1,721,001 research outputs found
Leukotrienes Are Dispensable for Vaginal Neutrophil Recruitment as Part of the Immunopathological Response During Experimental Vulvovaginal Candidiasis.
Full text linkRecruitment of polymorphonuclear neutrophils (PMNs) into the vaginal lumen is the hallmark of an acute immunopathologic inflammatory response during vulvovaginal candidiasis (VVC) caused by Recurrent VVC (RVVC) remains a chronic health burden in affected women worldwide despite the use of antifungal therapy. Based on the role leukotrienes (LTs) play in promoting inflammation, leukotriene receptor antagonists (LTRAs) targeted for LTB (etalocib) or LTC, LTD and LTE (zafirlukast or montelukast) have been shown to reduce inflammation of epithelial tissues. An open-label pilot study using long-term regimens of zafirlukast in women with RVVC indicated the potential for some relief from recurrent episodes. To investigate this clinical observation further, we evaluated the effects of LT antagonistic agents and LT deficiency on the immunopathogenic response in a mouse model of VVC. Results showed that mice given daily intraperitoneal injections of individual LTRAs, starting 2days prior to vaginal inoculation with and continuing through 14days post-inoculation, had no measurable reduction in PMN migration. The LTRAs were also ineffective in reducing levels of the hallmark vaginal inflammatory markers (S100A8, IL-1β) and tissue damage (LDH) associated with the immunopathogenic response. Finally, LT-deficient 5-lipoxygenase knockout mice showed comparable levels of vaginal fungal burden and PMN infiltration to wild-type mice following inoculation with a vaginal (ATCC 96113) or laboratory (SC5314) isolate. These results indicate that despite some clinical evidence suggestive of off-target efficacy of LTRAs in RVVC, LTs and associated signaling pathways appear to be dispensable in the immunopathogenesis of VVC
Proteomics and functional analysis of outer membrane vesicle from Pseudomonas aeruginosa under SOS
Full text linkThe author has granted permission for their work to be available to the general public.The bacterial SOS response and outer membrane vesicle (OMV) production are conserved among Gram-negative bacteria, both contributing to virulence and biofilm development. SOS may link to OMV generation. To investigate such a connection, we conducted functional and proteomic analyses of OMVs from Pseudomonas aeruginosa. Functionally, OMVs mediated macrophage cytotoxicity and contributed to biofilm formation. OMV production and the cytotoxicity increased when the OMV-producing cells were SOS-induced by ciprofloxacin treatment, but decreased when LexA repressed SOS. Proteomic analysis revealed that proteins whose genes are known to be expressed during SOS were found predominantly in OMV proteins (OMVps) produced by the SOS-induced cells. These OMVps thus is termed SOS-OMVps. Many cytotoxic and biofilm-related proteins were also found in SOS-OMVps while some in the SOS-independent OMVps. Therefore, SOS appears to play a role in the antibiotic-stimulated OMV production, which contributes to cytotoxicity and biofilm formation.Integrative Biolog
Effect of Site-specific Changes in the Regulatory Functions of BosR
No full textThe author has granted permission for their work to be available to the general public.Borrelia burgdorferi, the causative agent of Lyme disease has a limited set of proteins involved in resistance to oxidative stress. Regulation of some of these proteins is mediated by Borrelia oxidative stress Regulator (BosR), a zinc-dependent DNA binding protein that recognizes motifs upstream of NapA, CoADR and SodA. We generated a bosR deficient strain in B.burgdorferi strain B31 lacking lp25(ML23) followed by restoration of minimal region of lp25 needed for infectivity using a borrelial shuttle vector (pBBE22). The bosR deficient strain was incapable of colonization of C3H/HeN mice following needle inoculation indicating that the regulatory functions of bosR is essential for infectivity. We complemented the BosR mutant with a native copy in cis, and generated site-specific substitutions in the CXXC motifs (bosRC114S-N-K-C117S; bosRC153S-N-N-C156S,) and conserved histidines (bosRH37A, bosRH 111A) present in BosR. The cis-complemented strain exhibited levels of BosR similar to the parental strain and the levels of Outer surface protein C reflected the parental phenotype. Interestingly, site-specific changes in the first CXXC motif (bosRC114S-N-K-C117S) resulted in levels of BosR similar to that of the cis complemented strain but had dramatically lower levels of OspC. Replacement of the conserved histidine at position 111 with an alanine resulted in not only a reduction in the levels of BosR but also in the levels of OspC. There was no changes in the levels of bb0646 which is co-transcribed with bosR (bb0647) as well as in the levels of a variety of other oxidatives stress response proteins such as SodA and NapA. The levels of P66 was similar in all strains serving as a control for levels of proteins loaded in each lane and as a measure of the physiological status of the spirochetes. A set of key defined site-specific mutants in B. burgdorferi has been generated and these strains will help in further analysis of the interactions of BosR with the members of its regulon. Moreover, the regulatory functions of BosR are complex and the phenotypic analysis of mutants carrying site-specific changes in bosR critical for coordinating metals such as Zn2+ would provide insights into the role of BosR in the patho-physiology of B.burgdorferi.Integrative Biolog
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Polyamine uptake and utilization and characterization of potA (bb0642) and potdD ( bb0639) in Borrelia burgdorferi
No full textThis item is available only to currently enrolled UTSA students, faculty or staff. To download, navigate to Log In in the top right-hand corner of this screen, then select Log in with my UTSA ID.Lyme disease is a multi-phasic, systemic infection caused by the spirochetal bacterium Borrelia burgdorferi. It is the major arthropod-borne disease in the US and is transmitted to mammals, including humans, by Ixodid ticks. This spirochetal pathogen has a limited metabolic capability, and therefore is intimately dependent on its hosts for survival. In silico analysis revealed that B. burgdorferi encodes for a series of co-transcribed genes (potA-D) for the uptake of polyamines, ubiquitous molecules necessary for many normal cell functions. The terminal open reading frame (ORF) of the operon encodes for potD, a substrate binding protein. potA and potD were cloned into E. coli expression vector pET23a, and the two recombinant proteins with a C-terminal 6-Histidine tag were expressed, purified, and used to generate mono-specific anti-serum in mice. These critical reagents have been used to determine that PotA is expressed at higher levels in the tick vector than vertebrate host, while PotD is expressed at the same levels in B. burgdorferi in both tick and mammalian host conditions highlighting a complex and potentially important function. Moreover, increasing concentrations of select polyamines causes a decreased levels of both PotA and PotD. Protease accessibility assays performed on two strains of B. burgdorferi, A3 and MSK5, show that PotD is not surface exposed, indicating a similar organization to the Pot system of E. coli. A plasmid construct allowing for deletion of potD from the B. burgdorferi chromosome has been generated and used to transform B. burgdorferi. Analysis of this deletion strain will be an extremely useful tool in delineating the role of polyamine transport in B. burgdorferi.Integrative Biolog
Evaluation of genomic binding locations of the Candida albicans transcription factor Brg1 during early hyphal induction
No full textThis item is available only to currently enrolled UTSA students, faculty or staff. To download, navigate to Log In in the top right-hand corner of this screen, then select Log in with my UTSA ID.Candia albicans is a common commensal organism and an opportunistic fungal pathogen of humans. Its ability to cause disease is dependent on a morphological change from yeast to true hyphae. It is widely known that the transcriptional repressor Nrg1 is a negative regulator of hypha formation and when this protein is overexpressed, the organism stays in the yeast form under hyphae inducing conditions. Recent studies have demonstrated that the transcription factor Brg1 is a positive regulator of hypha formation and its role is critical in driving the change from yeast to true hyphae. Previous work in our laboratory uncovered that Brg1 and Nrg1 function in a genetic feedback loop and, specifically that BRG1 expression leads to rapid down-regulation of NRG1 transcript levels. It was subsequently shown that this down-regulation was independent of the promoter and 3’ UTR regions of NRG1 and entirely dependent on the coding sequence of the gene. Finally, we were able to detect the presence of an NRG1 antisense transcript that is induced in wild-type strains but not in brg1Δ mutants, which fail to filament under hyphal growth conditions. Currently, it is not known whether Brg1 acts directly or indirectly on the NRG1 coding sequence to up-regulate the antisense NRG1 transcript production. The purpose of this study was to determine whether Brg1 binds to the coding sequence of NRG1 during the early stages of hyphal induction. In order to do this, a novel Candida albicans strain was constructed in which both BRG1 alleles are tagged with a 13XMyc sequence. The tagged Brg1 transcription factor remains functional in its ability to drive filamentation and can be purified through immunoprecipitation. Quantitative PCR analysis on DNA immunoprecipitated from this strain suggests that Brg1 does not bind directly to a Brg1 consensus motif that was identified within the NRG1 coding sequence. However, now that we have the novel Myc-tagged Candida albicans strain, it will be possible to conduct a more comprehensive analysis of Brg1 binding sites during the early stages of hyphal induction using ChIP-Seq. That information will be very useful in helping us to unravel the cellular machinery through which Brg1 controls the pivotal virulence attribute of hyphal formation and may identify new targets for antifungal drug development.Integrative Biolog
Molecular analysis of proteins involved in phosphate uptake and regulation in Borrelia burgdorferi
No full textThis item is available only to currently enrolled UTSA students, faculty or staff. To download, navigate to Log In in the top right-hand corner of this screen, then select Log in with my UTSA ID.Lyme disease is a multi-phasic, systemic infection caused by the spirochetal bacterium <italic>Borrelia burgdorferi</italic>. It is the major arthropod-borne disease in the US and is transmitted to mammals, including humans, by Ixodid ticks. This spirochetal pathogen has a limited metaboliccapability, and therefore is intimately dependent on its hosts for survival. Regulatory mechanisms allowing for drastic adaptive changes between the tick and mammalian host are key to understanding the pathogenesis of <italic>B. burgdorferi</italic>. PhoU regulates genes of the Pho regulon, including a phosphate transporter and two component regulatory systems (TCRS) responsible for the uptake and expression of genes necessary for the maintenance of inorganic phosphate (Pi). In silico analysis of B. burgdorferi does not demonstrate the presence of a Pho TCRS, though there are open reading frames (ORFs) homologous to members of a phosphate transport system present in other bacteria. PstA, PstB, PstC, and PstS are additional proteins involved in Pi-specific transport under the regulation of PhoU. Pi levels play a large role in bacterial virulence as phosphorylation is required for the activation of the two known TCRS present in B. burgdorferi, responsible for adaptive gene response. Recombinant PhoU, PstB, and PstS have been over-expressed, purified to homogeneity and used to generate anti-sera in mice. A deletion construct was designed by 2-step PCR to replace phoU with an antibiotic resistance cassette and the native gene was also placed under the control of an IPTG-inducible promoter on a shuttle-vector for over-expression in B. burgdorferi. Since B. burgdorferi has only a limited set of ORFs corresponding to regulators of gene expression, we hypothesize that proteins involved in maintaining phosphate homeostasis such as PhoU and other associated Pst proteins, are required for the coupling the metabolic states of <italic>B. burgdorferi</italic> to its host-specific virulence attributes.Integrative Biolog
Antibiotic-inducile biofilm formation is regulated by SOS-regulated motility of Pseudomonas aeruginosa
No full textThis item is available only to currently enrolled UTSA students, faculty or staff. To download, navigate to Log In in the top right-hand corner of this screen, then select Log in with my UTSA ID.Biofilms are multicellular communities of unicellular organisms encoded by an extracellular polymeric matrix. Bacterial biofilm formation can be induced by antimicrobial and DNA damage agents. These agents trigger an SOS response which is sensed by an activated RecA coprotease that stimulates the auto-cleavage of LexA, a repressor of SOS. We tested to see if a quinolone antibiotic, Ciprofloxacin which is a DNA replication inhibitor is able to induce this SOS-inducible biofilm formation by conducting the 96-well assay and the newly developed biofilm lipid assay. We found that it appeared repressed by the non-cleavable LexA and can thus stimulate the biofilm formation by Pseudomonas aeruginosa. This stimulation involves the SOS response because the SOS repressor LexA controls the early development of biofilm formation. Proteomic analysis of the outer membrane fraction of biofilm cells suggest that the relative abundance of flagellum-related proteins is negatively affected by LexA. The functional and morphological analysis of flagellum-based motility verifies that the flagellum-propelled swimming motility is repressed by LexA and it controls flagellation. Hence, LexA controls the early development of antibiotic-inducible biofilm formation through repression of flagellum function.Integrative Biolog
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