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Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived SequencesLudwig, Tobias, Hoffmann, Ralf, Krizsan, Andor 16 January 2024 (has links)
The global spread of multi- and pan-resistant bacteria has triggered research to identify
novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages.
In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting
Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial
peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can
synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost
of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition
of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected
E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta
strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally,
plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed
the production of satisfactory peptide quantities. When these sequences were integrated into the
T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the
expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable
quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select
phage genome, expression was below detection limits and an effect on the growth of potentially
phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we
were able to show that apidaecins can be integrated into the T7Select phage genome to induce their
expression in host cells, but further research is required to optimize the engineered T7Select phages
for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible
when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures.
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Investigating the host and microbial determinants of Pseudomonas aeruginosa mucoid conversionLimoli, Dominique H. 29 December 2014 (has links)
No description available.
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ENVIRONMENTAL INFLUENCES ONAMPHIBIAN INNATE IMMUNE DEFENSE TRAITSKrynak, Katherine L. 03 September 2015 (has links)
No description available.
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Immune evasion tactics and immunopathology of mixed mucoid and nonmucoid <i>Pseudomonas aeruginosa</i> populations in cystic fibrosisMalhotra, Sankalp 27 July 2018 (has links)
No description available.
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Design and Study of Novel Antimicrobial Peptides with Proline SubstitutionHe, Jing January 2009 (has links)
No description available.
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PhoPQ- and PmrAB-mediated Lipopolysaccharide Modification and Cationic Antimicrobial Peptide Resistance in <i>Salmonella enterica</i> Serovars Typhimurium and TyphiRichards, Susan Michelle 16 December 2010 (has links)
No description available.
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Reflection Absorption Infrared Spectroscopic Studies of Surface Chemistry Relevant to Chemical and Biological Warfare Agent DefenseUzarski, Joshua Robert 26 February 2009 (has links)
Reflection absorption infrared spectroscopy was used as the primary analysis technique to study the interfacial chemistry of surfaces relevant to chemical and biological warfare agent defense. Many strategies utilized by the military to detect and decompose chemical and biological warfare agents involve their interaction with surfaces. However, much of the chemistry that occurs at the interface between the agents and surfaces of interest remains unknown. The surface chemistry plays an important role in efficacy of both detection and decontamination technology, and by obtaining a deeper understanding of that chemistry, researchers might be able to develop more sensitive detection devices and more effective decontamination strategies. Our efforts have focused on three different areas of surface chemistry relevant to chemical and biological warfare agent defense:
1) The development of a surface synthesis strategy to create and control the structure of antibacterial self-assembled monolayers (SAMs). Our work demonstrated a successful strategy for creating SAMs that contain long-chain quaternary ammonium groups, which were synthesized and subsequently characterized using RAIRS and X-ray photoelectron spectroscopy (XPS).
2) The determination of the surface conformation, orientation, and relative surface density of immobilized antimicrobial peptides. Our results revealed that the peptides consisted of tilted (50-60°), α-helices on the surface, regardless of solution conditions.
3) The design and construction of a new ultrahigh vacuum surface science instrument that allows for the study of gas-surface reactions with up to three gases simultaneously.
4) The study of the adsorption of chemical warfare agent simulants to silica nanoparticulate films. Our work demonstrated that the adsorbate structure was dependent on the number of hydrogen-bonding groups, and the adsorption consists of a pressure-dependent two part mechanism.
The results presented here will help increase the understanding of the surface chemistry of three interfaces relevant to chemical and biological defense. Future researchers may apply the new information to develop more effective detection and decontamination strategies for chemical and biological warfare agents. / Ph. D.
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Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting HIV-1 RRE RNA and MicrobialsWynn, Jessica Elaine 29 August 2016 (has links)
The interaction of the protein Rev with Rev Response Element (RRE) RNA is critical to the HIV-1 life cycle as this complex is required for the export of singly-spliced and unspliced mRNAs from the nucleus to the cytoplasm. Disruption of this interaction is considered to be a powerful strategy towards the development of HIV-1 therapeutics. Therefore, we have developed several branched peptide libraries containing unnatural amino acids to target the high-affinity binding site of RRE RNA (RRE IIB), with the idea that branching in peptides can provide multivalent contacts with folded RNA structures and boost binding affinity and selectivity for the target. Unnatural amino acids were incorporated into the library design to encourage non-canonical interactions with the RNA and to improve proteolytic stability.
The on-bead high-throughput screening of our first branched peptide library (46,656 sequences) against HIV-1 RRE RNA generated hit peptides with binding affinities in the low micromolar range. We demonstrated that branching in the peptide is required for efficient binding and selectivity towards the RNA, and that the peptides bind a large surface area of RRE IIB. Introduction of boronic acids into branched peptides boosted selectivity of the peptides for RRE IIB, and proved to be a novel and tunable mode of binding towards RNA. Additionally, we revealed that these branched peptide boronic acids (BPBAs) were cell permeable and non-toxic. One BPBA (BPBA3) bound RRE IIB selectively and was able to inhibit HIV-1 replication in vitro, revealing enzymatic cleavage of the RNA upon binding.
A second generation BPBA library that introduced acridinyl lysine as an intercalator (4,096 sequences) was screened against RRE IIB. Several hit compounds bound in the low nanomolar regime, and a significant number of compounds inhibited HIV-1 replication in vitro. These BPBAs were also found to severely inhibit the microbial growth of bacteria and fungus, with MICs as low as 1 µg/mL against Staphylococcus aureus, Candida albicans, and Escherichia coli. These compounds were also found to significantly inhibit biofilm formation and growth, and were non-hemolytic.
High-throughput screening of a third generation BPBA library containing all unnatural amino acids (46,656 sequences) revealed several hits that bound RRE IIB RNA in the nanomolar range. Sequence motifs present in the hit peptides suggested that the location and composition of amino acids within the branched peptide structure were important for recognizing the RNA target. In particular, lead compounds 2C5 and 4B3 demonstrated selectivity towards RRE, and footprinting experiments combined with SHAPE experiments revealed different interactions of the peptides with the RNA Toxicity assays revealed no impact on cell viability for the majority of hit sequences tested up to 100 µM, and several compounds also demonstrated inhibition of HIV-1 replication. / Ph. D.
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Cytokines as therapeutic targets in skin inflammationWittmann, Miriam, McGonagle, D., Werfel, T. January 2014 (has links)
No / This review focuses on treatment targets for the most common inflammatory skin diseases, eczema and psoriasis with an emphasis on cytokines expressed in the uppermost layer of the skin which is easily accessible for diagnostic and therapeutic approaches. Recently, a significant body of research has highlighted the influence of the skin barrier and the patients’ microbiome on skin inflammatory responses and we will comment on their impact on mediator regulation. Itch is a prominent dermatology symptom which is influenced by cytokines and can via itch–scratch cycle impact on the skin barrier and mediator expression associated with damage. Taking the contribution of pruritus and superficial skin damage into account, we address cytokines as targets for stratified treatment approaches in subgroups of eczema and psoriasis.
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Mode of action studies of defensin peptides from native South African Brassicaceae speciesBarkhuizen, Helmien 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Plant defensin peptides have become promising and attractive candidates to be used as
antifungal agents in agricultural biotechnology. These peptides have a broad spectrum
antifungal activity and play a vital role in the innate immune system of plants. Plant diseases
caused by fungi are a major contributor to the decrease in the quality and safety of agricultural
products. Due to the dangerous effects and negative environmental impact of pesticides, an
effective, safe, natural and durable method to control crop pathogens has therefore become one
of the major concerns in modern agriculture. Although these peptides are promising and
attractive candidates, their precise mechanism of action is to date still unknown. Several
common observations have been made. These include the antagonistic effect of cations on the
activity of plant defensins. It is of vital importance to understand the underlying mechanism of
the cation-antagonistic effect on the antifungal potency of defensin peptides in order to evaluate
the possible contribution to defence reactions against microorganisms in planta.
To this end we set out to characterize the effect of cations in the form of biological salts,
NaCl, KCl, MgCl2 and CaCl2 on the structural stability and activity in terms of growth inhibition,
morphological effects and permeabilization. In order to perform these characterization
experiments, a production method resulting in a greater yield and involving simple and rapid
purification methods was required. Heliophila coronopifolia peptides have previously been
produced in a bacterial system, however the purification methods were tedious resulting in poor
yields. Pichia pastoris was selected as production system as several other plant defensins have
been successfully produced in this eukaryotic system. Hc-AFP1 and Hc-AFP3 was successfully
produced using the Pichia production system and rendered active peptides. Hc-AFP2 and Hc-
AFP4 was, however, not produced correctly, due to a post-translational modification event
leading to the cyclization of the N-terminal glutamine to generate pyroglutamic acid. This
modification negatively influenced the activity of these peptides. An active Hc-AFP2 could be
produced by replacing the production buffer with a reduced ionic buffer.
The effect of divalent and monovalent cations on the secondary structure of Hc-AFP1 was
evaluated by circular dichroism spectroscopy. These cations induced a conformational change
in the secondary structure of Hc-AFP1, with NaCl and MgCl2 inducing a more defined secondary
structure and KCl and CaCl2 inducing a less defined secondary structure. Monovalent cations
caused a slight reduction in the growth inhibition activity of Hc-AFP1 on Botrytis cinerea,
however, characteristic hyperbranching and other morphogentic effects were still visible.
Divalent cations had a greater antagonistic effect on the activity of Hc-AFP1, completely
abolishing the growth inhibitory activity of the peptide, but the induced morphological effects on
hyphae remained present. The activity of Hc-AFP1 to permeabilize B. cinerea hyphae was not
influenced by the addition of cations, however it was in fact increased to up to 10-fold. However,
since the growth inhibition activity of Hc-AFP1 was reduced in the presence of the biological
salts indicates that permeabilization is not the sole activity responsible for growth inhibition
caused by Hc-AFP1. This peptide probably has an alternative/primary target and more complex
MOA. This is the first known report of the investigation of the influence of cations on the
structure of plant defensin peptides. It is clear that cations induce a secondary structural
conformational change in Hc-AFP1. This may be linked to the antagonism on the activity of this
peptide. This study provides significant progress towards the structure-function analysis of plant
defensins. / AFRIKAANSE OPSOMMING: Plantdefensinpeptiede word beskou as belowende en aantreklike kandidate vir gebruik as
swammiddles in agribiotegnologie. Hierdie peptiede beskik oor breë spektrum antifungiese
aktiwiteit en speel ‘n essensiële rol in die ingebore immuunsisteem van plante. Plant siektes wat
deur swamme veroorsaak word dra betekenisvol by tot die afname in die kwaliteit en veiligheid
van landbouprodukte. As gevolg van die skadelike effekte en negatiewe omgewingsimpak van
plaagdoders, het effektiewe, veilige, natuurlike en duursame metodes om gewaspatogene te
beheer, van die belangrikste vraagstukke van moderne landbou geword. Alhoewel hierdie
peptiede belowende en aantreklike kandidate is vir die toepassing, is hulle presiese meganisme
van aksie tot vandag toe steeds onbekend. Verskeie algemene waarnemings is egter al
gemaak. Dit sluit die antagonistiese effek van katione op die aktiwiteit van
plantdefensinpeptiede in. Dit is kernbelangrik om die onderliggende meganisme van die
katioon-antagonistiese effek op die antifungiese effektiwiteit te verstaan om die moontlike
bydrae van die peptiede tot die verdedigingsreaksies teen mikro-organismes in planta te
evalueer.
Met die doel voor oë het ons gemik om die effek van katione, spesifiek in die vorm van die
biologiese soute NaCl, KCl, MgCl2 en CaCl2, op die strukturele stabiliteit en aktiwiteit in terme
van groei inhibisie, morfologiese effekte en permeabilisasie te karakteriseer. Om uiteindelik
hierdie karakterisasie eksperimente uit te voer was dit nodig om ‘n metode met ‘n groter
opbrengs en wat vinnige suiwering van die peptied ondersteun, te optimiseer. Heliophila
coronopifolia peptiede was voorheen in ‘n bakteriese sisteem geproduseer, maar die
suiweringsmetodes was tydsaam en het gelei tot ‘n swak opbrengs. Pichia pastoris is dus
geselekteer as die produksie sisteem aangesien verskeie ander plantdefensinpeptiede al
suksesvol geproduseer is in hierdie eukariotiese sisteem. Hc-AFP1 and Hc-AFP3 is suksesvol
vervaardig in die Pichia sisteem en het aktiewiteit getoon. Hc-AFP2 and Hc-AFP4 kon egter nie
korrek vervaardig word nie as gevolg van ‘n na-vertalingsverandering wat gelei het tot die
siklisering van die N-terminale glutamien, om piroglutamiensuur te lewer. Hierdie verandering
het die aktiwiteit van die peptied negatief beinvloed. ‘n Aktiewe Hc-AFP2 kon wel vervaardig
word deur die produksiebuffer te vervang met ‘n lae-ionise buffer.
Die effek van divalente en monovalente katione op die sekondêre struktuur van Hc-AFP1 is
ge-evalueer deur van sirkulêre dikroisme spektroskopie gebruik te maak. Hierdie katione het ‘n
vouingsverandering in die sekondêre struktuur van Hc-AFP1 geïnduseer, NaCl and MgCl2 het ‘n
meer gedefinieërde sekondêre struktuur induseer, terwyl KCl and CaCl2 ‘n minder gedefinieërde
sekondêre struktuur geinduseer het. Monovalente katione het ‘n effense vermindering in die
groei-inhibisie aktiwiteit van Hc-AFP1 op Botrytis cinerea veroorsaak, alhoewel kenmerkende
hife-oorvertakking en ander morfologiese effekte nogsteeds sigbaar was. Divalente katione het
‘n sterker antagonistiese effek gehad op die aktiwiteit van Hc-AFP1, waar dit totaal en al die
groei-inhibisie aktiwiteit van die peptied vernietig het, alhoewel die geïnduseerde morfologiese
effekte op die hiffes steeds sigbaar was . Die aktiwiteit van Hc-AFP1 om B. cinerea hyphae te
permeabiliseer is nie negatief beinvloed deur die byvoeging van katione nie, tewens dit het die
aktiwiteit tot 10-voudig verhoog. Aangesien die groei-inhibisie aktiwiteit van Hc-AFP1 nie
verminder is in die teenwoordigheid van die biologiese soute nie, dui dit aan dat permeabilisasie
nie die enigste aktiwiteit is wat die groei inhibisie veroorsaak het nie. Die peptied het dus
moontlik ‘n alternatiewe of primêre teiken en ‘n meer komplekse meganisme van aksie. Dit is
die eerste verslag wat die invloed van katione op die struktuur van plantdefensinpeptiede
ondersoek het. Dit is duidelik dat katione ‘n sekondêre strukturele vouingsverandering in Hc-AFP1 induseer. Hierdie verandering mag dalk bydra tot die antagonistiese uitwerking op die
aktiwiteit van hierdie peptied. Hierdie studie het betekensisvolle vordering gemaak met die
analise van die struktuur-funksie interaksie van plantdefensinpeptiede. / The National Research Foundation (NRF), Institute of Wine Biotechnology (IWBT),
THRIP and Winetech for financial assistance.
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