Identifikation der für die Kontrolle der Typ III Sekretion und Virulenz von Yersinia pseudotuberculosis relevanten Regulationsmechanismen

The virulence strategy of pathogenic Yersinia relies on a program encoded on the virulence plasmid (pYV) and includes the adhesion factor YadA and the Ysc-Yop type III secretion system (T3SS). This program enables translocation of effector proteins into the host cell, subverting the host immune defense. Transcription of genes encoding for this program is activated by the regulator LcrF. LcrF production occurs only at host temperatures, preventing expression of yadA and ysc-yop outside the host. Host cell contact or low calcium signals trigger lcrF expression, resulting in massive synthesis of these virulence factors. Regulatory cascades adjusting the lcrF expression were investigated. In the absence of secretion signal, lcrF expression is downregulated by a negative feedback loop. The translocon component YopD accumulates inside the pathogen and prevents LcrF synthesis. YopD exerts a negative effect on the lcrF mRNA stability. Moreover, YopD was shown to positive affect RNase E and PNPase by interacting with the 5'-UTRs of their mRNAs. These enzymes, in turn, were found to repress LcrF synthesis. YopD was shown to reduce the pYV copy number, decreasing the lcrF amount. This work unraveled the mechanism of CsrA-mediated transcriptional repression of LcrF synthesis upon calcium depletion. The global regulator CsrA was shown to repress the transcriptional activator of LcrF, RcsB. CsrA was found to reduce the rcsB mRNA stability, decreasing lcrF transcription. Calcium limitation was shown to act as a global signal, inducing rearrangements in the Yersinia transcriptome mainly towards expression of the pYV-encoded virulence factors. This signal induces secretion of Yops, including YopD, increasing the pYV copy number and abolishing feedback inhibition of LcrF synthesis. Under calcium limitation and upon host cell contact CsrA was shown to stimulate lcrF translation initiation. CsrA was also found to increase the lcrF mRNA stability by repression of RNase E and PNPase. This work indicated that CsrA elevates the LcrF amount by increasing the pYV copy number. The present work demonstrated that LcrF is the only pYV-encoded factor required for induction of yadA expression upon host cell contact. Apart from CsrA, the OmpR/EnvZ ESRS, and the outer membrane porin OmpF have been suggested as promising factors that might be involved in sensing and/or transmission of the host cell contact signal to LcrF synthesis.Die Virulenzstrategie von pathogenen Yersinia beruht auf einem auf dem Virulenzplasmid (pYV) kodiertem Programm, dass aus dem Adhäsionsfaktor YadA und dem Ysc-Yop-Typ-III-Sekretionssystem (T3SS) besteht. Das ermöglicht die direkte Translokation von Effektorproteinen in die Wirtszelle und inhibiert die Immunabwehr des Wirts. Die Transkription der Gene, die für das Programm kodieren, wird durch den Regulator LcrF aktiviert. Die LcrF-Synthese erfolgt nur bei Wirtstemperaturen. Das verhindert die Expression der yadA- und ysc-yop-Gene außerhalb des Wirts. Der Kontakt zu Wirtszellen oder die Calciumlimitierung induzieren die lcrF Expression und lösen eine massive Synthese von antiphagozytischen Virulenzfaktoren aus. Die regulatorische Kaskaden, die die LcrF Synthese kontrollieren, wurden untersucht. In Abwesenheit eines Sekretionssignals wird die lcrF-Expression über eine negative Rückkopplungsschleife verringert. Unter diesen Bedingungen akkumuliert die Translokon-Komponente YopD im Pathogen und verhindert die LcrF-Synthese durch einen negativen Effekt auf die lcrF- Transkriptstabilität. Außerdem, beeinflusst YopD positiv die RNase E und PNPase, über Interaktion mit den 5'-UTRs ihrer mRNAs. Diese Enzyme reduzieren wiederum die LcrF-Synthese. Zusätzlich, reduziert YopD die pYV-Kopienzahl, wodurch die lcrF-Menge verringert wird. Der globale Regulator CsrA beeinflusst negativ den Transkriptionsaktivator von LcrF, RcsB. CsrA reduziert die rcsB-Transkriptstabilität, was die lcrF-Transkription verringert. Die Calciumlimitierung ist ein globales Signal, dass großflächige Veränderungen im Yersinia-Transkriptom hauptsächlich zur Expression der pYV-kodierten Virulenzfaktoren auslöst. Dieses Signal induziert die Yops-Sekretion, einschließlich YopD. Das erhöht die pYV-Kopienzahl und hebt die Rückkopplungshemmung der LcrF-Synthese auf. Unter Calciumlimitierung oder bei Kontakt mit Wirtszellen induziert CsrA die lcrF-Translationsinitiation. CsrA erhöht auch die Stabilität des lcrF-Transkripts durch die negative Regulation von RNase E und PNPase. Außerdem, steigert CsrA die LcrF-Menge durch Erhöhen der pYV-Kopienzahl. Diese Arbeit zeigte, dass LcrF der einzige für die Zellkontakt-abhängige Induktion der yadA-Expression benötigte pYV-kodierte Faktor ist. Abgesehen von CsrA wurden das OmpR/EnvZ ESRS sowie das Membranporin OmpF als vielversprechende Faktoren vorgeschlagen, die an der Wahrnehmung und/oder Übertragung des Wirtskontaktsignals zur LcrF-Synthese beteiligt sein könnten

Kidney efficiency index quantitative parameter of a dynamic renal scintigraphy. II. usefulness in the diagnosis of obstructive nephropathy

BACKGROUND: One of the main indications for DRS is a diagnosis of obstructive uro-/nephropathy. In standard practice,this study includes the assessment of sequential scintigraphic images, renographic curves and such quantitative parametersas TMAX, T1/2 and split function of each kidney (SF). Due to the relative nature of SF and limitations of diagnostic capabilities ofTMAX and T1/2, DRS was expanded to include new quantitative parameters describing kidney function in absolute values. Thisstudy aims to evaluate the usefulness of kidney efficiency index (KEi) — new, in-house developed parameter proportional tothe average clearance function of the kidney.MATERIAL AND METHODS: The study included 156 people aged 18–84 (average 51) years. The first group, from whichnormative values of new parameters were determined, consisted of 20 healthy volunteers. The second group consisted of 136patients selected retrospectively, based on archived scintigraphic data. “Normalcy rate” (percentage of normal results amongselected 62 patients with a low likelihood of obstructive uro-/nephropathy) was used to evaluate the reliability of KEi. A comparativedifferential analysis of obstructive uro-/nephropathy, based on standard and new DRS parameters, was performedon selected 74 patients (92 kidneys) with single functioning kidney or bilateral obstructive uropathy, where SF is unreliable.RESULTS: Normative values: KEi ≥ 8; Normalcy rate for KEi: 95%. In comparison with standard DRS evaluation, application of KEichanged the diagnosis in 1/3 of assessed kidneys (from uropathy to nephropathy in 27/92 kidneys and vice versa in 4 kidneys).CONCLUSIONS: KEi enables reproducible, quantitative assessment of absolute kidney function without any modificationsof the standard DRS protocol. Its values can be compared between independent studies (e.g. follow-up examinations). KEicorrected the diagnosis of obstructive uro-/nephropathy in cases of single functioning kidney or bilateral obstructive uropathy

Determination of the electron's solvation site on D₂O/Cu(111) using Xe overlayers and femtosecond photoelectron spectroscopy

We investigate the binding site of solvated electrons in amorphous D2O clusters and D2O wetting layers adsorbed on Cu(111) by means of two-photon photoelectron (2PPE) spectroscopy. On the basis of different interactions of bulk- or surface-bound solvated electrons with rare gas atoms, titration experiments using Xe overlayers reveal the location of the electron solvation sites. In the case of flat clusters with a height of 2-4 bilayers adsorbed on Cu(111), solvated electrons are found to reside at the ice - vacuum interface, whereas a bulk character is determined for solvated electrons in wetting layers. Furthermore, time-resolved experiments are performed to determine the origin of the transition between these different solvation sites with increasing D2O coverage. We employ an empirical model calculation to analyse the rate of electron transfer back to the substrate and the energetic stabilization of the solvated electrons, which allows further insight into the binding site for clusters. We find that the solvated electrons reside at the edges of the clusters. Therefore, we attribute the transition from surface- to bulk-solvation to the coalescence of the clusters to a closed ice film occurring at a nominal coverage of 2-3 BL, while the distance of the binding sites to the metal-ice interface is maintained

1,N 6 -α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase

1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein – an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A → C and A → T transversions and, less frequently, A → G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II), and αKG concentrations for this reaction were determined. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S stereoisomer seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA

Higher-order mean-field theory of chiral waveguide QED

Waveguide QED with cold atoms provides a potent platform for the study of non-equilibrium, many-body, and open-system quantum dynamics. Even with weak coupling and strong photon loss, the collective enhancement of light-atom interactions leads to strong correlations of photons arising in transmission, as shown in recent experiments. Here we apply an improved mean-field theory based on higher-order cumulant expansions to describe the experimentally relevant, but theoretically elusive, regime of weak coupling and strong driving of large ensembles. We determine the transmitted power, squeezing spectra and the degree of second-order coherence, and systematically check the convergence of the results by comparing expansions that truncate cumulants of few-particle correlations at increasing order. This reveals the important role of many-body and long-range correlations between atoms in steady state. Our approach allows to quantify the trade-off between anti-bunching and output power in previously inaccessible parameter regimes. Calculated squeezing spectra show good agreement with measured data, as we present here.Comment: 16+9 pages, 9+2 figure

Effects of electron irradiation on structure and bonding of SF₆ on Ru(0001)

Electron stimulated desorption ion angular distribution (ESDIAD) and temperature programmed desorption (TPD) techniques have been employed to study radiation-induced decomposition of fractional monolayer SF₆ films physisorbed on Ru(0001) at 25 K. Our focus is on the origin of F⁺ and F⁻ ions, which dominate ESD from fractional monolayers. F⁻ ions escape only in off-normal directions and originate from undissociated molecules. The origins of F⁺ ions are more complicated. The F⁺ ions from electron stimulated desorption of molecularly adsorbed SF₆ desorb in off-normal directions, in symmetric ESDIAD patterns. Electron beam exposure leads to formation of SFx (x = 0 - 5) fragments, which become the source of positive ions in normal and off-normal directions. Electron exposure > 10¹⁶ cm⁻ ² results in decomposition of the entire adsorbed SF₆ layer

Kidney Efficiency Index — quantitative parameter of a dynamic renal scintigraphy. I. Theory and preliminary verification

BACKGROUND: One of the basic clinical indications for dynamic renal scintigraphy (DRS) is a diagnosis of obstructiveuropathy and/or nephropathy. Currently, a basic quantitative criterion for diagnosing nephropathy is the percentage of individualkidney’s contribution in the global uptake of a radiopharmaceutical from the blood (so-called Split Function - SF).From a clinical point of view, a parameter evaluating a radiopharmaceutical uptake and reflecting the efficiency of a specifickidney, determined independently of the total uptake of both kidneys, would be much more useful. Based on a Rutland theory,a kidney uptake constant K proportional to a radiotracer uptake by individual kidney was introduced and applied to DRS with99mTc-ethylene-1-dicysteine (99mTc-EC). In addition, a kidney efficiency index (KEi) was also worked out as a new parameterobtained by dividing the uptake constant K by the surface of the ROI of a given kidney, which can be interpreted as the average“efficiency” of clearance of a kidney.MATERIAL AND METHODS: K and KEi values were verified in 72 studies selected retrospectively from patients referredroutinely for DRS, with available current level of blood creatinine, used for calculation of estimated GFR (eGFR) according toa CKD-EPI formula. After splitting of eGFR values into individual kidneys according to SF, single kidney eGFR values (SKeGFR)were obtained and then used as a verification method for SF, K and KEi values.RESULTS: Correlation between SF and SKeGFR values, rsp = 0.64, was significantly weaker (p < 0.0022) than the correlationof SKeGFR values with K uptake constants and KEi indices: 0.90 and 0.84, respectively.CONCLUSIONS: Uptake constant K and KEi, as quantitative parameters, give the opportunity to analyze a function of eachkidney separately and in an absolute way. KEi also allows for a reliable assessment of kidneys of atypical sizes (larger or smallerthan average). It also gives the opportunity to create normative values for this parameter and may be useful in a number ofclinical situations where the diagnostic effectiveness of such a relative parameter as SF, is severely limited, e.g. in assessinga large kidney with hydronephrosis or while differing a cirrhotic from hypoplastic (i.e. a small but properly functioning) kidney

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: the role of AlkB protein in repair of 3,N4-ethenocytosine and 3,N4-α-hydroxyethanocytosine

Etheno () adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate -adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of -adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (C.G. Cupples, J.H. Miller. Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 5345-5349) which allowed to monitor Lac+ revertants, the latter arose by GCAT or GCTA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in E. coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of C proceeds via a relatively stable intermediate, 3,N4--hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and mug, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA+, alkB+, and mug+ controls. Considering the levels of CAA-induced Lac+ revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of C and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs C and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and εC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein