Journal of Microbiology & Biology Education, Microbiology and Molecular Biology Reviews. Tous les décès depuis 1970, évolution de l'espérance de vie en France, par département, commune, prénom et nom de famille ! While translucent colonies were occasionally detected in the OpaD+var Gc population, OpaD+nv Gc colonies were uniformly opaque (Fig. The translucent Opa proteins confer a poorly opaque phenotype on Gc colonies grown on commonly used media, and Opa expression can be reliably detected only by immunoblot (38). Création de site internet, Référencement Google, Publicité sur les réseaux sociaux. Expression of Opa proteins, particularly OpaCEA, affects PMN antibacterial activities and Gc survival after exposure to PMNs (4). Therefore, we assessed the ability of adherent, primed PMNs to internalize genetically Opa-deficient Gc by immunofluorescence. Other value added services offered by the Electronic Resources Section (ERS) include cd-burning, printing and document scanning. (B to G) Colonies of parental (B, E), ΔopaBEGK (C, F), and Opaless (D, G) Gc were imaged using white light transilluminated with a mirrored (B to D) or frosted (E to G) substage for opacity-associated phenotypes. Moreover, it is not always possible to identify Opa expression by colony photoopacity. Phenotypically translucent ΔopaBEGK Gc did not express any Opa proteins by Western blotting (Fig. Interactions of Opaless Gc with primary human PMNs. As overlap extension PCR products from opaF, opaG, and opaJ could not be generated, fragments A and B were separately cloned into pSmart or pCR-Blunt TOPO. Prior to this work, a derivative of FA1090 in which all 11 opa genes had been deleted, the ΔopaA-K mutant, was constructed (50, 53). 2C and F), and the opaque subpopulation expressed Opa proteins by Western blotting (Fig. After dilution to an optical density at 550 nm (OD550) of 0.07, optical densities were measured, and CFU/ml were enumerated from each culture every hour for 3 h. Growth curves were not extended past 3 h since Gc begins to undergo autolysis as it approaches the stationary phase. Arrows point to translucent colonies in the OpaD+var population. Lun-Ven 9h30-19h [email protected] nous contacter. Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00171-13. TPL is one of the world's busiest urban public library systems, with 100 branches across the city and over 10 million books, movies, and other items to borrow or download online. NLB promotes reading, learning and information literacy by providing a trusted, accessible and globally-connected library and information service through the National Library and a comprehensive network of Public Libraries. Identification of surface proteins of, Up-regulation of human neutrophil receptors for, The specific innate immune receptor CEACAM3 triggers neutrophil bactericidal activities via a Syk kinase-dependent pathway, Opa (protein II) influences gonococcal organization in colonies, surface appearance, size and attachment to human fallopian tube tissues, Multiple gonococcal pilin antigenic variants are produced during experimental human infections, An alternative DNA structure is necessary for pilin antigenic variation in, Gene splicing and mutagenesis by PCR-driven overlap extension, Gonococci exit apically and basally from polarized epithelial cells and exhibit dynamic changes in type IV pili, Purification and visualization of lipopolysaccharide from Gram-negative bacteria by hot aqueous-phenol extraction, Loss of both Holliday junction processing pathways is synthetically lethal in the presence of gonococcal pilin antigenic variation, Experimental gonococcal genital tract infection and opacity protein expression in estradiol-treated mice, Anaerobic growth of gonococci does not alter their Opa-mediated interactions with human neutrophils, Investigation of oxidative stress defenses of, A strain-specific catalase mutation and mutation of the metal-binding transporter gene, Experimental gonococcal infection in male volunteers: cumulative experience with, Estradiol-treated female mice as surrogate hosts for, Construction of Opa-positive and Opa-negative strains of, Macropinocytosis: an endocytic pathway for internalising large gulps, Antigonococcal activity of human neutrophil cathepsin G, Studies on gonococcus infection. Status and Prospect for the Sustainable Development of the Maritime Education Program of LB By: Dr. Rolando Lontoc Sr. (2007) Show Abstract. (ii) Liquid medium.Gc was grown in GCBL modified with Kellogg's supplements I and II and 0.042% sodium bicarbonate as previously described (33). Among these isolates was variable OpaD+ Gc (OpaD+var). Bacterial survival at each concentration was calculated as the ratio of the number of CFU recovered after 45 min divided by the number of CFU present at the start of the experiment and is presented relative to survival in the absence of polymyxin B (100%). Creation of Opaless FA1090 Gc. 3A. It's a collection of multiple types of lists used during security assessments, collected in one place. MarketingTracer SEO Dashboard, created for webmasters and agencies. One mutant clone served as the parent for the next round of transformation, until all 11 opa genes were sequentially deleted. OpaD+nv and OpaD+var Gc expressed similar amounts of Opa protein (Fig. Substance Search. (C) Lysates prepared from equal CFU of ΔopaBEGK Gc, OpaD+var Gc, Opaless Gc, and OpaD+nv Gc were separated by SDS-PAGE and Western blotted with a pan-Opa antibody. To begin to understand the mechanism underlying the increased killing of OpaD+ Gc by PMNs, we measured bacterial survival after exposure to the model cationic peptide polymyxin B. Other options New and used from $6.99. Parental and ΔopaBEGK Gc induced minor amounts of ROS production from PMNs, but the oxidative burst was smaller and delayed compared to PMNs infected with S. aureus (Fig. Your source for achieving better health through nutrition. Its powerful stain-blocking formula also blocks most pet and smoke odors and creates an … Loss of opa is indicated by the faster-migrating PCR product for each primer set. Neisseria meningitidis is an inhabitant of the nasopharynx, from which it is transmitted from person to person or disseminates in blood and becomes a harmful pathogen. 3A). Saba R, Störchel PH, Aksoy-Aksel A, Kepura F, Lippi G, Plant TD, Schratt GM. FA1090 1-81-S2 Gc predominantly expressing OpaB was previously described (35). 2B to G) and yielded equivalent numbers of CFU per colony after 24 h of growth on solid medium (see Fig. Hier gehts direkt zum Katalog. Opa+ Gc engagement of CEACAM1 also leads to B cell death (67). - danielmiessler/SecLists Powered by Bow and Baan From each transformation, 400 to 600 colonies were screened for the opa deletion by PCR, using the conditions listed in Table S3 in the supplemental material. (i) Phase-ON Opa+ Gc in the ΔopaBEGK background (OpaD+var).FA1090 ΔopaBEGK Gc was passaged on GCB. A third possibility is that Opa+ bacteria are more sensitive to PMN antimicrobial proteins. 1A). Bacteria were resuspended in 100% freshly isolated normal human serum for 30 min. Opaless Gc and Opa-expressing derivatives provide a robust system for studies of Opa biology and Gc pathogenesis. Over 2,000 bacteria were examined for their localization. To block actin-dependent phagocytosis, cytochalasin D (10 μg/ml) (Sigma) was added to PMNs 30 min prior to infection and kept in the infection medium for the duration of the experiment. Complementation of Opaless Gc with a nonvariable, constitutively expressed allele of FA1090 opaD. WeBoost, votre agence de marketing digital située à Montreuil, vous aide à obtenir plus de prospects qualifiés à un coût abordable. We previously showed that OpaB+ Gc survives less well than Opa− bacteria after PMN exposure (35). All Rights Reserved. These and other approaches will help reveal the contribution of Opa proteins, individually or in combination, to Gc-host interactions and enhance understanding of how these interactions influence the course of human disease. Gc genomes contain approximately 11 opa loci, encoding 7 to 9 unique Opa proteins (7–9). In contrast, we reported that predominantly Opa− Gc can be internalized by adherent, IL-8-treated human PMNs (35). Gc-PMN coculture.All human subject research was conducted in accordance with a protocol approved by the University of Virginia Institutional Review Board for Health Sciences Research. Polymyxin B survival.Mid-logarithmic phase Gc was exposed to increasing concentrations of polymyxin B (Alexis Biochemicals) as described in reference 49, except that incubations occurred in 0.5× GCBL. Notably, unopsonized Opaless Gc was internalized by adherent, chemokine-primed, primary human PMNs, by an actin-dependent process. 11 rue des Grandes Cultures. Best Shrink Wrap Size: 57 x 55. Um effizient in Opac plus zu recherchieren, muss Ihr Rechner am Hochschulnetz angemeldet sein. Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. We do not retain these email addresses. XIII. These derivatives provide a robust system for assessing the role of Opa proteins in Gc biology. 3C. S3 in the supplemental material). In contrast, PMNs exposed to Opaless Gc were identical to uninfected PMNs and generated no ROS (Fig. While it is possible that we selected for a mutation that allows for Gc growth in the absence of all Opa expression, opa-deficient derivatives of strain MS11 Gc and strain H44/76 Neisseria meningitidis also show no in vitro growth defect (19, 59). Chemiluminescent blots were imaged with a ChemiDoc XRS+ system (Bio-Rad). We're doing our best to make sure our content is useful, accurate and safe. 5D to G). My Cloud Web access. OpaD expressors are isolated from the male urethra and mouse genital tract after infection with FA1090 Gc, and OpaD+ Gc stimulates a strong oxidative burst in PMNs (25, 30, 50, 52, 53) (Fig. 3F11 recognizes the terminal N-acetyllactosamine of the Gc oligosaccharide α chain of LOS (18). SLUth searches for print books, ebooks, journals, journal articles, audio and video media, microfilm, and more including the full text for many ebooks and journal articles. Consultez les chiffres clés, l'identité, les dirigeants de toutes les entreprises immatriculées au RNCS (Registre National du Commerce des Sociétés). Bottle Height: 2.75". © 2020 Visvesvaraya Technological University. 4.4 out of 5 stars 2,707. Parental and ΔopaBEGK Gc were predominantly Opa− by Western blotting and colony opacity (data not shown). Opaless Gc can serve as a recipient for Opa variants expressed by other strains of Neisseria or Opa proteins synthesized in vitro, in order to evaluate these proteins' receptor binding profiles and other biochemical properties. S1A, C, and D), and all derivatives retained the piliated colony morphology of the parent (Fig. (C) Opaless Gc, OpaD+var Gc, and OpaD+nv Gc were exposed to polymyxin B at the indicated concentrations. One such pressure may come from the requirement for Gc to attach to the apical surface of the mucosal epithelium and be internalized by epithelial cells (63). Sensitivity of OpaD+ Gc to killing by human PMNs but not polymyxin B. These results confirm that OpaD is a strong stimulator of PMN ROS production. Fiore R, Schratt G. EMBO J. Another possibility is that PMNs are more adept at binding and engulfing OpaD+ bacteria, leading to increased bacterial killing. We gratefully acknowledge members of the Criss laboratory for their input, especially Jonathan Handing for advice on polymyxin B. This bottle is made of HDPE plastic. New Look, Same Trusted Formula, KILZ Original 13 New Look, Same Trusted Formula, KILZ Original 13 oz. Die Verfügbarkeit der einzelnen Titel wird in der Trefferliste angezeigt. Bacterial survival was calculated as the CFU after 45 min of exposure, divided by the input CFU. Copyright © 2020 American Society for Microbiology | Privacy Policy | Website feedback, Print ISSN: 0021-9193; Online ISSN: 1098-5530, Department of Microbiology, Immunology, and Cancer Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia, USA, Constitutively Opa-Expressing and Opa-Deficient, Sign In to Email Alerts with your Email Address. (A) ROS production was measured in human PMNs exposed to S. aureus, ΔopaBEGK Gc (Opa− Gc), or ΔopaBEGK Gc phase-ON for OpaA, OpaD, OpaF, or OpaI, as described for Fig. Generating Opaless Gc in strain FA1090.To delete the opa genes from piliated Gc of strain FA1090, the majority of each open reading frame was replaced in frame with a restriction enzyme recognition sequence (Fig. The University of Technology Sydney Library provides support for the teaching, learning and research needs of the students and staff at UTS. Scale bar = 10 μm. Gc growth measurements. The dopamine-regulated microRNA, miR-181a, controls GluA2 surface expression in hippocampal neurons. OpaD+nv and OpaD+var Gc induced similar amounts of ROS production in PMNs, whereas Opaless Gc did not induce detectable ROS (Fig. Copyright © 2013, American Society for Microbiology. All Content in this webport is for the using stakeholder of VTU Only. Expression of one or more Opa proteins in parental and ΔopaBEGK opaque Gc is indicated by a heat-modifiable shift in reactivity with the Opa antibody. (B) The 5′ end of FA1090 opaD was mutated to replace the phase-variable CTCTT repeats (line above the DNA sequences) with nucleotides encoding the same amino acids (written below the DNA sequences) yet unable to phase vary (black boxes). Lumière, isolation, design, finesse, couleur. *, P < 0.05 (Student's two-tailed t test) for OpaB+ Gc compared to the parent, ΔopaBEGK, or Opaless Gc at matched time points. Hope you guys can identify them as in the past. Given the small-colony phenotype of a previously constructed FA1090 opa deletion strain (50), we measured the in vitro growth of FA1090 parental, ΔopaBEGK, and Opaless Gc. For subsequent experiments, parental Gc was subcloned to yield an isolate that was predominantly Opa negative (Opa−) by Western blotting and colony morphology (data not shown). Thus, Gc can express anywhere from zero to theoretically all Opa proteins. The study aimed to trace the education graduates of Lyceum of the Philippines-University-Batangas from 2008 to 2012. Weitere Infos hier. Combien de temps vous reste-t-il ? The product was cloned into pCR-Blunt TOPO (Invitrogen) or pSmart (Lucigen) and transformed into chemically competent TOP10 E. coli, following the manufacturers' instructions, except for opaC, where the overlap extension product was confirmed by DNA sequencing and then directly transformed into Gc (see below). Opa proteins can also recognize lacto-N-neotetraose moieties on LOS (18, 19). The presence of Gc at human mucosal surfaces initiates the recruitment of abundant polymorphonuclear leukocytes (neutrophils; PMNs) to the site of infection (4). 10 - 70173 Stuttgart - Telefon +49 711-212-4454 (A) Lysates from equal CFU of parental Gc, phenotypically translucent ΔopaBEGK (ΔopaBEGK translucent) Gc, phenotypically opaque ΔopaBEGK (ΔopaBEGK opaque) Gc, and Opaless Gc were incubated at 37°C or 100°C and then separated by SDS-PAGE and immunoblotted with a pan-Opa antibody. CFU/ml were enumerated from the culture before and after serum exposure, and bacterial survival was expressed as (CFU/ml)t = 30 min/(CFU/ml)t = 0 min × 100%. Although most colonies of parental Gc were phenotypically translucent, the population contained some Opa-expressing bacteria. Martin's 400032-XXX Bleedproof White, 1.0 oz. 181 p. 22 cm Date: 2010 Availability: Copies available: College Library Filipiniana Section [ PE1130 .B928 2010 ] (3), Remind your doctor if you take medications or have a health condition that can affect your results. The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). The 4B12 pan-Opa antibody hybridoma was obtained from Christof Hauck (University of Konstanz). It is a branch library that services to students enrolled on various programs such as BSCS, BSIT, BSIS, BSBA, BSENT& BSA, that extends to faculty members, non-teaching staffs and administrators of Mapúa-Makati. While Opa variation may enable bacterial evasion of host immunity and adaptation to new hosts or sites within a host, it complicates the assessment of how Opa expression influences specific aspects of Gc pathogenesis. 01 81 80 15 71. Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. Opaless Gc survived exposure to primary human PMNs and suppressed the PMN oxidative burst akin to parental, Opa− bacteria. These results indicate that opa-deficient Gc, like Opa phase-OFF parental Gc, retains the ability to suppress PMN ROS production. 5C). Opaque colonies arising in the predominantly translucent culture were streak purified and stored at −80°C. CFU arising after nonselective transformation were screened by PCR for replacement of the full-length opa with the deletion construct. No differences were observed in protein or LOS composition among parental, ΔopaBEGK, and Opaless Gc (see Fig. We therefore engineered a derivative of Gc strain FA1090 in which all opa genes were deleted in frame, termed Opaless. Targeting of the Arpc3 actin nucleation factor by miR-29a/b regulates dendritic spine morphology. PCR analysis showed the four intended opa genes and no others were deleted in ΔopaBEGK Gc, while all 11 opa genes were deleted in Opaless Gc (Fig. We are an international, independent publisher headquartered in Berlin. Taken together, these results show that opa genes can be successfully deleted from the FA1090 Gc chromosome to yield a strain incapable of expressing any Opa proteins. Moreover, during extended growth in liquid culture, bacteria of a different Opa expression state stochastically emerge (our unpublished observations). In support of this notion, we and others have shown that expression of CEACAM-binding Opa proteins (for instance, FA1090 OpaB) enhances the association of Gc with PMNs (30–32, 35). We conclude that expression of the OpaCEA OpaD renders FA1090 Gc more sensitive to PMNs. 4). SLUth Search Plus. In each subsequent round of opa deletion, Southern blotting and/or PCR was used to verify that all opa deletions that were previously introduced were retained. Opa proteins have high isoelectric points (pI of mature OpaD = 9.83 [available via http://web.expasy.org]), which may help the negatively charged bacterial surface resist binding of positively charged antimicrobial peptides. NLB manages the National Library, 26 Public Libraries and the National Archives. Retrouver les marchés publics des collectivités locales, leurs établissements publics et des bailleurs sociaux. Loss of Opa expression did not affect Gc growth. Screening and validation of opa gene deletions in FA1090 Gc.Deletion constructs were sequentially introduced into FA1090 Gc by natural transformation on a solid medium (43). The latest wellness and longevity research. For freshness, you possibly need to use a sturdy kitchen clip or something similar. Therefore, we conclude that Opa expression is not required for Gc growth in vitro. TISS is a unique institution that brings together high quality scholars and practitioners from Social, Economic, Political, Physical, Habitat, Engineering, Health, and Environmental Sciences to create interdisciplinary teaching and research programmes combined with field action to address the most critical current and emerging issues of the nation. Using Opaless and OpaD+nv Gc, we found that constitutively Opa− and Opa+ Gc behave similarly to their phase-varying counterparts in survival after challenge with primary human PMNs and in modulating the PMN oxidative burst. The three strains produced similarly sized colonies (Fig. . The parent of Opaless and OpaD+ nonvariable (OpaD+nv) Gc has a point mutation in the G-quadruplex region upstream of pilE that abrogates pilin antigenic variation (40) (FA1090 1-81-S2 P+nv), to ensure all bacteria express the same pilin. De Gruyter publishes first-class scholarship and has done so for more than 270 years. Pendant la crise sanitaire, 70 % des parents ont ressenti plus de fatigue que d’habitude 22/12/2020 08:45:34. Thus, reintroduction of a nonvarying opa gene into Opaless Gc allows for constitutive Opa protein expression, without affecting Gc growth. Another pressure may stem from the human adaptive immune system. Julie Boyer. Interior Primer, Sealer, and Stain Blocker in a fast-drying aerosol formula. Given the potent ROS response of PMNs to OpaD+ Gc, one possibility is that Opa+ bacteria are killed by PMNs' extensive oxidative arsenal. Assuming the reduced ROS in response to OpaD+var Gc is due to a minor fraction of Opa− bacteria, these findings highlight the advantage of using Gc expressing a nonvarying opaD allele to examine Opa-dependent bacterial interactions with host cells. (A) Primary human PMNs were exposed to Opaless Gc, OpaD+var Gc, or OpaD+nv Gc, and ROS production was measured as described for Fig. Experiments were performed 3 times. Finally, the contribution of putative (non-Opa) Gc virulence factors can be investigated in constitutively Opa+ or Opa− genetic backgrounds, to determine if and how the presence of Opa affects their function. 2A). We conclude that the complete loss of opa genes from FA1090 Gc does not affect bacterial viability, growth rate, or protein and LOS expression. The deletions were designed to replace most of the opa open reading frame (ORF) with an HpaI restriction site (underlined in the primer sequence in Table S2 in the supplemental material). The FA1090 genome has been sequenced and annotated (www.genome.ou.edu/gono.html; http://stdgen.northwestern.edu), the locations of all 11 opa genes in the FA1090 chromosome are known (8) (see Table S1 in the supplemental material), and the colony opacity phenotype for each FA1090 Opa protein has been elucidated (25, 53). Extensive database of ORAC food values. When investigating the contribution of Opa proteins to Gc interactions with PMNs and other cells, the high rate of Opa phase variation means that any Gc culture in which one Opa phenotype predominates will always include bacteria of a different Opa expression state. --jpgordon ∇∆∇∆ 1 July 2005 03:31 (UTC) I'm pretty sure this whole post is a joke. FA1090 has been used extensively in human urethral challenge studies (57) and the murine genital tract model of gonorrhea (58). PH Bibliothek Ludwigsburg, Ludwigsburg (Ludwigsburg, Germany). However, the FA1090 ΔopaA-K mutant has a significant growth defect on solid and in liquid media (53) (our unpublished observations). Powered by Bow and Baan Approximately 60% of PMN-associated Opaless Gc was intracellular, a percentage statistically indistinguishable from the Opa− parental or ΔopaBEGK Gc (Fig. Gefällt 1.295 Mal. 2A), and opaque Gc colonies were detected in cultures of parental Gc (Fig. Enseignement Formation et Conseil chez LB FORMATION Montpellier. Percent internalization (E) was calculated as the number of intracellular Gc divided by the total number of cell-associated Gc. Murièle Jacquier, Ph.D. Murièle Jacquier, Ph.D Chargée d’enseignement en didactique de la physique chez Université de Genève Auvergne-Rhône-Alpes, France. The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). The heat-inactivated normal human serum (Sigma) used to coat the coverslips was unable to opsonize Gc (K. Daily and A. Criss, unpublished observations). 1B). In addition, antibodies have been raised against individual FA1090 Opa proteins (23, 24). My Cloud Web access. (B) Opaless Gc, OpaD+var Gc, and OpaD+nv Gc were exposed to adherent, IL-8-primed PMNs, and bacterial survival was calculated as described for Fig. Hinweise: Mit einem * können Sie beliebige Wortendungen mit suchen. Manage and improve your online marketing. OpaD-expressing Gc survived significantly less well after PMN challenge compared with Opaless bacteria (Fig. Opaless Gc and an intermediate isolate in which the genes encoding translucent Opa proteins were deleted (ΔopaBEGK) behaved similarly to Opa phase-OFF bacteria in in vitro growth and survival after exposure to primary human PMNs. S2C). Opaless Gc behaves similarly to phenotypically Opa. To examine the survival of genetically opa-deficient Gc after PMN challenge, adherent, IL-8-primed human PMNs were infected with ΔopaBEGK and Opaless Gc. PMN preparations routinely contained >95% granulocytes, as assessed by phase-contrast microscopy, and were >99% viable, as assessed by trypan blue exclusion.