DNA extraction, detection of stx1 and stx2 genes was accomplished by multiplex PCR.  For confirmation of the PCR amplicon, DNA sequencing was used. Antibiotic sensitivity tests were performed by disk diffusion method. Results:  Among the positive strains, 13 strains contained stx2 genes, 4 strains contained Stx/Stx1 genes and 4 strains harbored both Stx/Stx1 and Stx2. The DNA extracted from other Gram-negative bacteria was not protected by the relevant parts of these toxins. Sequencing of the amplified fragments indicated the correct toxin sequences.  The sensitivity for identification of Stx/Stx1 gene was 1.56 pg/ µl and for Stx2 was 1.08 pg/µl. The toxin positive strains were all sensitive to Cefixime, Gentamicin, Amikacin, Ceftriaxone, and Nitrofurantoin. Conclusion: This method is fast and accurate for detection of bacteria producing shiga toxin and can be used to identify different types of shiga toxin.

Serum inhibin B in healthy pubertal and adolescent boys

DEFF Research Database (Denmark)

Andersson, A M; Juul, A; Petersen, J H

1997-01-01

Inhibin B levels were measured in serum from 400 healthy Danish prepubertal, pubertal, and adolescent males, aged 6-20 yr, in a cross-sectional study using a recently developed immunoassay that is specific for inhibin B, the physiologically important inhibin form in men. In addition, serum levels...

Aflatoxin B1 use in radioimmunoassay

International Nuclear Information System (INIS)

Liu Yinkun; Zhang Xiaying; Chen Ruiqun; Gu Tianjue

1987-01-01

Antibodies against Aflatoxin B 1 (AFB 1 ) were obtained after multiple-site injections of bovine serum albumin-AFB 1 conjugate into rabbits. The greatest specific binding effciency of antibody for AFB 1 is 70∼80%. The sensitivity of radioimmunoassay (RIA) for AFB 1 is between 0.46∼2.3 ng. The retrievel rate of AFB 1 by RIA from intentionally contaminated serum and rice is between 70∼80%. Detailed methods for the preparation of conjugate, immune serum, and methods for antibody titer determination are described

The EGG 57-CO B-12 absorption test, in the evaluation of patients with low serum B-12

International Nuclear Information System (INIS)

Sinow, R.M.; Carmel, R.; Siegel, M.E.

1985-01-01

The Schilling Test (ST) is the standard test for diagnosing vitamin B-12 malabsorption (MA). However, patients with subtle gastric dysfunction may have normal ST, but impaired absorption of B-12 given with food. The authors have adapted an Egg B-12 Absorption Test (EBAT) in which 57-Co cyanocobalamin (57-Co B-12) is mixed with scrambled egg to evaluate this phenomenon in patients with low serum B-12, normal ST and possible subtle MA. Lyophilized egg yolk is reconstituted and mixed with 57-Co- B-12 of equal dose to that of ST. The authors studied 46 individuals: 13 controls, 5 patients with known pernicious anemia (PA), in addition to 28 patients with low serum B-12 levels and normal ST. ST/EBAT ratios were calculated. Twenty-eight test patients excreted 13.3% on ST and 3.5% on EBAT. Mean ST/EBAT ratio was 8.2 (1.4-35.9). Five had EBAT results in the PA range (<1%) and ST/EBAT ratios (14.4-35.9) that were significantly elevated. This group is also evaluated with pepsinogen I/II ratios, gastric analysis, deoxyuridine suppression tests, anti intrinsic factor, and antiparietal cell antibodies. The authors' results indicate that the EBAT can differentiate between PA and non PA patients, and that some patients with low serum B-12 levels and normal ST may, in fact, have subtle MA. The EBAT, combined with ST/EBAT ratio, may provide a means for identifying this group of patients

Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes Are Strong Determinants of the Serum Triglyceride and Cholesterol Levels of Japanese Alcoholic Men.

Science.gov (United States)

Yokoyama, Akira; Yokoyama, Tetsuji; Matsui, Toshifumi; Mizukami, Takeshi; Kimura, Mitsuru; Matsushita, Sachio; Higuchi, Susumu; Maruyama, Katsuya

2015-01-01

Elevated serum triglyceride (TG) and high-density-lipoprotein cholesterol (HDL-C) levels are common in drinkers. The fast-metabolizing alcohol dehydrogenase-1B encoded by the ADH1B*2 allele (vs. ADH1B*1/*1 genotype) and inactive aldehyde dehydrogenase-2 encoded by the ALDH2*2 allele (vs. ALDH2*1/*1 genotype) modify ethanol metabolism and are prevalent (≈90% and ≈40%, respectively) in East Asians. We attempted to evaluate the associations between the ADH1B and ALDH2 genotypes and lipid levels in alcoholics. The population consisted of 1806 Japanese alcoholic men (≥40 years) who had undergone ADH1B and ALDH2 genotyping and whose serum TG, total cholesterol, and HDL-C levels in the fasting state had been measured within 3 days after admission. High serum levels of TG (≥150 mg/dl), HDL-C (>80 mg/dl), and low-density-lipoprotein cholesterol (LDL-C calculated by the Friedewald formula ≥140 mg/dl) were observed in 24.3%, 16.8%, and 15.6%, respectively, of the subjects. Diabetes, cirrhosis, smoking, and body mass index (BMI) affected the serum lipid levels. Multivariate analysis revealed that the presence of the ADH1B*2 allele and the active ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) for a high TG level (2.22 [1.67-2.94] and 1.39 [0.99-1.96], respectively), and decreased the OR for a high HDL-C level (0.37 [0.28-0.49] and 0.51 [0.37-0.69], respectively). The presence of the ADH1B*2 allele decreased the OR for a high LDL-C level (0.60 [0.45-0.80]). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs for high TG levels and lowest OR for a high HDL-C level. The genotype effects were more prominent in relation to the higher levels of TG (≥220 mg/dl) and HDL-C (≥100 mg/dl). The fast-metabolizing ADH1B and active ALDH2, and especially a combination of the two were strongly associated with higher serum TG levels and lower serum HDL-C levels of alcoholics. The fast-metabolizing ADH1B was associated with lower serum LDL

Alcohol Dehydrogenase-1B (rs1229984 and Aldehyde Dehydrogenase-2 (rs671 Genotypes Are Strong Determinants of the Serum Triglyceride and Cholesterol Levels of Japanese Alcoholic Men.

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Akira Yokoyama

Full Text Available Elevated serum triglyceride (TG and high-density-lipoprotein cholesterol (HDL-C levels are common in drinkers. The fast-metabolizing alcohol dehydrogenase-1B encoded by the ADH1B*2 allele (vs. ADH1B*1/*1 genotype and inactive aldehyde dehydrogenase-2 encoded by the ALDH2*2 allele (vs. ALDH2*1/*1 genotype modify ethanol metabolism and are prevalent (≈90% and ≈40%, respectively in East Asians. We attempted to evaluate the associations between the ADH1B and ALDH2 genotypes and lipid levels in alcoholics.The population consisted of 1806 Japanese alcoholic men (≥40 years who had undergone ADH1B and ALDH2 genotyping and whose serum TG, total cholesterol, and HDL-C levels in the fasting state had been measured within 3 days after admission.High serum levels of TG (≥150 mg/dl, HDL-C (>80 mg/dl, and low-density-lipoprotein cholesterol (LDL-C calculated by the Friedewald formula ≥140 mg/dl were observed in 24.3%, 16.8%, and 15.6%, respectively, of the subjects. Diabetes, cirrhosis, smoking, and body mass index (BMI affected the serum lipid levels. Multivariate analysis revealed that the presence of the ADH1B*2 allele and the active ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval for a high TG level (2.22 [1.67-2.94] and 1.39 [0.99-1.96], respectively, and decreased the OR for a high HDL-C level (0.37 [0.28-0.49] and 0.51 [0.37-0.69], respectively. The presence of the ADH1B*2 allele decreased the OR for a high LDL-C level (0.60 [0.45-0.80]. The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs for high TG levels and lowest OR for a high HDL-C level. The genotype effects were more prominent in relation to the higher levels of TG (≥220 mg/dl and HDL-C (≥100 mg/dl.The fast-metabolizing ADH1B and active ALDH2, and especially a combination of the two were strongly associated with higher serum TG levels and lower serum HDL-C levels of alcoholics. The fast-metabolizing ADH1B was associated with lower serum LDL

Impact of Chronic Hepatitis C Virus Genotype 1b Infection on Triglyceride Concentration in Serum Lipoprotein Fractions

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Tomohisa Nagano

2015-08-01

Full Text Available Reduced low-density lipoprotein (LDL cholesterol level is a characteristic feature of dyslipidemia in chronic hepatitis C virus (HCV infection. However, abnormality in serum triglyceride (TG has not been fully investigated. To clarify the impact of HCV genotype 1b (G1b infection and advanced fibrosis on serum TG profiles, TG concentrations in lipoprotein fractions were examined in fasting sera from 185 subjects with active or cleared HCV infection by high-performance liquid chromatography. Serum lipoproteins were fractionated into four classes: chylomicron, very low-density lipoprotein (VLDL, LDL, and high-density lipoprotein (HDL. Then, the significance of HCV G1b infection on TG levels in each lipoprotein fraction was determined using multiple regression models. We found that active HCV G1b infection was positively associated with high HDL-TG levels and low VLDL-TG levels, independent of other factors included in the regression model. In VLDL sub-fractions, active HCV infection was only found to be associated with low levels of large VLDL-TG. Similarly, advanced liver fibrosis in chronic HCV G1b infection was associated with high levels of LDL-TG, HDL-TG, and small VLDL-TG, independent of other clinical factors. These findings indicate that active HCV G1b infection and advanced fibrosis are closely associated with abnormal serum TG profiles.

Quantitative Measurement of Serum Hepatitis B Surface Antigen Using an Immunoradiometric Assay in Chronic Hepatitis B

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Kwon, Hyun Woo; Lee, Ho Young; Kim, Seog Gyun; Kim, Won; Jung, Wong Jin; Kang, Keon Wook; Chung, June Key; Lee, Myung Chul; Lee, Dong Soo [Seoul National Univ. Seoul (Korea, Republic of)

2011-03-15

Measurement of serum hepatitis B virus surface antigen (HBsAg) levels is important for the management of chronic hepatitis D patients in terms of monitoring response to antiviral therapy. This study aimed to evaluate the diagnostic performance of a new diagnostic kit, which quantitatively measures serum HBsAg level using an immunoradiometric assay (IRMA) based method. Measurements were compared with those obtained using a chemiluminescent microparticle immunoassay (CMIA) based method. The blood samples of 96 patients with chronic hepatitis B were used in this study. Copy numbers of serum hepatitis B virus (HBV) DNA were determined in 23 of these samples. The correlation between and the concordance of IRMA and CMIA results were determined using Pearson's correlation coefficients. P values of 0.05 were considered to be statistically significant throughout. Laboratory diagnoses based on CMIA. Furthermors, serum HBsAg levels by IRMA were found to be highly correlated with those determined by CMIA (correlation coefficient R{sup 2=}0.838, P<0.001). Serum HBsAg level and serum HBV DNA copies were found to be linearly related by both methods (R{sup 2=}0.067, P=0.316 by IRMA, and R{sup 2=}0.101, P=0.215 by CMIA). The diagnostic performance of the investigated IRMA method of determining HBsAg levels was found to be comparable with that of a CMIA based method in chronic hepatitis B patients.

Clinical significance of determination of plasma Leptin and serum Hcy, S100B and NSE levels in patients with alzheimer disease

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Dou Huanzhi; Lu Meng

2011-01-01

To explore the clinical significance of changes of plasma leptin and serun Hcy, S100B and NSE levels in patients with Alzheimer Disease (AD). The plasma leptin and serum NSE levels in 32 AD patients and 30 controls were determined by using RIA, and the serum Hcy and S100B levels were measured by using CLIA. The results showed that the plasma leptin and serun Hcy, S100B and NSE levels in AD patients were significantly higher than these in controls (P<0.01). The plasma leptin levels in AD patients was mutually positively correlated with serum Hcy, S100B and NSE levels (r=0.5982, 0.4762, 0.6014, P<0.01). The detection of plasma leptin and serum Hcy, S100B and NSE levels may be helpful for the prediction of treatment efficiency in patients with Alzheimer disease. (authors)

Dynamic change of serum protein S100b and its clinical significance in patients with traumatic brain injury

Institute of Scientific and Technical Information of China (English)

CHEN Da-qing; ZHU Lie-lie

2005-01-01

Objective: To analyze the dynamic change of serum protein S100b in patients with traumatic brain injury and its clinical value in assessing brain damage. Methods: According to Glasgow coma scale (GCS), 102 cases of traumatic brain injury were divided into mild brain injury group (GCS≥13, n=31, Group A), moderate brain injury group (8B) and severe brain injury group (GCS≤8, n=34, Group C). Serial S100b concentrations were analyzed by enzyme-linked immunosorbent assay (ELISA) in blood samples taken on admission, 12 h, 24 h, 48 h, 72 h and 7 days after traumatic brain injury. Results: The severe brain injury group showed significantly higher concentration of serum S100b, with earlier increase and longer duration, than the mild and moderate brain injury groups. The patients with higher S100b exhibited lower GCS scores and poor clinical prognosis. The increase in S100b could emerge before clinical image evidence indicated so. Conclusions: Serum S100b can be used as a sensitive index for assessment and prediction of traumatic brain injury severity and prognosis.

Quantitative Measurement of Serum Hepatitis B Surface Antigen Using an Immunoradiometric Assay in Chronic Hepatitis B

International Nuclear Information System (INIS)

Kwon, Hyun Woo; Lee, Ho Young; Kim, Seog Gyun; Kim, Won; Jung, Wong Jin; Kang, Keon Wook; Chung, June Key; Lee, Myung Chul; Lee, Dong Soo

2011-01-01

Measurement of serum hepatitis B virus surface antigen (HBsAg) levels is important for the management of chronic hepatitis D patients in terms of monitoring response to antiviral therapy. This study aimed to evaluate the diagnostic performance of a new diagnostic kit, which quantitatively measures serum HBsAg level using an immunoradiometric assay (IRMA) based method. Measurements were compared with those obtained using a chemiluminescent microparticle immunoassay (CMIA) based method. The blood samples of 96 patients with chronic hepatitis B were used in this study. Copy numbers of serum hepatitis B virus (HBV) DNA were determined in 23 of these samples. The correlation between and the concordance of IRMA and CMIA results were determined using Pearson's correlation coefficients. P values of 0.05 were considered to be statistically significant throughout. Laboratory diagnoses based on CMIA. Furthermors, serum HBsAg levels by IRMA were found to be highly correlated with those determined by CMIA (correlation coefficient R 2= 0.838, P 2= 0.067, P=0.316 by IRMA, and R 2= 0.101, P=0.215 by CMIA). The diagnostic performance of the investigated IRMA method of determining HBsAg levels was found to be comparable with that of a CMIA based method in chronic hepatitis B patients

Effects of Irbesartan combined Atorvastatin on serum levels of Cys C, Hcy, TNF-α, ET, TGF-b1 in patients with early diabetic nephropathyn

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Abudula.Reziwanguli

2017-10-01

Full Text Available Objective: To observe the effects of Irbesartan combined with Atorvastatin on early diabetic nephropathy patients’ serum Cys C, Hcy, TNF-α, ET and TGF-b1 levels. Methods: A total of 60 early diabetic nephropathy patients were randomly divided into observation group (30 cases and control group (30 cases. Observation group: Irbesartan combined with Atorvastatin; control group: patients were treated only by Irbesartan. Recording and comparing the levels of Cys C, Hcy, TNF-α, ET and TGF-b1 before and after treatment. Results: (1 Before treatment, there was no statistically significant difference in the serum FBG, TG, Scr, BUN levels between the two groups. After treatment, compared with the same group before treatment, the serum TG, Scr, BUN levels of the two groups were significantly lower, and those levels of observation group were significantly better than the control group, the difference between two groups was statistically significant; (2 Before treatment, there was no statistically significant difference in the serum Cys C, Hcy, TNF-α, ET, TGF-b1 levels between the two groups. After treatment, compared with the same group before treatment, the serum Cys C, Hcy, TNF-α, ET, TGF-b1 levels of the two groups were significantly lower, and those levels of observation group were significantly better than the control group, the difference between two groups was statistically significant. Conclusion: Irbesartan combined with Atorvastatin for early diabetic nephropathy patients can reduce the levels of serum Cys C, Hcy, TNF-α, ET, TGF-b1 and be beneficial to protect their nephritic function.

Effects of subinhibitory concentrations of antimicrobial agents on Escherichia coli O157:H7 Shiga toxin release and role of the SOS response.

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Nassar, Farah J; Rahal, Elias A; Sabra, Ahmad; Matar, Ghassan M

2013-09-01

Treatment of Escherichia coli O157:H7 by certain antimicrobial agents often exacerbates the patient's condition by increasing either the release of preformed Shiga toxins (Stx) upon cell lysis or their production through the SOS response-triggered induction of Stx-producing prophages. Recommended subinhibitory concentrations (sub-MICs) of azithromycin (AZI), gentamicin (GEN), imipenem (IMI), and rifampicin (RIF) were evaluated in comparison to norfloxacin (NOR), an SOS-inducer, to assess the role of the SOS response in Stx release. Relative expression of recA (SOS-inducer), Q (late antitermination gene of Stx-producing prophage), stx1, and stx2 genes was assessed at two sub-MICs of the antimicrobials for two different strains of E. coli O157:H7 using reverse transcription-real-time polymerase chain reaction. Both strains at the two sub-MICs were also subjected to Western blotting for LexA protein expression and to reverse passive latex agglutination for Stx detection. For both strains at both sub-MICs, NOR and AZI caused SOS-induced Stx production (high recA, Q, and stx2 gene expression and high Stx2 production), so they should be avoided in E. coli O157:H7 treatment; however, sub-MICs of RIF and IMI induced Stx2 production in an SOS-independent manner except for one strain at the first twofold dilution below MIC of RIF where Stx2 production decreased. Moreover, GEN caused somewhat increased Stx2 production due to its mode of action rather than any effect on gene expression. The choice of antimicrobial therapy should rely on the antimicrobial mode of action, its concentration, and on the nature of the strain.

Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood-brain barrier.

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Meisen, Iris; Rosenbrück, Regina; Galla, Hans-Joachim; Hüwel, Sabine; Kouzel, Ivan U; Mormann, Michael; Karch, Helge; Müthing, Johannes

2013-06-01

Shiga toxin (Stx) 2e, released by certain Stx-producing Escherichia coli, is presently the best characterized virulence factor responsible for pig edema disease, which is characterized by hemorrhagic lesions, neurological disorders and often fatal outcomes. Although Stx2e-mediated brain vascular injury is the key event in development of neurologic signs, the glycosphingolipid (GSL) receptors of Stx2e and toxin-mediated impairment of pig brain endothelial cells have not been investigated so far. Here, we report on the detailed structural characterization of Stx2e receptors globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), which make up the major neutral GSLs in primary porcine brain capillary endothelial cells (PBCECs). Various Gb3Cer and Gb4Cer lipoforms harboring sphingenine (d18:1) or sphinganine (d18:0) and mostly a long-chain fatty acid (C20-C24) were detected. A notable batch-to-batch heterogeneity of primary endothelial cells was observed regarding the extent of ceramide hydroxylation of Gb3Cer or Gb4Cer species. Gb3Cer, Gb4Cer and sphingomyelin preferentially distribute to detergent-resistant membrane fractions and can be considered lipid raft markers in PBCECs. Moreover, we employed an in vitro model of the blood-brain barrier (BBB), which exhibited strong cytotoxic effects of Stx2e on the endothelial monolayer and a rapid collapse of the BBB. These data strongly suggest the involvement of Stx2e in cerebral vascular damage with resultant neurological disturbance characteristic of edema disease.

The prognostic value of serum S100B in patients with cutaneous melanoma: a meta-analysis.

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Mocellin, Simone; Zavagno, Giorgio; Nitti, Donato

2008-11-15

S100B protein detected in the serum of patients with cutaneous melanoma has been long reported as a prognostic biomarker. However, no consensus exists on its implementation in the routine clinical setting. This study aimed to comprehensively and quantitatively summarize the evidence on the suitability of serum S100B to predict patients' survival. Twenty-two series enrolling 3393 patients with TNM stage I to IV cutaneous melanoma were reviewed. Standard meta-analysis methods were applied to evaluate the overall relationship between S100B serum levels and patients' survival (meta-risk). Serum S100B positivity was associated with significantly poorer survival (hazard ratio [HR] = 2.23, 95% CI: 1.92-2.58, p < 0.0001). Between-study heterogeneity was significant, which appeared to be related mainly to dissemination bias and the inclusion of patients with stage IV disease. Considering stage I to III melanoma (n = 1594), the meta-risk remained highly significant (HR = 2.28, 95% CI: 1.8-2.89; p < 0.0001) and studies' estimates were homogeneous. Subgroup analysis of series reporting multivariate survival analysis supported S100B as a prognostic factor independent of the TNM staging system. Our findings suggest that serum S100B detection has a clinically valuable independent prognostic value in patients with melanoma, with particular regard to stage I-III disease. Further investigation focusing on this subset of patients is justified and warranted before S100B can be implemented in the routine clinical management of melanoma. (c) 2008 Wiley-Liss, Inc.

Blockade of the SNARE protein syntaxin 1 inhibits glioblastoma tumor growth.

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Fausto Ulloa

Full Text Available Glioblastoma (GBM is the most prevalent adult brain tumor, with virtually no cure, and with a median overall survival of 15 months from diagnosis despite of the treatment. SNARE proteins mediate membrane fusion events in cells and are essential for many cellular processes including exocytosis and neurotransmission, intracellular trafficking and cell migration. Here we show that the blockade of the SNARE protein Syntaxin 1 (Stx1 function impairs GBM cell proliferation. We show that Stx1 loss-of-function in GBM cells, through ShRNA lentiviral transduction, a Stx1 dominant negative and botulinum toxins, dramatically reduces the growth of GBM after grafting U373 cells into the brain of immune compromised mice. Interestingly, Stx1 role on GBM progression may not be restricted just to cell proliferation since the blockade of Stx1 also reduces in vitro GBM cell invasiveness suggesting a role in several processes relevant for tumor progression. Altogether, our findings indicate that the blockade of SNARE proteins may represent a novel therapeutic tool against GBM.

Clinical significance of determination of changes of serum SF, folic acid and vitamin B12 levels in pregnant women of different gestational stages

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Jiang Hua

2007-01-01

Objective: To explore the changes of serum SF, FA and Vitamin Bn levels in pregnant women of different gestational stages. Methods: Levels of serum SF, fore acid and Vitamin B 12 were detected with RIA in 132 pregnant women of different gestational stages and 35 controls (non-pregant women). Results: Serum SF, folic acid and VitB 12 levels were significantly lower in pregnant women of 2nd and 3rd trimester than those in controls (P l2 levels were low in late pregnancy predisposing to development anemia. (authors)

Glycan Encapsulated Gold Nanoparticles Selectively Inhibit Shiga Toxins 1 and 2

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Kulkarni, Ashish A.; Fuller-Schaefer, Cynthia; Korman, Henry; Weiss, Alison A.; Iyer, Suri S.

2010-01-01

Shiga toxins (Stx) released by Escherichia coli O157:H7 and Shigella dysentriae, cause life-threatening conditions that include hemolytic-uremic syndrome (HUS), kidney failure and neurological complications. Cellular entry is mediated by the B subunit of the AB5 toxin, which recognizes cell surface glycolipids present in lipid raft like structures. We developed gold glyconanoparticles that present a multivalent display similar to the cell surface glycolipids to compete for these toxins. These...

Bad Phages in Good Bacteria: Role of the Mysterious orf63 of λ and Shiga Toxin-Converting Φ24B Bacteriophages

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Aleksandra Dydecka

2017-08-01

Full Text Available Lambdoid bacteriophages form a group of viruses that shares a common schema of genome organization and lifecycle. Some of them can play crucial roles in creating the pathogenic profiles of Escherichia coli strains. For example, Shiga toxin-producing E. coli (STEC acquired stx genes, encoding Shiga toxins, via lambdoid prophages (Stx phages. The results obtained so far present the evidence for the relation between the exo-xis region of the phage genome and lambdoid phage development, however molecular mechanisms of activities of the exo-xis genes' products are still unknown. In view of this, we decided to determine the influence of the uncharacterized open reading frame orf63 of the exo-xis region on lambdoid phages development using recombinant prophages, λ and Stx phage Φ24B. We have demonstrated that orf63 codes for a folded protein, thus, it is a functional gene. NMR spectroscopy and analytical gel filtration were used to extend this observation further. From backbone chemical shifts, Orf63 is oligomeric in solution, likely a trimer and consistent with its small size (63 aa., is comprised of two helices, likely intertwined to form the oligomer. We observed that the deletion of phage orf63 does not impair the intracellular lambdoid phage lytic development, however delays the time and decreases the efficiency of prophage induction and in consequence results in increased survival of E. coli during phage lytic development. Additionally, the deletion of phage orf63 negatively influences expression of the major phage genes and open reading frames from the exo-xis region during prophage induction with hydrogen peroxide. We conclude, that lambdoid phage orf63 may have specific functions in the regulation of lambdoid phages development, especially at the stage of the lysis vs. lysogenization decision. Besides, orf63 probably participates in the regulation of the level of expression of essential phage genes and open reading frames from the exo

Bacterial community affects toxin production by Gymnodinium catenatum.

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Maria E Albinsson

Full Text Available The paralytic shellfish toxin (PST-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01 and grown with: 1 complex bacterial communities derived from each of the two parent cultures; 2 simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3 a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell of clonal offspring (134-197 fmol STX cell(-1 was similar to the parent cultures (169-206 fmol STX cell(-1, however cultures grown with single bacterial types contained less toxin (134-146 fmol STX cell(-1 than offspring or parent cultures grown with more complex mixed bacterial communities (152-176 fmol STX cell(-1. Specific toxin production rate (fmol STX day(-1 was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell(-1 day(-1 did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX

Bacterial community affects toxin production by Gymnodinium catenatum.

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Albinsson, Maria E; Negri, Andrew P; Blackburn, Susan I; Bolch, Christopher J S

2014-01-01

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134-197 fmol STX cell(-1)) was similar to the parent cultures (169-206 fmol STX cell(-1)), however cultures grown with single bacterial types contained less toxin (134-146 fmol STX cell(-1)) than offspring or parent cultures grown with more complex mixed bacterial communities (152-176 fmol STX cell(-1)). Specific toxin production rate (fmol STX day(-1)) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell(-1) day(-1)) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX

Occurrence of verotoxigenic E.coli in cow feces and antimicrobial resistance of the isolates in cattle farms in Shahrekord area

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Mojtaba Bonyadian

2017-09-01

Full Text Available Introduction:Escherichia coli is a common bacterium in the intestinal microflora of warm-blooded animals. They are routinely shed into the environment through feces and can contaminate water and soil, and, consequently fruits and vegetables .Enterohemorrhagic E. coli strains are recently emerged group of food-borne pathogens that are a significant public health threat. This group causes bloody diarrhea and hemolytic uremic syndrome (HUS, and the disease is prevalent in developed countries. The purpose of this study was to isolate and identify the E.coli O157: H7 and other verotoxigenic ones and major virulence genes (rfbE, eaeA, stx1, stx2 in fecal swab samples by PCR in Shahrekord area. Materials and methods: In Spring and Summer 2015, 400 cow fecal swab samples were collected from farms in Shahrekord area. Bacteriological and biochemical examinations were done for detection of E.coli. PCR assay was done for identification of O157:H7 serotype and other verotoxigenic E. coli using rfbE, eae, stx1 and stx2 genes. Results: E. coli O157:H7 was not detected in any strains tested. But PCR showed that out of 384 E.coli strain, 104(27/08% isolates carried stx1 gene, 36(9/37% carried stx2 gene and 16 (4.16% carried both stx1 and stx2 genes. Intimin (eaeA gene was detected in 280(72/91% of the isolates. Among verotoxigenic strain antibiotic resistance to Tetracycline 87/1%, Ampiciline 51/62%, Cefotaxime 48/38%, Gentamycin 25/81%,, Ciprofeloxacin 3/22% and Sulfamethoxazol 3/22% were observed. Discussion and conclusion: According to the results, although the serotype O157: H7 did not isolate from the feces of cattle but other verotoxigenic strains that showed high resistance  to antibiotic were isolated so it is a risk for human health.

Affinity purification and partial characterization of the zonulin/zonula occludens toxin (Zot) receptor from human brain.

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Lu, R; Wang, W; Uzzau, S; Vigorito, R; Zielke, H R; Fasano, A

2000-01-01

The intercellular tight junctions (TJs) of endothelial cells represent the limiting structure for the permeability of the blood-brain barrier (BBB). Although the BBB has been recognized as being the interface between the bloodstream and the brain, little is known about its regulation. Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot) elaborated by Vibrio cholerae, both modulate intercellular TJs by binding to a specific surface receptor with subsequent activation of an intracellular signaling pathway involving phospholipase C and protein kinase C activation and actin polymerization. Affinity column purification revealed that human brain plasma membrane preparations contain two Zot binding proteins of approximately 55 and approximately 45 kDa. Structural and kinetic studies, including saturation and competitive assays, identified the 55-kDa protein as tubulin, whereas the 45-kDa protein represents the zonulin/Zot receptor. Biochemical characterization provided evidence that this receptor is a glycoprotein containing multiple sialic acid residues. Comparison of the N-terminal sequence of the zonulin/Zot receptor with other protein sequences by BLAST analysis revealed a striking similarity with MRP-8, a 14-kDa member of the S-100 family of calcium binding proteins. The discovery and characterization of this receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB.

Incidência de fumonisina B1, aflatoxinas B1, B2, G1 e G2, ocratoxina A e zearalenona em produtos de milho Occurrence of fumonisin B1, aflatoxins B1, B2, G1, and G2, ochratoxin A and zearalenone in corn products

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Luciane Mie Kawashima

2006-09-01

Full Text Available Levantamentos de ocorrência de micotoxinas em alimentos foram realizados nas últimas duas décadas nas regiões Sudeste e Sul do Brasil. Levantamentos em alimentos comercializados em outras regiões têm-se limitado a aflatoxinas em amendoim e castanhas do Brasil. O presente trabalho pesquisou a presença de fumonisina B1, aflatoxinas B1, B2, G1 e G2, ocratoxina A e zearalenona em 74 amostras de produtos a base de milho adquiridas no comércio da cidade de Recife, PE, durante o período de 1999 a 2001. Fumonisina B1 foi determinada por cromatografia líquida de alta eficiência com detecção por fluorescência e as demais toxinas foram determinadas por cromatografia em camada delgada. Fumonisina B1 foi encontrada em 94,6% das amostras em concentrações variando de 20 a 8600 µg/kg. Apenas 5 amostras continham aflatoxina B1 e o teor máximo encontrado foi 20 µg/kg. Duas amostras ultrapassaram o limite de 20 µg/kg para a somatória das aflatoxinas B1, B2, G1 e G2 (farinha de milho pré-cozida com 21,5 µg/kg e quirera (xerém com 23,3 µg/kg. As aflatoxinas G1 e G2, ocratoxina A e zearalenona não foram detectadas em nenhuma das amostras. Todas as amostras contaminadas com aflatoxinas também apresentaram fumonisina B1.Research concerning the presence of mycotoxin in food has been conducted in the Southwest and South regions of Brazil over the last two decades. Research in other regions has been limited to aflatoxin in peanuts and Brazil nuts. The aim of this work is to study the presence of fumonisin B1, aflatoxins B1, B2, G1, and G2, ochratoxin A and zearalenone in 74 samples of corn products acquired in shops and food markets in the city of Recife (PE from 1999 to 2001. Fumonisin B1 was determined by high performance liquid chromatography and fluorescence was detected. The other toxins were determined by thin layer chromatography. Fumonisin B1 was found in 94.6% of the samples in levels from 20 to 8600 µg/kg. Only 5 samples contained

Ia-restricted B-B cell interaction. I. The MHC haplotype of bone marrow cells present during B cell ontogeny dictates the self-recognition specificity of B cells in the polyclonal B cell activation by a B cell differentiation factor, B151-TRF2

International Nuclear Information System (INIS)

Ono, S.; Takahama, Y.; Hamaoka, T.

1987-01-01

We have demonstrated that B cell recognition of Ia molecules is involved in polyclonal B cell differentiation by B151-TRF2. The present study was undertaken to examine the Ia recognition specificity of B151-TRF2-responsive B cells in fully major histocompatibility complex (MHC)-allogeneic P1----P2, semiallogeneic P1----(P1 x P2)F1, and double donor (P1 + P2)----(P1 x P2)F1 and (P1 + P2)----P1 radiation bone marrow chimeras. The B cells from both P1----P2 and P1----(P1 x P2)F1 chimeras could give rise to in vitro immunoglobulin M-producing cells upon stimulation with B151-TRF2 comparable in magnitude to that of normal P1 B cells, and their responses were inhibited by anti-I-AP1 but not by anti-I-AP2 monoclonal antibody even in the presence of mitomycin C-treated T cell-depleted P2 spleen cells as auxiliary cells. In contrast, the B151-TRF2 responses of P1 B cells isolated from both (P1 + P2)----(P1 x P2)F1 and (P1 + P2)----P1 double bone marrow chimeras became sensitive to the inhibition of not only anti-I-AP1 but also anti-I-AP2 monoclonal antibody only when the culture was conducted in the presence of P2 auxiliary cells, demonstrating that they adaptively differentiate to recognize as self-structures allogeneic as well as syngeneic Ia molecules. Moreover, the experiments utilizing B cells from H-2-congenic mice and B cell hybridoma clones as auxiliary cells revealed that B151-TRF2-responsive B cells recognize Ia molecules expressed on B cells. Taken together, these results demonstrate that B151-TRF2-responsive B cells recognize Ia molecules expressed by B cells as self-structures and that their self-recognition specificity is dictated by the MHC haplotype of bone marrow cells present during the B cell ontogeny but not by the MHC haplotype of a radiation-resistant host environment

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils

International Nuclear Information System (INIS)

Carmo, Lívia A.S.; Dias, Felipe F.; Malta, Kássia K.; Amaral, Kátia B.; Shamri, Revital; Weller, Peter F.; Melo, Rossana C.N.

2015-01-01

Background: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Methods: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. Results: STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. Conclusions: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. - Highlights: • First demonstration of the Qa-SNARE syntaxin-17 (STX17) in human eosinophils. • High

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils

Energy Technology Data Exchange (ETDEWEB)

Carmo, Lívia A.S.; Dias, Felipe F.; Malta, Kássia K.; Amaral, Kátia B. [Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG (Brazil); Shamri, Revital; Weller, Peter F. [Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States); Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br [Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG (Brazil); Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (United States)

2015-10-01

Background: SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Methods: Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. Results: STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. Conclusions: The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. - Highlights: • First demonstration of the Qa-SNARE syntaxin-17 (STX17) in human eosinophils. • High

The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

Science.gov (United States)

Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

2016-09-01

Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

Heat shock factors HsfB1 and HsfB2b are involved in the regulation of Pdf1.2 expression and pathogen resistance in Arabidopsis.

Science.gov (United States)

Kumar, Mukesh; Busch, Wolfgang; Birke, Hannah; Kemmerling, Birgit; Nürnberger, Thorsten; Schöffl, Friedrich

2009-01-01

In order to assess the functional roles of heat stress-induced class B-heat shock factors in Arabidopsis, we investigated T-DNA knockout mutants of AtHsfB1 and AtHsfB2b. Micorarray analysis of double knockout hsfB1/hsfB2b plants revealed as strong an up-regulation of the basal mRNA-levels of the defensin genes Pdf1.2a/b in mutant plants. The Pdf expression was further enhanced by jasmonic acid treatment or infection with the necrotrophic fungus Alternaria brassicicola. The single mutant hsfB2b and the double mutant hsfB1/B2b were significantly improved in disease resistance after A. brassicicola infection. There was no indication for a direct interaction of Hsf with the promoter of Pdf1.2, which is devoid of perfect HSE consensus Hsf-binding sequences. However, changes in the formation of late HsfA2-dependent HSE binding were detected in hsfB1/B2b plants. This suggests that HsfB1/B2b may interact with class A-Hsf in regulating the shut-off of the heat shock response. The identification of Pdf genes as targets of Hsf-dependent negative regulation is the first evidence for an interconnection of Hsf in the regulation of biotic and abiotic responses.

Serum Sialic Acid Concentration and Content in ApoB-Containing Lipoproteins in Liver Diseases.

Science.gov (United States)

Gudowska, Monika; Gruszewska, Ewa; Cylwik, Bogdan; Panasiuk, Anatol; Filisiak, Robert; Szmitkowski, Maciej; Chrostek, Lech

2016-01-01

The great significance for the metabolism of lipoproteins is the composition of carbohydrate chain of apolipoproteins, where sialic acid (SA) is located. In VILDL and LDL sialic acid is attached to apolipoprotein B. The sialylation of serum proteins including apolipoprotein B can be affected in the course of liver diseases. Therefore, the aim of this study was to assess the effect of liver diseases on the concentration and content of SA in ApoB-containing lipoproteins. The tested group consisted of 165 patients (118 males, 47 females) with liver diseases: alcoholic cirrhosis, non-alcoholic cirrhosis, chronic hepatitis, toxic hepatitis, chronic viral hepatitis, and liver cancer. ApoB-containing lipoproteins were isolated by a turbidimetric procedure and SA concentration was measured according to an enzymatic method. There was a significant increase in the serum concentration of SA in ApoB-containing lipoproteins in viral hepatitis. Although the serum concentration of ApoB was not significantly different between specific liver diseases, the serum levels of SA in ApoB-containing lipoproteins appeared to be different. There is an association between SA concentration and triglycerides in alcoholic cirrhosis and viral hepatitis. Also, in viral hepatitis SA concentration correlated negatively with HDL-cholesterol. The content of SA in ApoB-containing lipoproteins in alcoholic cirrhosis and viral hepatitis was significantly higher than that in the control group, but did not differ between diseases. This study may explain the variations in serum lipids and lipoproteins in liver diseases. It seems that the reason for these abnormalities is the changes in the concentration of sialic acid in ApoB-containing lipoproteins.

B700, a murine melanoma-specific antigen, binds Vitamin D3; conservation of binding among albuminoid molecules

International Nuclear Information System (INIS)

Farzaneh, N.K.; Walden, T.L. Jr.; Hearing, V.J.; Gersten, D.M.

1990-01-01

B700, a murine melanoma-specific antigen, is a member of the serum albumin protein family. Other members of this family include serum albumin (SMA), a-fetoprotein (AFP), vitamin D binding protein (DBP), and C700. The primary structure and biochemical functions of B700, as well as its in vivo metabolic fate are largely unknown. The authors examined the functional characteristics of MSA, AFP, and DBP, and for their ability to specifically bind [ 3 H]-1,25-dihydroxy-vitamin D 3 . Scatchard analysis revealed a single binding site for B700 with a Kd of 51,000 M and a Bmax of 4.51 x 10 -7 . There is no significant difference between the Kd and Bmax values among the albuminoid proteins. However, differences in the binding sites could be distinguished by competition of the 1,25-dihydroxy vitamin D 3 with other steroids. 2nM of vitamin D 3 , vitamin D 2 , or estrogen competed for the specific binding of 1,25-dihydroxy vitamin D 3 by B700 but not by DBP. The MSA binding site for 1,25 dihydroxy vitamin D 3 more closely resembles that of DBP than B700. These data indicate that the binding function of the albuminoid proteins has been conserved in the B700 melanoma antigen

Shiga toxin 1 interaction with enterocytes causes apical protein mistargeting through the depletion of intracellular galectin-3

Energy Technology Data Exchange (ETDEWEB)

Laiko, Marina; Murtazina, Rakhilya; Malyukova, Irina [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Zhu, Chengru; Boedeker, Edgar C. [Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87131 (United States); Gutsal, Oksana [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); O' Malley, Robert; Cole, Robert N. [Department of Biochemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Tarr, Phillip I. [Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110 (United States); Murray, Karen F. [Department of Pediatrics, Children' s Hospital and Regional Medical Center, Seattle, WA 98105 (United States); Kane, Anne [The Tufts New England Medical Center, Boston, MA 02111 (United States); Donowitz, Mark [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Kovbasnjuk, Olga, E-mail: okovbas1@jhmi.edu [Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States)

2010-02-15

Shiga toxins (Stx) 1 and 2 are responsible for intestinal and systemic sequelae of infection by enterohemorrhagic Escherichia coli (EHEC). However, the mechanisms through which enterocytes are damaged remain unclear. While secondary damage from ischemia and inflammation are postulated mechanisms for all intestinal effects, little evidence excludes roles for more primary toxin effects on intestinal epithelial cells. We now document direct pathologic effects of Stx on intestinal epithelial cells. We study a well-characterized rabbit model of EHEC infection, intestinal tissue and stool samples from EHEC-infected patients, and T84 intestinal epithelial cells treated with Stx1. Toxin uptake by intestinal epithelial cells in vitro and in vivo causes galectin-3 depletion from enterocytes by increasing the apical galectin-3 secretion. This Shiga toxin-mediated galectin-3 depletion impairs trafficking of several brush border structural proteins and transporters, including villin, dipeptidyl peptidase IV, and the sodium-proton exchanger 2, a major colonic sodium absorptive protein. The mistargeting of proteins responsible for the absorptive function might be a key event in Stx1-induced diarrhea. These observations provide new evidence that human enterocytes are directly damaged by Stx1. Conceivably, depletion of galectin-3 from enterocytes and subsequent apical protein mistargeting might even provide a means whereby other pathogens might alter intestinal epithelial absorption and produce diarrhea.

Genetic determinants of serum vitamin B12 and their relation to body mass index

DEFF Research Database (Denmark)

Allin, Kristine H.; Friedrich, Nele; Pietzner, Maik

2017-01-01

for associations between (1) serum vitamin B12 levels and body mass index (BMI), (2) genetic variants and serum vitamin B12 levels, and (3) genetic variants and BMI. The effect of a genetically determined decrease in serum vitamin B12 on BMI was estimated by instrumental variable regression. Decreased serum......Lower serum vitamin B12 levels have been related to adverse metabolic health profiles, including adiposity. We used a Mendelian randomization design to test whether this relation might be causal. We included two Danish population-based studies (ntotal = 9311). Linear regression was used to test...... vitamin B12 associated with increased BMI (P vitamin B12 associated variants associated strongly with serum vitamin B12 (P vitamin B12...

Delayed clearance of serum HBsAg in compensated cirrhosis B

DEFF Research Database (Denmark)

Fattovich, G; Giustina, G; Sanchez-Tapias, J

1998-01-01

The aim of this study was to evaluate the incidence, prognostic factors and clinical significance of delayed clearance of serum HBsAg in compensated cirrhosis B.......The aim of this study was to evaluate the incidence, prognostic factors and clinical significance of delayed clearance of serum HBsAg in compensated cirrhosis B....

Clinical application of human serum apolipoprotein B ria

International Nuclear Information System (INIS)

He Rongxia

1988-01-01

The serum apolipoprotein B (Apo B) was measured in 89 normal subjects with radioimmunoassay method established by the authors, among them 50 patients with coronary heart disease (CHD), 19 patients with cerebrae-vascular accident (CVA) and 46 patients with hyperlipemia. Meanwhile the serum cholesterol and triglyceride were also measured. Although cholesterol, triglyceride, and Apo B levels in disease groups were all significantly higher than control group, there are more overlap between the control and disease group for cholesterol and triglyceride. The Apo B level was 723.9 +- 195.9 mg/L in control group, 1097 +- 236.0 mg/L in CHD group and in CVA group, and this difference was highly significant (P < 0.001). Besides, less overlap of the Apo B value between disease and countrol group was observed in both disease groups. When the Apo B was used as single parameter for the diagnosis CHD, the accuracy rate reached 82%. The results of this study indicated that measurement of Apo B can offer important prediction for coronary artery disease, especially in those having normal levels of plasma cholesterol. In conclusion, the study of apolipoprotein is more significant than lipid component in discriminating between atherosclerotic patients and normal persons

Evaluation of FlaB1, FlaB2, FlaB3, and Tp0463 of Treponema pallidum for serodiagnosis of syphilis.

Science.gov (United States)

Jiang, Chuanhao; Xiao, Jinhong; Xie, Yafeng; Xiao, Yongjian; Wang, Chuan; Kuang, Xingxing; Xu, Man; Li, Ranhui; Zeng, Tiebing; Liu, Shuanquan; Yu, Jian; Zhao, Feijun; Wu, Yimou

2016-02-01

Syphilis is a multistage disease caused by the invasive spirochete Treponema pallidum subsp. pallidum, and accurate diagnosis is important for the prevention and treatment of syphilis. Here, to identify appropriate diagnostic antigens for serodiagnosis of syphilis, 6 recombinant proteins were expressed in Escherichia coli and purified, including flagellins (FlaB1 [Tp0868], FlaB2 [Tp0792], and FlaB3 [Tp0870]), Tp0463, Tp0751, and Tp1038. The sensitivities were determined by screening sera from individuals with primary (n=82), secondary (n=115), latent (n=105), and congenital (n=65) syphilis. The specificities were determined by screening sera from uninfected controls (n=30) and potentially cross-reactive infections including Lyme disease (n=30), leptospirosis (n=5), and hepatitis B (n=30). Our data showed that FlaB1, FlaB2, FlaB3, Tp0463, and Tp1038 exhibited higher overall sensitivities and specificities for detecting IgG antibody, with 95.4% and 98.9%, 92.6% and 95.8%, 95.1% and 95.8%, 92.6% and 97.9%, and 95.9% and 98.9%, respectively. In contrast, Tp0751 demonstrated only an overall sensitivity of 39.2%. For comparison, the sensitivity and specificity of Architect Syphilis TP were determined to be 98.1% and 93.7%, respectively. In addition, FlaB1, FlaB2, FlaB3, and Tp0463 demonstrated excellent performance for detecting IgM antibody in primary and congenital syphilis, with sensitivities of 76.8% and 83.1%, 72.0% and 87.7%, 74.4% and 89.2%, and 64.6% and 75.3%, respectively. These results indicate that FlaB1, FlaB2, FlaB3, and Tp0463 could be as novel diagnostic candidates for serodiagnosis of syphilis. Copyright © 2016 Elsevier Inc. All rights reserved.

Characteristics of Clinical Shiga Toxin-Producing Escherichia coli Isolated from British Columbia

Directory of Open Access Journals (Sweden)

Kevin J. Allen

2013-01-01

Full Text Available Shiga toxin-producing Escherichia coli (STEC are significant public health threats. Although STEC O157 are recognized foodborne pathogens, non-O157 STEC are also important causes of human disease. We characterized 10 O157:H7 and 15 non-O157 clinical STEC derived from British Columbia (BC. Eae, hlyA, and stx were more frequently observed in STEC O157, and 80 and 100% of isolates possessed stx1 and stx2, respectively. In contrast, stx1 and stx2 occurred in 80 and 40% of non-O157 STEC, respectively. Comparative genomic fingerprinting (CGF revealed three distinct clusters (C. STEC O157 was identified as lineage I (LI; LSPA-6 111111 and clustered as a single group (C1. The cdi gene previously observed only in LII was seen in two LI O157 isolates. CGF C2 strains consisted of diverse non-O157 STEC while C3 included only O103:H25, O118, and O165 serogroup isolates. With the exception of O121 and O165 isolates which were similar in virulence gene complement to STEC O157, C1 O157 STEC produced more Stx2 than non-O157 STEC. Antimicrobial resistance (AMR screening revealed resistance or reduced sensitivity in all strains, with higher levels occurring in non-O157 STEC. One STEC O157 isolate possessed a mobile blaCMY-2 gene transferrable across genre via conjugation.

Ouabain Protects Human Renal Cells against the Cytotoxic Effects of Shiga Toxin Type 2 and Subtilase Cytotoxin

Directory of Open Access Journals (Sweden)

María M. Amaral

2017-07-01

Full Text Available Hemolytic uremic syndrome (HUS is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx-producing Escherichia coli (STEC. In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2 are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC and the human proximal tubule epithelial cell (HK-2 line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS.

Radioassays for quantitation of intact complement proteins C2 and B in human serum

Energy Technology Data Exchange (ETDEWEB)

Oglesby, T J; Ueda, A; Volanakis, J E

1988-05-25

Availability of polyclonal and monoclonal antibodies recognizing determinants on the major cleavage fragments of complement proteins C2 and B enabled development of sensitive radioassays which can be used to quantitate the intact proteins in human sera. Changes in C2 and B concentrations indicative of classical or alternative pathway activation, or both, were seen in normal serum after incubation with complement activators. The authors determined the normal range of C2 concentration to be 11-35 ..mu..g/ml in 32 healthy individuals, and that of protein B to be 74-286 ..mu..g/ml. Sera from patients with systemic lupus erythematosus (SLE), septic shock, infections, and following orthopedic surgery were then assayed. Mean protein B concentration was significantly higher in SLE sera and in the infected and post-operative sera, and the mean C2 concentration in the septic shock group was significantly lower than the mean of healthy individuals. Intact C2 was not detected in known C2-deficient individuals. These assays allow parallel quantitation of the structurally and functionally homologous proteins of the classical (C2) and alternative (B) pathways, which is of interest in patients with genetic and acquired hypocomplementemia. 22 refs.; 3 figs.

B2B or Not to Be: Does B2B E-Commerce Increase Labour Productivity?

OpenAIRE

Bertschek, Irene; Fryges, Helmut; Kaiser, Ulrich

2004-01-01

We implement an endogeneous switching-regression model for labour productivity and firms' decision to use business-to-business (B2B) e-commerce. Our approach allows B2B usage to affect any parameter of the labour productivity equation and to properly take account of strategic complementarities between the input factors and B2B usage. Empirical evidence from 1,394 German firms shows that firms using B2B e-commerce have a significantly higher output elasticity with respect to ICT-investment and...

Population-specific regulation of Chmp2b by Lbx1 during onset of synaptogenesis in lateral association interneurons.

Directory of Open Access Journals (Sweden)

Jun Xu

Full Text Available Chmp2b is closely related to Vps2, a key component of the yeast protein complex that creates the intralumenal vesicles of multivesicular bodies. Dominant negative mutations in Chmp2b cause autophagosome accumulation and neurodegenerative disease. Loss of Chmp2b causes failure of dendritic spine maturation in cultured neurons. The homeobox gene Lbx1 plays an essential role in specifying postmitotic dorsal interneuron populations during late pattern formation in the neural tube. We have discovered that Chmp2b is one of the most highly regulated cell-autonomous targets of Lbx1 in the embryonic mouse neural tube. Chmp2b was expressed and depended on Lbx1 in only two of the five nascent, Lbx1-expressing, postmitotic, dorsal interneuron populations. It was also expressed in neural tube cell populations that lacked Lbx1 protein. The observed population-specific expression of Chmp2b indicated that only certain population-specific combinations of sequence specific transcription factors allow Chmp2b expression. The cell populations that expressed Chmp2b corresponded, in time and location, to neurons that make the first synapses of the spinal cord. Chmp2b protein was transported into neurites within the motor- and association-neuropils, where the first synapses are known to form between E11.5 and E12.5 in mouse neural tubes. Selective, developmentally-specified gene expression of Chmp2b may therefore be used to endow particular neuronal populations with the ability to mature dendritic spines. Such a mechanism could explain how mammalian embryos reproducibly establish the disynaptic cutaneous reflex only between particular cell populations.

Saxitoxins and okadaic acid group: accumulation and distribution in invertebrate marine vectors from Southern Chile.

Science.gov (United States)

García, Carlos; Pérez, Francisco; Contreras, Cristóbal; Figueroa, Diego; Barriga, Andrés; López-Rivera, Américo; Araneda, Oscar F; Contreras, Héctor R

2015-01-01

Harmful algae blooms (HABs) are the main source of marine toxins in the aquatic environment surrounding the austral fjords in Chile. Huichas Island (Aysén) has an history of HABs spanning more than 30 years, but there is limited investigation of the bioaccumulation of marine toxins in the bivalves and gastropods from the Region of Aysén. In this study, bivalves (Mytilus chilenses, Choromytilus chorus, Aulacomya ater, Gari solida, Tagelus dombeii and Venus antiqua) and carnivorous gastropods (Argobuccinum ranelliformes and Concholepas concholepas) were collected from 28 sites. Researchers analysed the accumulation of STX-group toxins using a LC with a derivatisation post column (LC-PCOX), while lipophilic toxins (OA-group, azapiracids, pectenotoxins and yessotoxins) were analysed using LC-MS/MS with electrospray ionisation (+/-) in visceral (hepatopancreas) and non-visceral tissues (mantle, adductor muscle, gills and foot). Levels of STX-group and OA-group toxins varied among individuals from the same site. Among all tissue samples, the highest concentrations of STX-group toxins were noted in the hepatopancreas in V. antiqua (95 ± 0.1 μg STX-eq 100 g(-1)), T. dombeii (148 ± 1.4 μg STX-eq 100 g(-1)) and G. solida (3232 ± 5.2 μg STX-eq 100 g(-1); p concholepas (81 ± 0.7 μg STX-eq 100 g(-1)) and T. dombeii (114 ± 1.2 μg STX-eq 100 g(-1)). The highest variability of toxins was detected in G. solida, where high levels of carbamate derivatives were identified (GTXs, neoSTX and STX). In addition to the detected hydrophilic toxins, OA-group toxins were detected (OA and DTX-1) with an average ratio of ≈1:1. The highest levels of OA-group toxins were in the foot of C. concholepas, with levels of 400.3 ± 3.6 μg OA eq kg(-1) (p mantle > adductor muscle for the STX-group toxins and foot > digestive gland for the OA-group toxins. These results gave a better understanding of the variability and compartmentalisation of STX-group and OA-group toxins in different

The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

Science.gov (United States)

Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

2009-02-01

In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

TIM-1 signaling in B cells regulates antibody production

International Nuclear Information System (INIS)

Ma, Juan; Usui, Yoshihiko; Takeda, Kazuyoshi; Harada, Norihiro; Yagita, Hideo; Okumura, Ko; Akiba, Hisaya

2011-01-01

Highlights: → TIM-1 is highly expressed on anti-IgM + anti-CD40-stimulated B cells. → Anti-TIM-1 mAb enhanced proliferation and Ig production on activated B cell in vitro. → TIM-1 signaling regulates Ab production by response to TI-2 and TD antigens in vivo. -- Abstract: Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3 + anti-CD28-stimulated CD4 + T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.

TIM-1 signaling in B cells regulates antibody production

Energy Technology Data Exchange (ETDEWEB)

Ma, Juan [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Usui, Yoshihiko [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Ophthalmology, Tokyo Medical University, 6-7-1 Nishi-shinjuku-ku, Tokyo 160-0023 (Japan); Takeda, Kazuyoshi [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Harada, Norihiro [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Department of Respiratory Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Research Institute for Diseases of Old Ages, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Yagita, Hideo; Okumura, Ko [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Akiba, Hisaya, E-mail: hisaya@juntendo.ac.jp [Department of Immunology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

2011-03-11

Highlights: {yields} TIM-1 is highly expressed on anti-IgM + anti-CD40-stimulated B cells. {yields} Anti-TIM-1 mAb enhanced proliferation and Ig production on activated B cell in vitro. {yields} TIM-1 signaling regulates Ab production by response to TI-2 and TD antigens in vivo. -- Abstract: Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3{sup +} anti-CD28-stimulated CD4{sup +} T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.

Simple serum markers for significant liver inflammation in chronic hepatitis B patients with an alanine aminotransferase level lower than 2 times upper limit of normal

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LI Qiang

2016-06-01

Full Text Available ObjectiveTo investigate the simple serum markers for significant liver inflammation in chronic hepatitis B (CHB patients with an alanine aminotransferase (ALT level of ＜2 times upper limit of normal (ULN. MethodsThe clinical data of 278 CHB patients with ALT ＜2×ULN (ULN=40 U/L were analyzed retrospectively. Significant liver inflammation was defined as a liver inflammatory activity grade (G ≥2. The t-test was used for comparison of normally distributed continuous data between groups, and the Kruskal-Wallis rank sum test was used for non-normally distributed continuous data; the chi-square test was used for comparison of categorical data between groups. Multivariate logistic regression analysis was used to identify independent predictors for significant liver inflammation in CHB patients with ALT ＜2×ULN. The receiver operating characteristic (ROC curve was used to evaluate the diagnostic value of serum markers in significant liver inflammation. ResultsOf the 278 CHB patients enrolled, 175 (62.9% had no significant liver inflammation (G0-1 group and 103 (37.1% had significant liver inflammation (G2-4 group. There were significant differences in ALT, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase (GGT, albumin, globulin, prothrombin time (PT, platelet, absolute neutrophil count, hyaluronic acid (HA, glycocholic acid, precollagen Ⅲ, and collagen type Ⅳ(ⅣC between the two groups (all P＜0.05. The multivariate regression analysis showed that GGT, PT, ⅣC, and HA were independent predictors for significant liver inflammation in CHB patients with ALT＜2×ULN (OR=1.015, 1.600, 1.151, and 1.014, P=0.008, 0.021, 0.003, and 0.018. The areas under the ROC curve for GGT, PT, IVC, and HA to diagnose significant liver inflammation were 0.804, 0.722, 0.707, and 0.632, respectively. The cut-off value of 48.5 U/L for GGT to predict significant liver inflammation had a specificity of 90.3% and a negative

Prevalence and characterization of Shiga Toxin-producing and enteropathogenic Escherichia coli in shellfish-harvesting areas and their watersheds

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Balière eCharlotte

2015-12-01

Full Text Available During a two-year study, the presence of Shiga-toxin producing E. coli (STEC and enteropathogenic E. coli (EPEC was investigated in shellfish (n=238, seawater (n=12 and surface sediment (n=39 collected from three French coastal shellfish-harvesting areas and freshwaters (n=216 in their watersheds. PCR detection of Shiga toxin- (stx1/stx2 and intimin- (eae genes following enrichment from these samples revealed the presence of least one of the stx genes in 30.3% of shellfish batches, 85.9% of freshwater, 41.7% of seawater, and 28.2% of sediment samples, while the eae gene was observed in 74.8%, 100%, 100%, and 43.6% of shellfish batches, freshwater, seawater, and sediment samples, respectively. Twenty-eight STEC and 89 EPEC strains were isolated and analyzed in order to determine their serotype, phylogroup, and genetic relatedness and to evaluate the presence of the saa and ehxA genes encoding the STEC autoagglutinating adhesin and the enterohemolysin A, respectively. Finally, the ability to form biofilms and antimicrobial susceptibility were investigated for a selection of strains. Eighteen serotypes were identified among the STEC isolates and 57 among the EPEC isolates. A high diversity was observed within these strains, as 79 different PFGE patterns and 48 distinguishable sequence types were identified. Strains were found to belong mainly to phylogroups B1 and B2 and virulence was observed to be low as more than 85% of the strains possessed only stx1, stx2 or eae genes. One STEC and several EPEC strains belonged to three of the five highly pathogenic serogroups (i.e., O26, O103, and O145. The subset of strains tested for their capacity to form biofilms was mainly strongly to moderately adherent and more strains formed a strong biofilm at 18°C than at 30°C. Finally, more than 85% of analyzed strains were found to be sensitive to the 16 tested antibiotics. These data suggest the low risk of human infection by STEC if shellfish from these

Comparison of monoclonal antibodies and rabbit antisera in B2 microglobulin (B2m) radioimmunoassay

International Nuclear Information System (INIS)

Vincent, C.

1981-01-01

Human B2m is a globular protein devoid of carbohydrates and is composed of 100 aminoacids with an intrachain disulfide bridge in position 25-81. Its aminoacid sequence and three dimensional structure shows a partial homology with the constant domains of immunoglobulins. B2m has been detected at the surface of nearly all cell types with the exception of erythrocytes and trophoblastic cells and also in all biological fluids. On the cell surface B2m is found non-covalently associated with HLA molecules, and in serum and urine only a small part of the B2m is associated with HLA heavy chain, the remaining is found as free B2m. The aim of this paper is firstly to try to demonstrate the presence of two types of epitopes one of them specific of free B2m and secondly to compare monoclonal antibodies with rabbit antisera for B2m radioimmunoassay. (Auth.)

Aislamiento y caracterización de Escherichia coliO157:H7 a partir de carne molida de bovino en Lima-Perú

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Carmen R. Méndez

2013-12-01

Full Text Available El objetivo del estudio fue aislar y caracterizar E. coliO157:H7 a partir de carne molida fresca de bovino obtenida en diferentes mercados de abastos. Se analizaron 195 muestras; para el aislamiento y enumeración de E. coli se utilizó la técnica del Numero Más probable mediante tubos múltiples; para el aislamiento y caracterización de E. coliO157:H7 el enriquecimiento selectivo y el análisis bioquímico, las colonias características se confirmaron mediante pruebas serológicas. Para determinar la presencia de shigatoxina (stx1, stx2 e intimina (eae A se empleó la técnica de PCR multiplex en tiempo real y para enterohemolisina la prueba de hemólisis. El 87.18% de la muestras fue positivo para E. coliy el 77.95% presentó un recuento igual o superior a 50 NMP/g. Se obtuvieron 3 (1.54% cepas de E. coliO157:H7, una stx1 +/ stx2 +/ eaeA – y enterohemolisina -, una stx1 +/ stx2 -/ eaeA + y enterohemolisina – y la otra stx1 -/ stx2 -/ eaeA – y enterohemolisina +. También se obtuvieron 4 cepas (2.05% de E. coli O157 no H7, ninguna presentó factores de virulencia. El estudio reveló el riesgo potencial de que E. coliO157:H7 afecte a la población de Lima.

Specific heat of MgB_2 after irradiation

OpenAIRE

Wang, Yuxing; Bouquet, Frederic; Sheikin, Ilya; Toulemonde, Pierre; Revaz, Bernard; Eisterer, Michael; Weber, Harald W.; Hinderer, Joerg; Junod, Alain

2002-01-01

We studied the effect of disorder on the superconducting properties of polycrystalline MgB_2 by specific-heat measurements. In the pristine state, these measurements give a bulk confirmation of the presence of two superconducting gaps with 2 Delta 0 / k_B T_c = 1.3 and 3.9 with nearly equal weights. The scattering introduced by irradiation suppresses T_c and tends to average the two gaps although less than predicted by theory. We also found that by a suitable irradiation process by fast neutr...

Human CRISP-3 binds serum alpha(1)B-glycoprotein across species

DEFF Research Database (Denmark)

Udby, Lene; Johnsen, Anders H; Borregaard, Niels

2010-01-01

CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular...

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

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Brasher, Megan I; Martynowicz, David M; Grafinger, Olivia R; Hucik, Andrea; Shanks-Skinner, Emma; Uniacke, James; Coppolino, Marc G

2017-09-29

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Bu-2470, a new peptide antibiotic complex. II. Structure determination of Bu-2470 A, B1, B2a and B2b.

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Sugawara, K; Yonemoto, T; Konishi, M; Matsumoto, K; Miyaki, T; Kawaguchi, H

1983-06-01

The structures of Bu-2470 A, B1, B2a, and B2b have been determined. Bu-2470 A is a simple octapeptide having no fatty acid moiety, while Bu-2470 B1, B2a and B2b are octapeptides that have been acylated with a beta-hydroxy C11 or C10 fatty acid. The octapeptide structure of Bu-2470 components was found identical with that of octapeptin C1, hence generic names of octapeptin C0, C2, C3 and C4 are proposed for Bu-2470 A, B1, B2a and B2b, respectively.

Serum S100B: a potential biomarker for suicidality in adolescents?

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Tatiana Falcone

Full Text Available BACKGROUND: Studies have shown that patients suffering from depression or schizophrenia often have immunological alterations that can be detected in the blood. Others reported a possible link between inflammation, a microgliosis and the blood-brain barrier (BBB in suicidal patients. Serum S100B is a marker of BBB function commonly used to study cerebrovascular wall function. METHODS: We measured levels of S100B in serum of 40 adolescents with acute psychosis, 24 adolescents with mood disorders and 20 healthy controls. Patients were diagnosed according to DSM-IV TR criteria. We evaluated suicidal ideation using the suicidality subscale of the Brief Psychiatric Rating Scale for Children (BPRS-C. RESULTS: Serum S100B levels were significantly higher (p<0.05 and correlated to severity of suicidal ideation in patients with psychosis or mood disorders, independent of psychiatric diagnosis. Patients with a BPRS-C suicidality subscores of 1-4 (low suicidality had mean serum S100B values +/- SEM of 0.152+/-0.020 ng/mL (n = 34 compared to those with BPRS-C suicidality subscores of 5-7 (high suicidality with a mean of 0.354+/-0.044 ng/mL (n = 30. This difference was statistically significant (p<0.05. CONCLUSION: Our data support the use of S100B as an adjunctive biomarker to assess suicidal risk in patients with mood disorders or schizophrenia.

A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

Science.gov (United States)

Zheng, X; Hu, B; Gao, S X; Liu, D J; Sun, M J; Jiao, B H; Wang, L H

2015-07-01

Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application. Copyright © 2015 Elsevier Ltd. All rights reserved.

The prevalence of Escherichia coli O157 and O157:H7 in ground beef and raw meatball by immunomagnetic separation and the detection of virulence genes using multiplex PCR.

Science.gov (United States)

Cadirci, Ozgür; Siriken, Belgin; Inat, Gökhan; Kevenk, Tahsin Onur

2010-03-01

The present study was conducted to investigate the presence of Escherichia coli O157 and O157:H7 strains and to detect the presence of the stx1, stx2, and eaeA genes in isolates derived from 200 samples (100 samples from fresh ground beef and 100 samples from raw meatball). The samples were purchased from the Samsun Province in Turkey, over a period of 1 year. Enrichment-based immunomagnetic separation and multiplex polymerase chain reaction were applied for these analyses. E. coli O157 was detected in five of the 200 (2.5%) samples tested (one isolated from ground beef and four from meatball samples), whereas E. coli O157: H7 was not detected in any sample. During the analysis, eight strains of E. coli O157 were obtained. The genes stx1, stx2, and eaeA were detected in two E. coli O157 isolates obtained from two meatball samples, whereas only the eaeA and the stx2 genes were detected in four E. coli O157 strains that were isolated from one meatball sample. None of the stx1, stx2, and eaeA was detected in the E. coli O157 isolates obtained from the ground beef and the one meatball samples. Copyright 2009 Elsevier Ltd. All rights reserved.

Shiga toxin-producing Escherichia coli in slaughtered pigs and pork products

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Lia Bardasi

2017-05-01

Full Text Available During the years 2015-2016, 83 faecal samples were collected at slaughter from pigs reared in farms located in Central- Northern Italy. During the years 2014-2016 a total of 562 pork products [465 not-ready-to-eat (NRTE and 97 ready-to-eat (RTE products] were collected from retail outlets, large retailers and processing plants. The samples were analysed according to ISO TS 13136:2012. Out of 83 swine faecal samples, 77 (92.8% resulted stx-positive by real time polymerase chain reaction (PCR, 5 stx2+ and 1 stx1+ Shiga toxin-producing Escherichia coli (STEC strains were isolated. Among the 465 NRTE samples, 65 (14.0% resulted stx-positive by real time PCR and 7 stx2+ STEC strains were isolated. The stx2 gene was detected more frequently than the stx1 gene both in faecal samples (90.4 vs 8.4% and in NRTE pork products (13.3 vs 1.3%. All the RTE samples included in the analysis resulted stxnegative. Among the samples resulted positive for stx and eae genes, serogroup-associated genes were detected at high frequency: O26 resulted the most frequent in faecal samples (81.3% and O145 in pork products (88.1%. The O157 serogroup resulted positive in 83.3 and 78.1% of pork products and faecal samples, respectively. Despite the frequent detection by real time PCR of genes indicating the possible presence of STEC strains belonging to the six serogroups, the bacteriological step did not confirm the isolation of any such strains.

High-dose vitamin B6 decreases homocysteine serum levels in patients with schizophrenia and schizoaffective disorders: a preliminary study.

Science.gov (United States)

Miodownik, Chanoch; Lerner, Vladimir; Vishne, Tali; Sela, Ben-Ami; Levine, Joseph

2007-01-01

Vitamin B6 plays an essential role in the normal functioning of the central nervous system. Normal homocysteine (Hcy) serum level is maintained by remethylation of Hcy to methionine by enzymes that require folic acid and vitamin B12 and by catabolism to cysteine by a vitamin B6-dependent enzyme. These findings may be consistent with the hypothesis that the vitamin B6 status may influence plasma Hcy levels. The aims of this preliminary study were (1) to determine whether a correlation exists between Hcy and vitamin B6 levels in patients with schizophrenia and schizoaffective disorders and (2) to investigate whether treatment with high-dose vitamin B6 may reduce Hcy levels in these patients. In this preliminary study, we enrolled 11 patients with schizophrenia or schizoaffective disorders (7 men and 4 women; mean age +/- SD, 50 +/- 12 years) receiving high doses of vitamin B6 treatment (1200 mg/d) for 12 weeks. Blood samples for the assessment of pyridoxal-5-phosphate and Hcy serum levels were obtained at baseline and after 12 weeks of treatment. Age was significantly positively correlated with Hcy levels at baseline (r = 0.392, P = 0.004). All other parameters, including diagnosis, disease duration, and pyridoxal-5-phosphate serum level, were not correlated with Hcy serum levels at baseline. After vitamin B6 treatment, Hcy serum levels significantly decreased (14.2 +/- 3.4 vs. 11.8 +/- 2.0 micromol/L, respectively, t = 2.679, P = 0.023); this decrease being statistically significant in men but not in women. High doses of vitamin B6 lead to a decrease in Hcy serum level in male patients with schizophrenia or schizoaffective disorder.

Low IDL-B and high LDL-1 subfraction levels in serum of ALS patients.

Science.gov (United States)

Delaye, J B; Patin, F; Piver, E; Bruno, C; Vasse, M; Vourc'h, P; Andres, C R; Corcia, P; Blasco, H

2017-09-15

Converging evidence highlights that lipid metabolism plays a key role in ALS pathophysiology. Dyslipidemia has been described in ALS patients and may be protective but peripheral lipoprotein subclasses have never been studied. We collected sera from 30 ALS patients and 30 gender and age-matched controls. We analyzed 11 distinct lipoprotein subclasses by linear polyacrylamide gel electrophoresis (Lipoprint, Quantimetrix Corporation, USA). We also measured lipoprotein (a), apolipoprotein B, and apolipoprotein E levels. ALS patients had significant higher total cholesterol, HDL-cholesterol, and LDL-cholesterol levels than controls (pALS patients than controls. Our preliminary work confirmed the association between ALS and dyslipidemia. The low IDL-B levels may explain the hepatic steatosis frequently reported in ALS. The high levels of the cholesterol-rich LDL-1 subfraction is consistent with previously reported hypercholesterolemia. This study describes, for the first time, the distribution of serum lipoproteins in ALS patients, with low IDL-B and high LDL-1 subfraction level. Copyright © 2017 Elsevier B.V. All rights reserved.

Serum Inhibin B and follicle-stimulating hormone levels as tools in the evaluation of infertile men: significance of adequate reference values from proven fertile men

DEFF Research Database (Denmark)

Andersson, A.-M.; Petersen, Jørgen Holm; Jørgensen, N.

2004-01-01

Inhibin B and FSH levels in 289 idiopathic infertile men were compared with reference materials consisting of 303 proven fertile men (reference group 1) and 307 healthy men from the general population with unknown fertility status (reference group 2). The diagnostic power of these two serum markers...... of spermatogenesis was evaluated by the use of receiver operating characteristic plot analysis, and an example of how both markers can be used simultaneously in a bivariate reference chart is presented. Inhibin B levels were significantly lower and FSH levels were significantly higher in the infertile men, compared...

Gene duplication, loss and selection in the evolution of saxitoxin biosynthesis in alveolates.

Science.gov (United States)

Murray, Shauna A; Diwan, Rutuja; Orr, Russell J S; Kohli, Gurjeet S; John, Uwe

2015-11-01

A group of marine dinoflagellates (Alveolata, Eukaryota), consisting of ∼10 species of the genus Alexandrium, Gymnodinium catenatum and Pyrodinium bahamense, produce the toxin saxitoxin and its analogues (STX), which can accumulate in shellfish, leading to ecosystem and human health impacts. The genes, sxt, putatively involved in STX biosynthesis, have recently been identified, however, the evolution of these genes within dinoflagellates is not clear. There are two reasons for this: uncertainty over the phylogeny of dinoflagellates; and that the sxt genes of many species of Alexandrium and other dinoflagellate genera are not known. Here, we determined the phylogeny of STX-producing and other dinoflagellates based on a concatenated eight-gene alignment. We determined the presence, diversity and phylogeny of sxtA, domains A1 and A4 and sxtG in 52 strains of Alexandrium, and a further 43 species of dinoflagellates and thirteen other alveolates. We confirmed the presence and high sequence conservation of sxtA, domain A4, in 40 strains (35 Alexandrium, 1 Pyrodinium, 4 Gymnodinium) of 8 species of STX-producing dinoflagellates, and absence from non-producing species. We found three paralogs of sxtA, domain A1, and a widespread distribution of sxtA1 in non-STX producing dinoflagellates, indicating duplication events in the evolution of this gene. One paralog, clade 2, of sxtA1 may be particularly related to STX biosynthesis. Similarly, sxtG appears to be generally restricted to STX-producing species, while three amidinotransferase gene paralogs were found in dinoflagellates. We investigated the role of positive (diversifying) selection following duplication in sxtA1 and sxtG, and found negative selection in clades of sxtG and sxtA1, clade 2, suggesting they were functionally constrained. Significant episodic diversifying selection was found in some strains in clade 3 of sxtA1, a clade that may not be involved in STX biosynthesis, indicating pressure for diversification

Detection and characterization of Shiga toxin-producing Escherichia coli from seagulls.

Science.gov (United States)

Makino, S; Kobori, H; Asakura, H; Watarai, M; Shirahata, T; Ikeda, T; Takeshi, K; Tsukamoto, T

2000-08-01

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from a seagull in Japan were examined. A total of 50 faecal samples was collected on a harbour bank in Hokkaido, Japan, in July 1998. Two different STEC strains, whose serotypes were O136:H16 and O153:H-, were isolated from the same individual by PCR screening; both of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing active Stx2 and Stx1, respectively. They harboured large plasmids, but did not carry the haemolysin or eaeA genes of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, pulsed-field gel electrophoresis analysis (PFGE), and the stx genes sequences, the isolates were different. Phylogenic analysis of the deduced Stx amino acid sequences demonstrated that the Stx toxins of seagull-origin STEC were closely associated with those of the human-origin, but not those of other animal-origin STEC. In addition, Stx2phi-K7 phage purified from O136 STEC resembled Stx2phi-II from human-origin O157:H7, and was able to convert non-toxigenic E. coli to STEC. These results suggest that birds may be one of the important carriers in terms of the distribution of STEC.

Erythrocyte-bound apolipoprotein B in relation to atherosclerosis, serum lipids and ABO blood group.

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Boudewijn Klop

Full Text Available INTRODUCTION: Erythrocytes carry apolipoprotein B on their membrane, but the determining factors of erythrocyte-bound apolipoprotein B (ery-apoB are unknown. We aimed to explore the determinants of ery-apoB to gain more insight into potential mechanisms. METHODS: Subjects with and without CVD were included (N = 398. Ery-apoB was measured on fresh whole blood samples using flow cytometry. Subjects with ery-apoB levels ≤ 0.20 a.u. were considered deficient. Carotid intima media thickness (CIMT was determined as a measure of (subclinical atherosclerosis. RESULTS: Mean ery-apoB value was 23.2% lower in subjects with increased CIMT (0.80 ± 0.09 mm, N = 140 compared to subjects with a normal CIMT (0.57 ± 0.08 mm, N = 258 (P = 0.007, adjusted P<0.001. CIMT and ery-apoB were inversely correlated (Spearman's r: -0.116, P = 0.021. A total of 55 subjects (13.6% were considered ery-apoB deficient, which was associated with a medical history of CVD (OR: 1.86, 95% CI 1.04-3.33; adjusted OR: 1.55; 95% CI 0.85-2.82. Discontinuation of statins in 54 subjects did not influence ery-apoB values despite a 58.4% increase in serum apolipoprotein B. Subjects with blood group O had significantly higher ery-apoB values (1.56 ± 0.94 a.u. when compared to subjects with blood group A (0.89 ± 1.15 a.u, blood group B (0.73 ± 0.1.12 a.u. or blood group AB (0.69 ± 0.69 a.u. (P-ANOVA = 0.002. CONCLUSION: Absence or very low values of ery-apoB are associated with clinical and subclinical atherosclerosis. While serum apolipoprotein B is not associated with ery-apoB, the ABO blood group seems to be a significant determinant.

A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples.

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Xiaohua He

Full Text Available BACKGROUND: Shiga toxin-producing Escherichia coli (STEC are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains. METHODS AND FINDINGS: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. CONCLUSIONS: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs and cats and genetic relationships with isolates from cattle, meat and humans.

Science.gov (United States)

Bentancor, A; Rumi, M V; Carbonari, C; Gerhardt, E; Larzábal, M; Vilte, D A; Pistone-Creydt, V; Chinen, I; Ibarra, C; Cataldi, A; Mercado, E C

2012-05-04

Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families. Copyright © 2011 Elsevier B.V. All rights reserved.

Major histocompatibility complex class I-related chain A/B (MICA/B) expression in tumor tissue and serum of pancreatic cancer: Role of uric acid accumulation in gemcitabine-induced MICA/B expression

International Nuclear Information System (INIS)

Xu, Xiulong; Rao, Geetha S; Groh, Veronika; Spies, Thomas; Gattuso, Paolo; Kaufman, Howard L; Plate, Janet; Prinz, Richard A

2011-01-01

Major histocompatibility complex class I-related chain A and B (MICA/B) are two stress-inducible ligands that bind the immunoreceptor NKG2D and play an important role in mediating the cyotoxicity of NK and T cells. In this study, we sought to study MICA/B expression in pancreatic cancer and to determine whether and how genotoxic drugs such as gemcitabine can affect MICA/B expression and natural killer cytotoxity. Seven pancreatic cancer cell lines were analyzed for MICA/B expression by flow cytometry and for their sensitivity to NK-92 cell killing by a 51 Cr release assay. MICA/B expression in tumor tissues and sera of pancreatic cancer was analyzed by immunohistochemical staining (IHC) and ELISA, respectively. Two MICA/B-positive cell lines were sensitive to the cytotoxic activity of NK-92 cells. Other two MICA/B-positive cell lines and three MICA/B-negative cell lines were resistant to NK-92 cell killing. MICA/B expression was positive in 17 of 25 (68%) pancreatic ductal adenocarcinomas but not in normal pancreatic ductal epithelial cells. Serum MICA/B levels were significantly elevated in patients with pancreatic adenocarcinomas but did not correlate with the stage of pancreatic cancer and patient survival. Gemcitabine therapy led to increased serum MICA levels in 6 of 10 patients with detectable serum MICA. Allopurinol, an inhibitor of xanthine oxidoreductase that converts xanthine to uric acid, blocked uric acid production, MICA/B expression, and sensitivity to NK-92 cell killing toward a PANC-1 cancer cell line exposed to radiation and two genotoxic drugs, gemcitabine and 5-fluorouracil. The levels of MICA/B expression in serum and tissue of pancreatic cancer are elevated. DNA damage-induced MICA/B expression is mediated through increased uric acid production

Major histocompatibility complex class I-related chain A/B (MICA/B expression in tumor tissue and serum of pancreatic cancer: Role of uric acid accumulation in gemcitabine-induced MICA/B expression

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Kaufman Howard L

2011-05-01

Full Text Available Abstract Background Major histocompatibility complex class I-related chain A and B (MICA/B are two stress-inducible ligands that bind the immunoreceptor NKG2D and play an important role in mediating the cyotoxicity of NK and T cells. In this study, we sought to study MICA/B expression in pancreatic cancer and to determine whether and how genotoxic drugs such as gemcitabine can affect MICA/B expression and natural killer cytotoxity. Methods Seven pancreatic cancer cell lines were analyzed for MICA/B expression by flow cytometry and for their sensitivity to NK-92 cell killing by a 51Cr release assay. MICA/B expression in tumor tissues and sera of pancreatic cancer was analyzed by immunohistochemical staining (IHC and ELISA, respectively. Results Two MICA/B-positive cell lines were sensitive to the cytotoxic activity of NK-92 cells. Other two MICA/B-positive cell lines and three MICA/B-negative cell lines were resistant to NK-92 cell killing. MICA/B expression was positive in 17 of 25 (68% pancreatic ductal adenocarcinomas but not in normal pancreatic ductal epithelial cells. Serum MICA/B levels were significantly elevated in patients with pancreatic adenocarcinomas but did not correlate with the stage of pancreatic cancer and patient survival. Gemcitabine therapy led to increased serum MICA levels in 6 of 10 patients with detectable serum MICA. Allopurinol, an inhibitor of xanthine oxidoreductase that converts xanthine to uric acid, blocked uric acid production, MICA/B expression, and sensitivity to NK-92 cell killing toward a PANC-1 cancer cell line exposed to radiation and two genotoxic drugs, gemcitabine and 5-fluorouracil. Conclusions The levels of MICA/B expression in serum and tissue of pancreatic cancer are elevated. DNA damage-induced MICA/B expression is mediated through increased uric acid production.

Both nuclear and cytoplasmic components are defective in oocytes of the B6.Y(TIR) sex-reversed female mouse.

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Amleh, A; Smith, L; Chen, H; Taketo, T

2000-03-15

In the mammalian gonadal primordium, activation of the Sry gene on the Y chromosome initiates a cascade of genetic events leading to testicular organization whereas its absence results in ovarian differentiation. An exception occurs when the Y chromosome of Mus musculus domesticus from Tirano, Italy (Y(TIR)), is placed on the C57BL/6J (B6) genetic background. The B6.Y(TIR) progeny develop only ovaries or ovotestes despite Sry transcription in fetal life. Consequently, the XY offspring with bilateral ovaries develop into apparently normal females, but their eggs fail to develop after fertilization. Our previous studies have shown that the primary cause of infertility can be attributed to oocytes rather than their surrounding somatic cells in the XY ovary. This study attempted to identify the defects in oocytes from the B6.Y(TIR) female mouse. We examined the developmental potential of embryos from XY and XX females after exchanging their nuclear components by microsurgery following in vitro maturation and fertilization. The results suggest that both nuclear and cytoplasmic components are defective in oocytes from XY females. In the XY fetal ovary, most germ cells entered meiosis and their autosomes appeared to synapse normally while the X and Y chromosomes remained unpaired during meiotic prophase. This lack of X-Y pairing probably caused aneuploidy in some secondary oocytes following in vitro maturation. However, normal numbers of chromosomes in the rest of the secondary oocytes indicate that aneuploidy alone can not explain the nuclear defect in oocytes. Copyright 2000 Academic Press.

Interaction of the B cell-specific transcriptional coactivator OCA-B and galectin-1 and a possible role in regulating BCR-mediated B cell proliferation.

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Yu, Xin; Siegel, Rachael; Roeder, Robert G

2006-06-02

OCA-B is a B cell-specific transcriptional coactivator for OCT factors during the activation of immunoglobulin genes. In addition, OCA-B is crucial for B cell activation and germinal center formation. However, the molecular mechanisms for OCA-B function in these processes are not clear. Our previous studies documented two OCA-B isoforms and suggested a novel mechanism for the function of the myristoylated, membrane-bound form of OCA-B/p35 as a signaling molecule. Here, we report the identification of galectin-1, and related galectins, as a novel OCA-B-interacting protein. The interaction of OCA-B and galectin-1 can be detected both in vivo and in vitro. The galectin-1 binding domain in OCA-B has been localized to the N terminus of OCA-B. In B cells lacking OCA-B expression, increased galectin-1 expression, secretion, and cell surface association are observed. Consistent with these observations, and a reported inhibitory interaction of galectin-1 with CD45, the phosphatase activity of CD45 is reduced modestly, but significantly, in OCA-B-deficient B cells. Finally, galectin-1 is shown to negatively regulate B cell proliferation and tyrosine phosphorylation upon BCR stimulation. Together, these results raise the possibility that OCA-B may regulate BCR signaling through an association with galectin-1.

Supply with the vitamins B1, B2 and B6 in carcinomas before and after radiotherapy

International Nuclear Information System (INIS)

Grimm, U.; Wulff, K.; Schmidt, W.

1983-01-01

In 108 breast cancer, 63 cervix carcinoma, 35 corpus carcinoma and 15 ovarial cancer patients the erythrocyte transketolase, gluthathione reductase and aspartate aminotransferase activity were determined as parameters for the supply with vitamin B 1 , B 2 and B 6 before and after radiotherapy. The effects of thiamine pyrophosphate determined in cancer patients were normal but the effects of flavin adenine dinucleotide and pyridoxal-5-phosphate were significantly increased compared to the controls. These results revealed radiation-induced disorders in the B 2 metabolism and tumor-induced disorders in the B 6 metabolism. Both disorders can be avoided by treatment with vitamin B complex. (author)

Different Effects of Six Antibiotics and Ten Traditional Chinese Medicines on Shiga Toxin Expression by Escherichia coli O157:H7

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Mei Ling Chen

2013-01-01

Full Text Available This study compared the effects of ten types of traditional Chinese medicines (TCMs and six different antibiotics on E. coli O157:H7 Shiga toxin gene (stx2 mRNA expression level based on real-time PCR and the expression level of Stx toxin using an ELISA quantitative assay. We also compared their effects on the induction of the SOS response. The results clearly indicated that all ten TCMs had negative results in the SOS response induction test, while most TCMs did not increase the levels of stx2 mRNA and the Stx toxin. Some TCMs did increase the mRNA levels of the stx2 gene and the Stx toxin level, but their increases were much lower than those caused by antibiotics. With the exception of cefotaxime, the six antibiotics increased the Stx toxin level and increased the stx2 gene mRNA level. With the exceptions of cefotaxime and tetracycline, the antibiotics increased the SOS induction response. These results suggest that TCMs may have advantages compared with antibiotics, when treating E. coli O157:H7; TCMs did not greatly increase Stx toxin production and release.

Complex inheritance of melanoma and pigmentation of coat and skin in Grey horses.

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Ino Curik

Full Text Available The dominant phenotype of greying with age in horses, caused by a 4.6-kb duplication in intron 6 of STX17, is associated with a high incidence of melanoma and vitiligo-like skin depigmentation. However, the progressive greying and the incidence of melanoma, vitiligo-like depigmentation, and amount of speckling in these horses do not follow a simple inheritance pattern. To understand their inheritance, we analysed the melanoma grade, grey level, vitiligo grade, and speckling grade of 1,119 Grey horses (7,146 measurements measured in six countries over a 9-year period. We estimated narrow sense heritability (h(2, and we decomposed this parameter into polygenic heritability (h(2 (POLY, heritability due to the Grey (STX17 mutation (h(2 (STX17, and heritability due to agouti (ASIP locus (h(2 (ASIP. A high heritability was found for greying (h(2 = 0.79, vitiligo (h(2 = 0.63, and speckling (h(2 = 0.66, while a moderate heritability was estimated for melanoma (h(2 = 0.37. The additive component of ASIP was significantly different from zero only for melanoma (h(2 (ASIP = 0.02. STX17 controlled large proportions of phenotypic variance (h(2 (STX17 = 0.18-0.55 and overall heritability (h(2 (STX17/h(2 = 0.28-0.83 for all traits. Genetic correlations among traits were estimated as moderate to high, primarily due to the effects of the STX17 locus. Nevertheless, the correlation between progressive greying and vitiligo-like depigmentation remained large even after taking into account the effects of STX17. We presented a model where four traits with complex inheritance patterns are strongly influenced by a single mutation. This is in line with evidence of recent studies in domestic animals indicating that some complex traits are, in addition to the large number of genes with small additive effects, influenced by genes of moderate-to-large effect. Furthermore, we demonstrated that the STX17 mutation explains to a large extent the

Serum inhibin B in polycystic ovary syndrome as a potential marker of ovarian dysfunction

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Ćetković Aleksandar

2008-01-01

Full Text Available INTRODUCTION Polycystic ovary syndrome (PCOS is one of the most common causes of anovulation, infertility and hyperandrogenism, and the prevalence of this condition in women of reproductive is 5-10%. The growth of early ovarian antral follicles is arrested and dominant follicle selection is disturbed in this syndrome. OBJECTIVE The aim of this study is to investigate whether inhibin B serum concentrations represent the extent of ovarian abnormalities in patients with PCOS. METHOD Inhibin B serum concentrations on the third day of spontaneous menstrual cycle and other endocrine characteristics were compared between 20 patients with PCOS and 19 healthy women in the control group. RESULTS Inhibin B concentrations were not significantly different between women with PCOS and women in the control group. In patients with PCOS there was statistically significant correlation between serum inhibin B and LH (r=0.514; p=0.021. There were no positive correlations between inhibin B and others endocrine parameters in patients with PCOS (FSH, E2, T, androstenedione. CONCLUSION Inhibin B serum concentrations on the third day of spontaneous menstrual cycle in women with PCOS are not different from the concentrations in healthy women. Serum Inhibin B levels in patients with PCOS are only slightly correlated with the endocrine markers of the disease so it could not represent the magnitude of ovarian dysfunction in this syndrome.

Incorporating epilepsy genetics into clinical practice

DEFF Research Database (Denmark)

Oates, Stephanie; Tang, Shan; Rosch, Richard

2018-01-01

seen in SCN8A, SCN2A, SCN1A, KCNQ2, HNRNPU, GRIN2A, SYNGAP1, STXBP1, STX1B, CDKL5, CHRNA4, PCDH19 and PIGT. Clinician prediction was poor. Clinicians and families rated the service highly. In neonates, the cost of investigations could be reduced from £9362 to £2838 by performing gene panel earlier...

Cannabinoid Receptor 2 (CB2 Plays a Role in the Generation of Germinal Center and Memory B Cells, but Not in the Production of Antigen-Specific IgG and IgM, in Response to T-dependent Antigens.

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Sreemanti Basu

Full Text Available The cannabinoid receptor 2 (CB2 has been reported to modulate B cell functions including migration, proliferation and isotype class switching. Since these processes are required for the generation of the germinal center (GC and antigen-specific plasma and memory cells following immunization with a T-dependent antigen, CB2 has the capacity to alter the quality and magnitude of T-dependent immune responses. To address this question, we immunized WT and CB2(-/- mice with the T-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP-chicken-gamma-globulin (CGG and measured GC B cell formation and the generation of antigen-specific B cells and serum immunoglobulin (Ig. While there was a significant reduction in the number of splenic GC B cells in CB2(-/- mice early in the response there was no detectable difference in the number of NP-specific IgM and IgG1 plasma cells. There was also no difference in NP-specific IgM and class switched IgG1 in the serum. In addition, we found no defect in the homing of plasma cells to the bone marrow (BM and affinity maturation, although memory B cell cells in the spleen were reduced in CB2(-/- mice. CB2-deficient mice also generated similar levels of antigen-specific IgM and IgG in the serum as WT following immunization with sheep red blood cells (sRBC. This study demonstrates that although CB2 plays a role in promoting GC and memory B cell formation/maintenance in the spleen, it is dispensable on all immune cell types required for the generation of antigen-specific IgM and IgG in T-dependent immune responses.

Matrix effect on paralytic shellfish toxins quantification and toxicity estimation in mussels exposed to Gymnodinium catenatum.

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Botelho, M J; Vale, C; Mota, A M; Rodrigues, S M; Costa, P R; Simões Gonçalves, M L S

2010-12-01

Paralytic shellfish toxins were quantified in whole tissues of the mussel Mytilus galloprovincialis exposed to blooms of the dinoflagellate Gymnodinium catenatum in Portuguese coastal waters. A validated liquid chromatography method with fluorescence detection, involving pre-chromatographic oxidation was used to quantify carbamoyl, N-sulfocarbamoyl and decarbamoyl toxins. In order to test for any matrix effect in the quantification of those toxins, concentrations obtained from solvent and matrix matched calibration curves were compared. A suppression of the fluorescence signal was observed in mussel extract or fraction in comparison to solvent for the compounds dcGTX2 + 3, GTX2 + 3 and GTX1 + 4, while an enhancement was found for C1 + 2, dcSTX, STX, B1, dcNEO and NEO. These results showed that a matrix effect varies among compounds. The difference of concentrations between solvent and matrix matched calibration curves for C1 + 2 (median = 421 ng g⁻¹) exceeded largely the values for the other quantified compounds (0.09-58 ng g⁻¹). Those differences were converted into toxicity differences, using Oshima toxicity equivalence factors. The compounds C1 + 2 and dcNEO were the major contributors to the differences of total toxicity in the mussel samples. The differences of total toxicity were calculated in ten mussel samples collected during a 10-week blooming period in Portuguese coastal lagoon. Values varied between 53 and 218 µg STX equivalents kg⁻¹. The positive differences mean that the estimated toxicity using solvent calibration curves exceed the values taking into account the matrix. For the toxicity interval 200-800 µg STX equivalents kg⁻¹ an increase was found between 44 and 28%.

Clinical value of serum vitamin B12 and folate in cerebrovascular disease

International Nuclear Information System (INIS)

Zhan Hao; Zhang Yongxue

2002-01-01

To study the clinical value of serum vitamin B 12 and folate in cerebrovascular disease, the concentration of serum vitamin B 12 and folate in 32 patients with cerebrovascular disease was measured by radioimmunoassay. The results showed that the changes in folate in all groups were not significant. The content of vitamin B 12 in multi-infarct dementia was markedly lower than that in cerebral infarction and cerebral hemorrhage. Moreover, the level of vitamin B 12 was lower in paralytic patients with muscular strength of grade 0-III. It can be concluded that serum vitamin B 12 level had association with intelligent disorder and paralytic degree

Liver-Specific Deletion of Protein-Tyrosine Phosphatase 1B (PTP1B) Improves Metabolic Syndrome and Attenuates Diet-Induced Endoplasmic Reticulum Stress

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Delibegovic, Mirela; Zimmer, Derek; Kauffman, Caitlin; Rak, Kimberly; Hong, Eun-Gyoung; Cho, You-Ree; Kim, Jason K.; Kahn, Barbara B.; Neel, Benjamin G.; Bence, Kendra K.

2009-01-01

OBJECTIVE—The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling; consequently, mice deficient in PTP1B are hypersensitive to insulin. Because PTP1B−/− mice have diminished fat stores, the extent to which PTP1B directly regulates glucose homeostasis is unclear. Previously, we showed that brain-specific PTP1B−/− mice are protected against high-fat diet–induced obesity and glucose intolerance, whereas muscle-specific PTP1B−/− mice have increased insulin sensitivity independent of changes in adiposity. Here we studied the role of liver PTP1B in glucose homeostasis and lipid metabolism. RESEARCH DESIGN AND METHODS—We analyzed body mass/adiposity, insulin sensitivity, glucose tolerance, and lipid metabolism in liver-specific PTP1B−/− and PTP1Bfl/fl control mice, fed a chow or high-fat diet. RESULTS—Compared with normal littermates, liver-specific PTP1B−/− mice exhibit improved glucose homeostasis and lipid profiles, independent of changes in adiposity. Liver-specific PTP1B−/− mice have increased hepatic insulin signaling, decreased expression of gluconeogenic genes PEPCK and G-6-Pase, enhanced insulin-induced suppression of hepatic glucose production, and improved glucose tolerance. Liver-specific PTP1B−/− mice exhibit decreased triglyceride and cholesterol levels and diminished expression of lipogenic genes SREBPs, FAS, and ACC. Liver-specific PTP1B deletion also protects against high-fat diet–induced endoplasmic reticulum stress response in vivo, as evidenced by decreased phosphorylation of p38MAPK, JNK, PERK, and eIF2α and lower expression of the transcription factors C/EBP homologous protein and spliced X box-binding protein 1. CONCLUSIONS—Liver PTP1B plays an important role in glucose and lipid metabolism, independent of alterations in adiposity. Inhibition of PTP1B in peripheral tissues may be useful for the treatment of metabolic syndrome and reduction of cardiovascular risk in addition to

Molecular characterization and phylogeny of Shiga toxin-producing E. coli (STEC) from imported beef meat in Malaysia.

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Abuelhassan, Nawal Nouridaim; Mutalib, Sahilah Abdul; Gimba, Fufa Ido; Yusoff, Wan Mohtar

2016-09-01

This study aimed at determining the presence and characterization of Escherichia coli and Shiga toxin-producing E. coli (STEC) from imported frozen beef meats. Seventy-four (74) frozen imported beef meat samples from two countries, India (42 samples) and Australia (32 samples), were collected and tested for E. coli. These samples were purchased from the frozen meat sections of five different supermarkets in different locations in Selangor, Malaysia, from April 2012 to October 2014. A total of 222 E. coli strains were isolated from the meat samples; 126 strains were isolated from country A (India), and 96 E. coli strains were from country of origin B (Australia), respectively. A total of 70 E. coli strains were identified and characterized. All E. coli strains were isolated into Fluorocult medium and identified using API 20E kit. All selected E. coli strains were characterized for Shiga toxin genes (stx1 and stx2). All biochemically identified E. coli in this study were further subjected to molecular detection through polymerase chain reaction (PCR) amplification and characterization using 16S ribosomal RNA (rRNA) gene of Shiga toxin-producing E. coli. Of the 70 E. coli strains, 11 strains were positive for both Shiga toxin genes (stx1 and stx2) and 11 (11/70) strains were positive for stx1 gene, while 25 (25/70) strains were positive for stx2 gene. The analysis of 16S rRNA gene of all the E. coli isolates in this study was successfully sequenced and analyzed, and based on sequence data obtained, a phylogenetic tree of the 16S rRNA gene was performed using Clustal W programme in MEGA 6.06 software. Phylogenetic tree showed that the E. coli isolates in our study cluster with the strain of E. coli isolated in other countries, which further confirm that the isolates of E. coli in this study are similar to those obtained in other studies. As a result, all the strains obtained in this study proved to be a strain of pathogenic E. coli, which may cause a serious outbreak

Elevated Serum Cyclophilin B Levels Are Associated with the Prevalence and Severity of Metabolic Syndrome

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Hang Zhang

2017-12-01

Full Text Available ObjectiveInflammation plays a central role in the pathogenesis of metabolic syndrome (MetS. Cyclophilin B (CypB can be constitutively secreted in response to inflammatory stimuli and oxidative stress, participating in tissue or systemic inflammation. We investigated the relationship between CypB and MetS in both humans and mice.MethodsSerum CypB levels were determined in 211 subjects with MetS and 292 subjects without MetS (non-MetS (133 healthy controls and 159 high-risk subjects with one to two MetS components. Additionally, CypB expression in metabolic organs was examined in mice fed with high-fat diet (HFD and genetically obese (ob/ob mice.ResultsSerum CypB level was significantly higher in MetS subjects compared with both groups of non-MetS subjects (193.80 ± 83.22 vs. 168.38 ± 65.01 vs. 124.26 ± 47.83 ng/mL, P < 0.001. Particularly, serum CypB level was significantly higher in subjects with hypertension, central obesity, diabetes mellitus or hyperglycemia, elevated levels of triglycerides, or reduced levels of high-density lipoprotein than in those without. Moreover, CypB was positively associated with the number of MetS components (r = 0.404, P < 0.001, indicating that a higher serum CypB level reflected more severe MetS. Multivariate regression revealed that a one SD increase in CypB was associated with an odds ratio of 1.506 (1.080–2.101, P = 0.016 for MetS prevalence after adjusting for age, gender, conventional risk factors, and medication. Stratified analyses by age and gender demonstrated that subjects >60 years old with higher CypB levels were more likely to have MetS, and the risk for MetS was higher and more significant in women compared with men. Additionally, CypB expression levels were lower at baseline and dramatically enhanced in metabolic organs (such as the liver and visceral and subcutaneous adipose tissue from HFD-induced obese mice and ob/ob mice.ConclusionIncreased CypB levels were

Noninvasive scoring system for significant inflammation related to chronic hepatitis B

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Hong, Mei-Zhu; Ye, Linglong; Jin, Li-Xin; Ren, Yan-Dan; Yu, Xiao-Fang; Liu, Xiao-Bin; Zhang, Ru-Mian; Fang, Kuangnan; Pan, Jin-Shui

2017-03-01

Although a liver stiffness measurement-based model can precisely predict significant intrahepatic inflammation, transient elastography is not commonly available in a primary care center. Additionally, high body mass index and bilirubinemia have notable effects on the accuracy of transient elastography. The present study aimed to create a noninvasive scoring system for the prediction of intrahepatic inflammatory activity related to chronic hepatitis B, without the aid of transient elastography. A total of 396 patients with chronic hepatitis B were enrolled in the present study. Liver biopsies were performed, liver histology was scored using the Scheuer scoring system, and serum markers and liver function were investigated. Inflammatory activity scoring models were constructed for both hepatitis B envelope antigen (+) and hepatitis B envelope antigen (-) patients. The sensitivity, specificity, positive predictive value, negative predictive value, and area under the curve were 86.00%, 84.80%, 62.32%, 95.39%, and 0.9219, respectively, in the hepatitis B envelope antigen (+) group and 91.89%, 89.86%, 70.83%, 97.64%, and 0.9691, respectively, in the hepatitis B envelope antigen (-) group. Significant inflammation related to chronic hepatitis B can be predicted with satisfactory accuracy by using our logistic regression-based scoring system.

Role of bacteriophages in STEC infections: new implications for the design of prophylactic and treatment approaches [v2; ref status: indexed, http://f1000r.es/437

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Jaime H. Amorim

2014-08-01

Full Text Available Shiga toxin (Stx is considered the main virulence factor in Shiga toxin-producing Escherichia coli (STEC infections. Previously we reported the expression of biologically active Stx by eukaryotic cells in vitro and in vivo following transfection with plasmids encoding Stx under control of the native bacterial promoter1,2. Since stx genes are present in the genome of lysogenic bacteriophages, here we evaluated the relevance of bacteriophages during STEC infection. We used the non-pathogenic E. coli C600 strain carrying a lysogenic 933W mutant bacteriophage in which the stx operon was replaced by a gene encoding the green fluorescent protein (GFP. Tracking GFP expression using an In Vivo Imaging System (IVIS, we detected fluorescence in liver, kidney, and intestine of mice infected with the recombinant E. coli strain after treatment with ciprofloxacin, which induces the lytic replication and release of bacteriophages. In addition, we showed that chitosan, a linear polysaccharide composed of d-glucosamine residues and with a number of commercial and biomedical uses, had strong anti-bacteriophage effects, as demonstrated at in vitro and in vivo conditions. These findings bring promising perspectives for the prevention and treatment of haemolytic uremic syndrome (HUS cases.

Sex steroids do not affect shigatoxin cytotoxicity on human renal tubular or glomerular cells

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Kohan Donald E

2002-08-01

Full Text Available Abstract Background The greater susceptibility of children to renal injury in post-diarrheal hemolytic-uremic syndrome (HUS may be related, at least in part, to heightened renal cell sensitivity to the cytotoxic effect of Shiga toxin (Stx, the putative mediator of kidney damage in HUS. We hypothesized that sexual maturation, which coincides with a falling incidence of HUS, may induce a relatively Stx-resistant state in the renal cells. Methods Cultured human glomerular endothelial (HGEN, human glomerular visceral epithelial (HGEC and human proximal tubule (HPT cells were exposed to Stx-1 after pre-incubation with progesterone, β-estradiol or testosterone followed by determination of cytotoxicity. Results Under basal conditions, Stx-1 potently and dose-dependently killed HPT and HGEC, but had relatively little effect on HGEN. Pre-incubation for 1, 2 or 7 days with physiologic or pharmacologic concentrations of progesterone, β-estradiol or testosterone had no effect on Stx-1 cytotoxicity dose-response on any cell type. In addition, no steroid altered Gb3 expression (Stx receptor by any cell type at any time point. Conclusion These data do not support the notion that hormonal changes associated with puberty induce an Stx-resistant state within kidney cells.

Gene Panel Testing in Epileptic Encephalopathies and Familial Epilepsies

DEFF Research Database (Denmark)

Møller, Rikke S.; Larsen, Line H.G.; Johannesen, Katrine M.

2016-01-01

-causing variant in 49 (23%) of the 216 patients. The variants were found in 19 different genes including SCN1A, STXBP1, CDKL5, SCN2A, SCN8A, GABRA1, KCNA2, and STX1B. Patients with neonatal-onset epilepsies had the highest rate of positive findings (57%). The overall yield for patients with EEs was 32%, compared...

Protection from hemolytic uremic syndrome by eyedrop vaccination with modified enterohemorrhagic E. coli outer membrane vesicles.

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Kyoung Sub Choi

Full Text Available We investigated whether eyedrop vaccination using modified outer membrane vesicles (mOMVs is effective for protecting against hemolytic uremic syndrome (HUS caused by enterohemorrhagic E. coli (EHEC O157:H7 infection. Modified OMVs and waaJ-mOMVs were prepared from cultures of MsbB- and Shiga toxin A subunit (STxA-deficient EHEC O157:H7 bacteria with or without an additional waaJ mutation. BALB/c mice were immunized by eyedrop mOMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB. Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs at 4 weeks. As parameters for evaluation of the OMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN/Creatinin (Cr were tested, as well as histopathology of renal tissue. In order to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice. Modified OMVs having penta-acylated lipid A moiety did not contain STxA subunit proteins but retained non-toxic Shiga toxin B (STxB subunit. Removal of the polymeric O-antigen of O157 LPS was confirmed in waaJ-mOMVs. The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses, suggesting that intact O157 LPS antigen can be a critical component for enhancing the immunogenicity of the mOMVs. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not. Collectively, for the first time, EHEC O157-derived mOMV eyedrop vaccine was experimentally evaluated as an efficient and safe means of vaccine development against EHEC O157:H7 infection-associated HUS.

Detección de Escherichia coli O157: H7 en carne picada fresca y hamburguesas congeladas Escherichia coli O157: H7 detection in fresh ground beef and hamburgers

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M. A. Marzocca

2006-03-01

Full Text Available Escherichia coli O157:H7 es un patógeno emergente asociado a enfermedades transmitidas por alimentos. En el año 1982 fue reconocido por primera vez, en los Estados Unidos, como causante de dos brotes de colitis hemorrágica. Hoy se sabe que la mayoría de los casos de síndrome urémico hemolítico son ocasionados por este microorganismo. El objetivo del trabajo fue detectar su presencia en carne picada fresca y hamburguesas congeladas, muestras obtenidas en puntos de venta de nuestra cadena de supermercados en un total de 37 y 43, respectivamente, en el período comprendido entre abril de 2003 y agosto de 2004. Las mismas fueron procesadas utilizando el caldo selectivo para enriquecimiento EC modificado conteniendo novobiocina, seguido de la aplicación de un método de inmunocaptura (TECRA E. COLI O157 IMMUNOCAPTURE TM ECOICM 20, y posterior aislamiento en agar MacConkey sorbitol suplementado con cefixima y telurito de potasio y un medio cromogénico. Las cepas sospechosas fueron caracterizadas a genotípicamente mediante la detección de los genes de virulencia stx1, stx2, eaeA y EHEC-hlyA por PCR e hibridación con sondas genéticas, b fenotípicamente mediante la determinación del serotipo, sensibilidad a los antimicrobianos por el método de difusión de Kirby-Bauer y producción de Stx por ensayo de citotoxicidad específica en células Vero. Se aisló E. coli O157:H7 en una sola muestra de carne picada fresca (2,7% caracterizada como gen eae (+/stx2/EHEC-hlyA.Escherichia coli O157:H7 is an emergent pathogen associated with food transmitted diseases. In 1982, Escherichia coli 0157:H7 was for the first time identified as the cause of two hemorrhagic colitis outbreaks in the United States. It is now well known that most cases of hemolytic uremic syndrome are caused by these bacteria. The objective of this work was to detect the microorganism in fresh ground beef and hamburgers. From April 2003 to August 2004 samples were taken at sale

HTLV-1 specific CD8+ T cell function augmented by blockade of 2B4/CD48 interaction in HTLV-1 infection.

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Chibueze Chioma Ezinne

Full Text Available CD8+ T cell response is important in the response to viral infections; this response though is regulated by inhibitory receptors. Expression of inhibitory receptors has been positively correlated with CD8+ T cell exhaustion; the consequent effect of simultaneous blockade of these inhibitory receptors on CD8+ T cell response in viral infections have been studied, however, the role of individual blockade of receptor-ligand pair is unclear. 2B4/CD48 interaction is involved in CD8+T cell regulation, its signal transducer SAP (signaling lymphocyte activation molecule (SLAM-associated protein is required for stimulatory function of 2B4/CD244 on lymphocytes hence, we analyzed 2B4/CD244 (natural killer cell receptor and SAP (signaling lymphocyte activation molecule(SLAM-associated protein on total CD8+ and HTLV-1 specific CD8+T cells in HTLV-1 infection and the effect of blockade of interaction with ligand CD48 on HTLV-1 specific CD8+ T cell function. We observed a high expression of 2B4/CD244 on CD8+ T cells relative to uninfected and further upregulation on HTLV-1 specific CD8+ T cells. 2B4+ CD8+ T cells exhibited more of an effector and terminally differentiated memory phenotype. Blockade of 2B4/CD48 interaction resulted in improvement in function via perforin expression and degranulation as measured by CD107a surface mobilization on HTLV-1 specific CD8+ T cells. In the light of these findings, we thus propose an inhibitory role for 2B4/CD48 interaction on CD8+T cell function.

Tissue-specific alternative splicing and expression of ATP1B2 gene ...

African Journals Online (AJOL)

After heat-stress, the expression levels of the different transcripts were lower in different tissues; however, the expression of the ATP1B2-complete transcript increased in heart and lung tissues. The results of this research provide some useful information for further studies into the function of the bovine ATP1B2 gene.

Hydroxysteroid sulfotransferase SULT2B1b promotes hepatocellular carcinoma cells proliferation in vitro and in vivo.

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Xiaoming Yang

Full Text Available Hydroxysteroid sulfotransferase 2B1b (SULT2B1b is highly selective for the addition of sulfate groups to 3β-hydroxysteroids. Although previous reports have suggested that SULT2B1b is correlated with cell proliferation of hepatocytes, the relationship between SULT2B1b and the malignant phenotype of hepatocarcinoma cells was not clear. In the present study, we found that SULT2B1 was comparatively higher in the human hepatocarcinoma tumorous tissues than their adjacent tissues. Besides, SULT2B1b overexpression promoted the growth of the mouse hepatocarcinoma cell line Hepa1-6, while Lentivirus-mediated SULT2B1b interference inhibited growth as assessed by the CCK-8 assay. Likewise, inhibition of SULT2B1b expression induced cell-cycle arrest and apoptosis in Hepa1-6 cells by upregulating the expression of FAS, downregulating the expression of cyclinB1, BCL2 and MYC in vitro and in vivo at both the transcript and protein levels. Knock-down of SULT2B1b expression significantly suppressed tumor growth in nude mouse xenografts. Moreover, proliferation rates and SULT2B1b expression were highly correlated in the human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721 and BEL-7402 cells. Knock-down of SULT2B1b inhibited cell growth and cyclinB1 levels in human hepatocarcinoma cells and suppressed xenograft growth in vivo. In conclusion, SULT2B1b expression promotes proliferation of hepatocellular carcinoma cells in vitro and in vivo, which may contribute to the progression of HCC.

Serum inhibin B levels during male childhood and puberty

DEFF Research Database (Denmark)

Andersson, A M; Skakkebaek, N E

2001-01-01

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. In males serum levels of inhibin B are detectable throughout life with prominent changes in the first year of life and during puberty. Serum inhibin B is normally detectable throughout childhood...... normal or near-normal levels are seen in cryptorchidism and disorders with preserved Sertoli cell function in spite of absence of germ cells or impaired androgen biosynthesis or action. During puberty a developmental change in the regulation of serum inhibin B occurs. In contrast to childhood inhibin B...

Expression and subcellular localization of the Qa-SNARE syntaxin17 in human eosinophils.

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Carmo, Lívia A S; Dias, Felipe F; Malta, Kássia K; Amaral, Kátia B; Shamri, Revital; Weller, Peter F; Melo, Rossana C N

2015-10-01

SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos. Copyright © 2015 Elsevier Inc. All rights reserved.

Association of high fetuin-B concentrations in serum with fertilization rate in IVF: a cross-sectional pilot study.

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Floehr, Julia; Dietzel, Eileen; Neulen, Josef; Rösing, Benjamin; Weissenborn, Ute; Jahnen-Dechent, Willi

2016-03-01

triplicate standard deviation was 3.3 ± 1.8%. IVF was declared as 'successful', if at least one oocyte was fertilized, and 'unsuccessful', if no oocyte could be fertilized. Patient age was 34.4 ± 4.4 years in successful IVF, and 35.4 ± 3.3 years in unsuccessful IVF. Serum and follicular fluid of patients undergoing controlled ovarian hyperstimulation were analyzed. Serum was drawn at the day of follicle aspiration. Serum fetuin-B and follicular fluid fetuin-B were not significantly different in six out of nine patients suggesting, in principle, free exchange of fetuin-B between serum and follicular fluid. Thus serum fetuin-B may be used as a proxy of follicular fluid fetuin-B. Serum fetuin-B increased during successful IVF cycles (n = 15, P inventors on the RWTH Aachen University patent application EP 13157317.2, 'Use of fetuin-B for culture of oocytes', applied for by RWTH Aachen University. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Detection of virulence genes and the phylogenetic groups of Escherichia coli isolated from dogs in Brazil

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Fernanda Morcatti Coura

2018-02-01

Full Text Available ABSTRACT: This study identified the virulence genes, pathovars, and phylogenetic groups of Escherichia coli strains obtained from the feces of dogs with and without diarrhea. Virulence genes and phylogenetic group identification were studied using polymerase chain reaction. Thirty-seven E. coli isolates were positive for at least one virulence factor gene. Twenty-one (57.8% of the positive isolates were isolated from diarrheal feces and sixteen (43.2% were from the feces of non-diarrheic dogs. Enteropathogenic E. coli (EPEC were the most frequently (62.2% detected pathovar in dog feces and were mainly from phylogroup B1 and E. Necrotoxigenic E. coli were detected in 16.2% of the virulence-positive isolates and these contained the cytotoxic necrotizing factor 1 (cnf1 gene and were classified into phylogroups B2 and D. All E. coli strains were negative for the presence of enterotoxigenic E. coli (ETEC enterotoxin genes, but four strains were positive for ETEC-related fimbriae 987P and F18. Two isolates were Shiga toxin-producing E. coli strains and contained the toxin genesStx2 or Stx2e, both from phylogroup B1. Our data showed that EPEC was the most frequent pathovar and B1 and E were the most common phylogroups detected in E. coli isolated from the feces of diarrheic and non-diarrheic dogs.

PD-L2 Regulates B-1 Cell Antibody Production against Phosphorylcholine through an IL-5-Dependent Mechanism.

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McKay, Jerome T; Haro, Marcela A; Daly, Christina A; Yammani, Rama D; Pang, Bing; Swords, W Edward; Haas, Karen M

2017-09-15

B-1 cells produce natural Abs which provide an integral first line of defense against pathogens while also performing important homeostatic housekeeping functions. In this study, we demonstrate that programmed cell death 1 ligand 2 (PD-L2) regulates the production of natural Abs against phosphorylcholine (PC). Naive PD-L2-deficient (PD-L2 -/- ) mice produced significantly more PC-reactive IgM and IgA. This afforded PD-L2 -/- mice with selectively enhanced protection against PC-expressing nontypeable Haemophilus influenzae , but not PC-negative nontypeable Haemophilus influenzae , relative to wild-type mice. PD-L2 -/- mice had significantly increased PC-specific CD138 + splenic plasmablasts bearing a B-1a phenotype, and produced PC-reactive Abs largely of the T15 Id. Importantly, PC-reactive B-1 cells expressed PD-L2 and irradiated chimeras demonstrated that B cell-intrinsic PD-L2 expression regulated PC-specific Ab production. In addition to increased PC-specific IgM, naive PD-L2 -/- mice and irradiated chimeras reconstituted with PD-L2 -/- B cells had significantly higher levels of IL-5, a potent stimulator of B-1 cell Ab production. PD-L2 mAb blockade of wild-type B-1 cells in culture significantly increased CD138 and Blimp1 expression and PC-specific IgM, but did not affect proliferation. PD-L2 mAb blockade significantly increased IL-5 + T cells in culture. Both IL-5 neutralization and STAT5 inhibition blunted the effects of PD-L2 mAb blockade on B-1 cells. Thus, B-1 cell-intrinsic PD-L2 expression inhibits IL-5 production by T cells and thereby limits natural Ab production by B-1 cells. These findings have broad implications for the development of therapeutic strategies aimed at altering natural Ab levels critical for protection against infectious disease, autoimmunity, allergy, cancer, and atherosclerosis. Copyright © 2017 by The American Association of Immunologists, Inc.

Clonal Diversity of Chilean Isolates of Enterohemorrhagic Escherichia coli from Patients with Hemolytic-Uremic Syndrome, Asymptomatic Subjects, Animal Reservoirs, and Food Products

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Rios, Maritza; Prado, Valeria; Trucksis, Michele; Arellano, Carolina; Borie, Consuelo; Alexandre, Marcela; Fica, Alberto; Levine, Myron M.

1999-01-01

To determine clonal relationship among Chilean enterohemorrhagic Escherichia coli (EHEC) strains from different sources (clinical infections, animal reservoirs, and food), 54 EHEC isolates (44 of E. coli O157, 5 of E. coli O111, and 5 of E. coli O26) were characterized for virulence genes by colony blot hybridization and by pulsed-field gel electrophoresis (PFGE). By colony blotting, 12 different genotypes were identified among the 44 E. coli O157 isolates analyzed, of which the genetic profile stx1+ stx2+ hly+ eae+ was the most prevalent. All human O157 strains that were associated with sporadic cases of hemolytic-uremic syndrome (HUS) carried both the stx1 and stx2 toxin-encoding genes and were eaeA positive. Only 9 of 13 isolates from human controls were stx1+ stx2+, and 8 carried the eaeA gene. Comparison of profiles obtained by PFGE of XbaI-digested genomic DNA showed a great diversity among the E. coli O157 isolates, with 37 different profiles among 39 isolates analyzed. Cluster analysis of PFGE profiles showed a wide distribution of clinical isolates obtained from HUS cases and asymptomatic individuals and a clonal relationship among O157 isolates obtained from HUS cases and pigs. Analysis of virulence genes showed that a correlation exists among strains with the genotype stx1+ stx2+ eae+ and pathogenic potential. A larger difference in the PFGE restriction patterns was observed among the EHEC strains of serogroups O26 and O111. These results indicate that several different EHEC clones circulate in Chile and suggest that pigs are an important animal reservoir for human infections by EHEC. Guidelines have been proposed for better practices in the slaughter of animals in Chile. PMID:9986852

Neuronal SH2B1 is essential for controlling energy and glucose homeostasis.

Science.gov (United States)

Ren, Decheng; Zhou, Yingjiang; Morris, David; Li, Minghua; Li, Zhiqin; Rui, Liangyou

2007-02-01

SH2B1 (previously named SH2-B), a cytoplasmic adaptor protein, binds via its Src homology 2 (SH2) domain to a variety of protein tyrosine kinases, including JAK2 and the insulin receptor. SH2B1-deficient mice are obese and diabetic. Here we demonstrated that multiple isoforms of SH2B1 (alpha, beta, gamma, and/or delta) were expressed in numerous tissues, including the brain, hypothalamus, liver, muscle, adipose tissue, heart, and pancreas. Rat SH2B1beta was specifically expressed in neural tissue in SH2B1-transgenic (SH2B1(Tg)) mice. SH2B1(Tg) mice were crossed with SH2B1-knockout (SH2B1(KO)) mice to generate SH2B1(TgKO) mice expressing SH2B1 only in neural tissue but not in other tissues. Systemic deletion of the SH2B1 gene resulted in metabolic disorders in SH2B1(KO) mice, including hyperlipidemia, leptin resistance, hyperphagia, obesity, hyperglycemia, insulin resistance, and glucose intolerance. Neuron-specific restoration of SH2B1beta not only corrected the metabolic disorders in SH2B1(TgKO) mice, but also improved JAK2-mediated leptin signaling and leptin regulation of orexigenic neuropeptide expression in the hypothalamus. Moreover, neuron-specific overexpression of SH2B1 dose-dependently protected against high-fat diet-induced leptin resistance and obesity. These observations suggest that neuronal SH2B1 regulates energy balance, body weight, peripheral insulin sensitivity, and glucose homeostasis at least in part by enhancing hypothalamic leptin sensitivity.

Profiling trait anxiety: transcriptome analysis reveals cathepsin B (Ctsb as a novel candidate gene for emotionality in mice.

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Ludwig Czibere

Full Text Available Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait "anxiety". We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB or low (LAB anxiety-related behavior, and also show signs of comorbid depression-like behavior. We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7, cathepsin B (Ctsb, muscleblind-like 1 (Mbnl1, metallothionein 1 (Mt1, solute carrier family 25 member 17 (Slc25a17, tribbles homolog 2 (Trib2, zinc finger protein 672 (Zfp672, syntaxin 3 (Stx3, ATP-binding cassette, sub-family A member 2 (Abca2, ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5, high mobility group nucleosomal binding domain 3 (Hmgn3 and pyruvate dehydrogenase beta (Pdhb. Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4.Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse model's behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females. Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy.

Serum S100B: A possible biomarker for severity of obstructive sleep apnea

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Eman Riad

2017-10-01

Conclusion: Serum S100B protein was significantly elevated in OSA patients and its serum levels correlated with the severity of the disease. Increased serum S100B could indicat brain injury and could be a potential serum biomarker for detection of early neurological complications in OSA patients that could improve the management and care of these patients.

Molecular characterization of Escherichia coli recovered from traditional milk products in Kashan, Iran

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Farhad Sharafati Chaleshtori

2017-10-01

Full Text Available Aim: Shiga toxigenic Escherichia coli (STEC strains as emerging groups of foodborne pathogens are responsible for most foodborne illnesses. The aim of this study was to determine the antibiotic resistance pattern in STEC isolated from traditional milk products and their molecular characterization. Materials and Methods: A total of 116 samples were randomly purchased from local markets in Kashan, Iran, and evaluated for the occurrence of STEC by culturing and molecular methods. The antibiotic resistance of obtained isolates was determined by Kirby Bauer method. Furthermore, isolates were assayed for the presence of Shiga toxins (stx1 and stx2 and intimin gene (eae. Results: The incidence of E. coli in 60 ice cream, 30 yoghurt, and 26 cheese samples was 8.33%, 10%, and 11.54%, respectively. The findings showed that 11 out of 11 (100% E. coli had both stx1 and stx2 while eae gene was not found in E. coli isolated of traditional milk products. For E. coli strains carrying stx1 and stx2, highest antibiotic sensitive levels were related to trimethoprim/sulfamethoxazole, norfloxacin, chloramphenicol, and ciprofloxacin, respectively. Conclusion: The results showed relationship between the presence of virulence factors and antimicrobial resistance. These results can be used for further studies on STEC as an emerging foodborne pathogen.

Immunoassay of paralytic shellfish toxins by moving magnetic particles in a stationary liquid-phase lab-on-a-chip.

Science.gov (United States)

Kim, Myoung-Ho; Choi, Suk-Jung

2015-04-15

In this study, we devised a stationary liquid-phase lab-on-a-chip (SLP LOC), which was operated by moving solid-phase magnetic particles in the stationary liquid phase. The SLP LOC consisted of a sample chamber to which a sample and reactants were added, a detection chamber containing enzyme substrate solution, and a narrow channel connecting the two chambers and filled with buffer. As a model system, competitive immunoassays of saxitoxin (STX), a paralytic shellfish toxin, were conducted in the SLP LOC using protein G-coupled magnetic particles (G-MPs) as the solid phase. Anti-STX antibodies, STX-horseradish peroxidase conjugate, G-MPs, and a STX sample were added to the sample chamber and reacted by shaking. While liquids were in the stationary state, G-MPs were transported from the sample chamber to the detection chamber by moving a magnet below the LOC. After incubation to allow the enzymatic reaction to occur, the absorbance of the detection chamber solution was found to be reciprocally related to the STX concentration of the sample. Thus, the SLP LOC may represent a novel, simple format for point-of-care testing applications of enzyme-linked immunosorbent assays by eliminating complicated liquid handling steps. Copyright © 2014 Elsevier B.V. All rights reserved.

Liver stiffness measurement-based scoring system for significant inflammation related to chronic hepatitis B.

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Mei-Zhu Hong

Full Text Available Liver biopsy is indispensable because liver stiffness measurement alone cannot provide information on intrahepatic inflammation. However, the presence of fibrosis highly correlates with inflammation. We constructed a noninvasive model to determine significant inflammation in chronic hepatitis B patients by using liver stiffness measurement and serum markers.The training set included chronic hepatitis B patients (n = 327, and the validation set included 106 patients; liver biopsies were performed, liver histology was scored, and serum markers were investigated. All patients underwent liver stiffness measurement.An inflammation activity scoring system for significant inflammation was constructed. In the training set, the area under the curve, sensitivity, and specificity of the fibrosis-based activity score were 0.964, 91.9%, and 90.8% in the HBeAg(+ patients and 0.978, 85.0%, and 94.0% in the HBeAg(- patients, respectively. In the validation set, the area under the curve, sensitivity, and specificity of the fibrosis-based activity score were 0.971, 90.5%, and 92.5% in the HBeAg(+ patients and 0.977, 95.2%, and 95.8% in the HBeAg(- patients. The liver stiffness measurement-based activity score was comparable to that of the fibrosis-based activity score in both HBeAg(+ and HBeAg(- patients for recognizing significant inflammation (G ≥3.Significant inflammation can be accurately predicted by this novel method. The liver stiffness measurement-based scoring system can be used without the aid of computers and provides a noninvasive alternative for the prediction of chronic hepatitis B-related significant inflammation.

Shiga toxin-producing Escherichia coli (STEC) O22:H8 isolated from cattle reduces E. coli O157:H7 adherence in vitro and in vivo.

Science.gov (United States)

Martorelli, L; Albanese, A; Vilte, D; Cantet, R; Bentancor, A; Zolezzi, G; Chinen, I; Ibarra, C; Rivas, M; Mercado, E C; Cataldi, A

2017-09-01

Shiga toxin-producing Escherichia coli (STEC) are a group of bacteria responsible for food-associated diseases. Clinical features include a wide range of symptoms such as diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome (HUS), a life-threatening condition. Our group has observed that animals naturally colonized with STEC strains of unknown serotype were not efficiently colonized with E. coli O157:H7 after experimental infection. In order to assess the basis of the interference, three STEC strains were isolated from STEC persistently-colonized healthy cattle from a dairy farm in Buenos Aires, Argentina. The three isolated strains are E. coli O22:H8 and carry the stx1 and stx2d genes. The activatable activity of Stx2d was demonstrated in vitro. The three strains carry the adhesins iha, ehaA and lpf O113 . E. coli O22:H8 formed stronger biofilms in abiotic surface than E. coli O157:H7 (eae+, stx2+) and displayed a more adherent phenotype in vitro towards HeLa cells. Furthermore, when both serotypes were cultured together O22:H8 could reduce O157:H7 adherence in vitro. When calves were intragastrically pre-challenged with 10 8 CFU of a mixture of the three STEC strains and two days later challenged with the same dose of the strain E. coli O157:H7 438/99, the shedding of the pathogen was significantly reduced. These results suggest that E. coli O22:H8, a serotype rarely associated with human illness, might compete with O157:H7 at the bovine recto-anal junction, making non-O157 carrying-calves less susceptible to O157:H7 colonization and shedding of the bacteria to the environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Specific heat of MgB2 after irradiation

International Nuclear Information System (INIS)

Wang Yuxing; Bouquet, Frederic; Sheikin, Ilya; Toulemonde, Pierre; Revaz, Bernard; Eisterer, Michael; Weber, Harald W; Hinderer, Joerg; Junod, Alain

2003-01-01

We studied the effect of disorder on the superconducting properties of polycrystalline MgB 2 by specific-heat measurements. In the pristine state, these measurements give a bulk confirmation of the presence of two superconducting gaps with 2Δ 0 /k B T c =1.3 and 3.9 with nearly equal weights. The scattering introduced by irradiation suppresses T c and tends to average the two gaps although less than predicted by theory. We also found that by a suitable irradiation process by fast neutrons, a substantial bulk increase of dH c2 /dT at T c can be obtained without sacrificing more than a few degrees in T c . The upper critical field of the sample after irradiation exceeds 28 T at T→0

Detection of Shiga Toxins by Lateral Flow Assay

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Kathryn H. Ching

2015-04-01

Full Text Available Shiga toxin-producing Escherichia coli (STEC produce shiga toxins (Stxs that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript, we report the development of a colorimetric lateral flow assay (LFA for the rapid detection of Stxs in <10 min using a pair of monoclonal antibodies that bind epitopes common to Stx1 and six Stx2 variants. This LFA provides a rapid and sensitive test for the detection of Stxs directly from STEC culture supernatants or at risk food samples with a 0.1 ng/mL limit of detection (LOD for Stx2a. This Stx LFA is applicable for use in the rapid evaluation of Stx production from cultured E. coli strains or as a tool to augment current methods as part of food safety testing.

Measurement of FA and VitB12 in serum by TRFIA and its clinical application

International Nuclear Information System (INIS)

Zhou Jinhong; Wang Huimin

2006-01-01

To investigate the correlation between diseases and the quantity of folic acid(FA) and vitamine B 12 (VitB 12 ) in serum, the quantity of FA and VitB 12 in serum was measured by TR- FIA. The results showed that the levels of FA and VitB 12 in 68 controlled subjects were 20.13±5.72 nmol/L and 240.45±58.23 pmol/L respectively. The levels of FA and VitB 12 in 6 patients with megaloblastic anemia, 26 with medium-terminal gestation femineity, 7 with liver cirrhosis, 10 with gastric ulcerand 31 with cerebral infarction were significant lower than those in the control group. The level of FA in 24 with malignant hematopathy and 57 with malignant tumor was significantly lower but VitB 12 between the 31 patients with iron deficiency anemia and 12 with CAA and the control group. The amount of the FA in 9 patients with chronic atrophic gastritis and 42 blood donors were lower than that in the control. The determination of FA and VitB 12 levels may be quite significant in the diagnosis, therapy and prognosis of diseases. Laboratories should establish the reference values which fit the locals specifically. (authors)

Inhibin B as a serum marker of spermatogenesis

DEFF Research Database (Denmark)

Jensen, T K; Andersson, A M; Hjollund, N H

1997-01-01

= -0.61, P sperm concentration (r = -0.40, P sperm concentration (r = 0.38, P sperm counts below 20 mill/mL among men who's inhibin B....... All blood samples were analyzed in the same laboratory. Median sperm concentration and the percentage of morphologically normal spermatozoa were significantly higher among men from centre A (56.0 mill/mL and 42.5%), compared with men from centre B (44.8 mill/mL and 39%). Men from centre B had...... reproductive health, may serve as a new marker of spermatogenesis, in addition to sperm concentration and serum FSH....

Towards a Molecular Definition of Enterohemorrhagic Escherichia coli (EHEC): Detection of Genes Located on O Island 57 as Markers To Distinguish EHEC from Closely Related Enteropathogenic E. coli Strains

Science.gov (United States)

Delannoy, Sabine; Beutin, Lothar

2013-01-01

Among strains of Shiga-toxin (Stx) producing Escherichia coli (STEC), seven serogroups (O26, O45, O103, O111, O121, O145, and O157) are associated with severe clinical illness in humans. These strains are also called enterohemorrhagic E. coli (EHEC), and the development of methods for their reliable detection from food has been challenging thus far. PCR detection of major EHEC virulence genes stx1, stx2, eae, and O-serogroup-specific genes is useful but does not identify EHEC strains specifically. Searching for the presence of additional genes issued from E. coli O157:H7 genomic islands OI-122 and OI-71 increases the specificity but does not clearly discriminate EHEC from enteropathogenic E. coli (EPEC) strains. Here, we identified two putative genes, called Z2098 and Z2099, from the genomic island OI-57 that were closely associated with EHEC and their stx-negative derivative strains (87% for Z2098 and 91% for Z2099). Z2098 and Z2099 were rarely found in EPEC (10% for Z2098 and 12% for Z2099), STEC (2 and 15%), and apathogenic E. coli (1% each) strains. Our findings indicate that Z2098 and Z2099 are useful genetic markers for a more targeted diagnosis of typical EHEC and new emerging EHEC strains. PMID:23325824

Correlation between serum vitamin B12 level and peripheral neuropathy in atrophic gastritis

Science.gov (United States)

Yang, Guo-Tao; Zhao, Hong-Ying; Kong, Yu; Sun, Ning-Ning; Dong, Ai-Qin

2018-01-01

AIM To explore the correlation between serum vitamin B12 level and peripheral neuropathy in patients with chronic atrophic gastritis (CAG). METHODS A total of 593 patients diagnosed with chronic gastritis by gastroscopy and pathological examination from September 2013 to September 2016 were selected for this study. The age of these patients ranged within 18- to 75-years-old. Blood pressure, height and weight were measured in each patient, and the body mass index value was calculated. Furthermore, gastric acid, serum gastrin, serum vitamin and serum creatinine tests were performed, and peripheral nerve conduction velocity and Helicobacter pylori (H. pylori) were detected. In addition, the type of gastritis was determined by gastroscopy. The above factors were used as independent variables to analyze chronic gastritis with peripheral neuropathy and vitamin B12 deficiency risk factors, and to analyze the relationship between vitamin B12 levels and peripheral nerve conduction velocity. In addition, in the treatment of CAG on the basis of vitamin B12, patients with peripheral neuropathy were observed. RESULTS Age, H. pylori infection, CAG, vitamin B9 and vitamin B12 were risk factors for the occurrence of peripheral nerve degeneration. Furthermore, CAG and H. pylori infection were risk factors for chronic gastritis associated with vitamin B12 deficiency. Serum vitamin B12 level was positively correlated with sensory nerve conduction velocity in the tibial nerve (R = 0.463). After vitamin B12 supplementation, patients with peripheral neuropathy improved. CONCLUSION Serum vitamin B12 levels in patients with chronic gastritis significantly decreased, and the occurrence of peripheral neuropathy had a certain correlation. CAG and H. pylori infection are risk factors for vitamin B12 deficiency and peripheral neuropathy. When treating CAG, vitamin B12 supplementation can significantly reduce peripheral nervous system lesions. Therefore, the occurrence of peripheral neuropathy

Correlation between serum vitamin B12 level and peripheral neuropathy in atrophic gastritis.

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Yang, Guo-Tao; Zhao, Hong-Ying; Kong, Yu; Sun, Ning-Ning; Dong, Ai-Qin

2018-03-28

To explore the correlation between serum vitamin B12 level and peripheral neuropathy in patients with chronic atrophic gastritis (CAG). A total of 593 patients diagnosed with chronic gastritis by gastroscopy and pathological examination from September 2013 to September 2016 were selected for this study. The age of these patients ranged within 18- to 75-years-old. Blood pressure, height and weight were measured in each patient, and the body mass index value was calculated. Furthermore, gastric acid, serum gastrin, serum vitamin and serum creatinine tests were performed, and peripheral nerve conduction velocity and Helicobacter pylori ( H. pylori ) were detected. In addition, the type of gastritis was determined by gastroscopy. The above factors were used as independent variables to analyze chronic gastritis with peripheral neuropathy and vitamin B12 deficiency risk factors, and to analyze the relationship between vitamin B12 levels and peripheral nerve conduction velocity. In addition, in the treatment of CAG on the basis of vitamin B12, patients with peripheral neuropathy were observed. Age, H. pylori infection, CAG, vitamin B9 and vitamin B12 were risk factors for the occurrence of peripheral nerve degeneration. Furthermore, CAG and H. pylori infection were risk factors for chronic gastritis associated with vitamin B12 deficiency. Serum vitamin B12 level was positively correlated with sensory nerve conduction velocity in the tibial nerve ( R = 0.463). After vitamin B12 supplementation, patients with peripheral neuropathy improved. Serum vitamin B12 levels in patients with chronic gastritis significantly decreased, and the occurrence of peripheral neuropathy had a certain correlation. CAG and H. pylori infection are risk factors for vitamin B12 deficiency and peripheral neuropathy. When treating CAG, vitamin B12 supplementation can significantly reduce peripheral nervous system lesions. Therefore, the occurrence of peripheral neuropathy associated with vitamin B12