Enterobactin export in escherichia coli via P43 (ents) and associated components

"December 2006"The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Vita.Thesis (Ph. D.) University of Missouri-Columbia 2006.Ferric iron, critical for the metabolic functions of many microorganisms, is generally insoluble at neutral pH or quickly complexed by host iron storage proteins. To acquire necessary ferric iron against harsh competition in the environment, iron-starved Escherichia coli synthesize, excrete and retrieve an iron-scavenging siderophore molecule termed enterobactin. Despite extensive characterization of the enterobactin system, the export machinery allowing enterobactin secretion to the extracellular environment has only recently been identified. E.coli membrane protein P43 (entS) in the enterobactin gene cluster encodes a Major Facilitator Superfamily (MFS) exporter. A P43 null mutant was unable to efficiently secrete enterobactin to the supernate, but did secrete elevated levels of enterobactin breakdown products as analyzed by TLC, HPLC, and cross-feeding assays. To further evaluate P43 function in enterobactin transport, inverted membrane vesicles were created using French press and incorporated with an iron-binding fluorescent dye, calcein-AM (CA). Differences in siderophore transport were observed between wild-type and the P43-mutant by monitoring CA fluorescence restoration following iron quenching and the addition of enterobactin. Using specific energy poisons in conjunction with this vesicle system, it was determined that proton motive force energy is utilized for this transport. Additional results demonstrate that siderophore transport from the periplasm to the external environment may be due to contributions from several other identified E.coli components, such as the multi-drug export system comprised of the outer membrane protein TolC and the translocase AcrAB. These data all demonstrate P43 provides a critical activity for the E.coli enterobactin secretion machinery and establish a mechanism for cellular release of siderophore.Includes bibliographical reference

Predictive ability of the ISS, NISS, and APACHE II score for SIRS and sepsis in polytrauma patients

Purpose: Systemic inflammatory response syndrome (SIRS) and sepsis as causes of multiple organ dysfunction syndrome (MODS) remain challenging to treat in polytrauma patients. In this study, the focus was set on widely used scoring systems to assess their diagnostic quality. Methods: A total of 512 patients (mean age: 39.2±16.2, range: 16-88years) who had an Injury Severity Score (ISS) ≥17 were included in this retrospective study. The patients were subdivided into four groups: no SIRS, slight SIRS, severe SIRS, and sepsis. The ISS, New Injury Severity Score (NISS), Acute Physiology and Chronic Health Evaluation II (APACHE II) scores, and prothrombin time were collected at admission. The Kruskal-Wallis test and χ2-test, multinomial regression analysis, and kernel density estimates were performed. Receiver operating characteristic (ROC) analysis is reported as the area under the curve (AUC). Data were considered as significant if p<0.05. Results: All variables were significantly different in all groups (p<0.001). The odds ratio increased with increasing SIRS severity for NISS (slight vs. no SIRS, 1.06, p=0.07; severe vs. no SIRS, 1.07, p=0.04; and sepsis vs. no SIRS, 1.11, p=0.0028) and APACHE II score (slight vs. no SIRS, 0.97, p=0.44; severe vs. no SIRS, 1.08, p=0.02; and sepsis vs. no SIRS, 1.12, p=0.0028). ROC analysis revealed that the NISS (slight vs. no SIRS, AUC 0.61; severe vs. no SIRS, AUC 0.67; and sepsis vs. no SIRS, AUC 0.77) and APACHE II score (slight vs. no SIRS, AUC 0.60; severe vs. no SIRS, AUC 0.74; and sepsis vs. no SIRS, AUC 0.82) had the best predictive ability for SIRS and sepsis. Conclusion: Quick assessment with the NISS or APACHE II score could preselect possible candidates for sepsis following polytrauma and provide guidance in trauma surgeons' decision-makin

Photothermal and immunological effects of immunologically modified graphene nanosystems for cancer treatment

Graphene oxide (GO) has been shown as a potential nanoadjuvant for biomedical applications. Particularly, the capability of GO in loading different therapeutic agents and in absorbing light of a broad wavelength range make GO an ideal nanoplatform for drug delivery and selective phototherapy. Chitosan (CS) is a naturally occurring compound with strong immunological stimulating effects. N-dihydrogalactochitosan, also referred to as glycated chitosan (GC), is an innovative and effective immunostimulant/adjuvant that further enhances the immunostimulatory and surfactant properties of CS. In this study, both CS and GC were used to functionalize GO to improve its biocompatibility and improve its immune-stimulating effect for cancer treatment. This work was designed to determine the main properties of GO/CS and GO/GC nanoparticles (NPs), when used in near-infrared photothermal therapy (PTT) for laser-initiated immunotherapy for cancer treatment. GO/CS and GO/GC NPs were synthesized by self-assembling in an aqueous solution via electrostatic interactions. GC allows for better dispersion and stabilization of NPs than CS. Compared with GO, GO/CS and GO/GC had bigger sizes and positive surface charges. GO showed high thermal conversion ability when irradiated with a near-infrared laser. In vitro studies were conducted to determine the effects of GO/CS + PTT and GO/GC + PTT on target pancreatic and melanoma tumor cells. Free GO was toxic to the cells whereas GO/CS and GO/GC showed limited cytotoxicity. The combination of NPs with PTT caused significant tumor cell death due to the GO-enhanced laser light absorption. Dendritic cells and macrophages were stimulated in vitro to investigate the potential of the NPs as immune-stimulating agents in inducing the secretion of the pro-inflammatory cytokines IL-6 and TNF-alpha. The responses of the immune cells to GC and GO were evaluated by phenotyping the surface markers by flow cytometry. Overall, GO/CS and GO/GC showed promising properties as stable, safe, and effective photothermal agents and immunostimulants for nano-ablative immunotherapy for cancer. Future in vitro and in vivo studies will be conducted to further understand the effects of GO/CS and GO/GC as nanostimulants/adjuvants for cancer treatment and vaccination. This and future work will lay the foundation for future clinical applications of these novel nanosystems. Keywords: graphene oxide (GO); chitosan (CS); N-dihydrogalactochitosan (glycated chitosan, GC); immunostimulant; laser immunotherapy (LIT); thermal effect; cell death

Involvement of TolC protein in the export of siderophore enterobactin in Escherichia coli

Abstract only availableTo acquire the necessary iron against harsh competition in the environment, iron starved bacteria synthesize, excrete and retrieve iron scavenging molecules termed siderophores, one of which is enterobactin. TolC protein may play a vital role in the secretion of enterobactin. Enterobactin molecules destined for secretion must cross both the inner (cytoplasmic) and outer membranes and the intervening periplasmic space, believed to be a distance of at least 130Å across. TolC resembles a trans-periplasmic tunnel embedded in the outer membrane of the cell. It is open to the external environment but is closed at its periplasmic entrance. In order for the cell to export enterobactin, TolC is recruited by substrate specific membrane complexes (translocases) in the periplasmic space and inner membrane. When TolC is recruited, the entrance is opened to allow substrate passage through a continuous machinery spanning the entire cell envelope, from the cytosol to the external environment. PCR primers specific for TolC were designed to amplify the TolC gene. The quality of the PCR product was confirmed using agarose gel electrophoresis. The TolC gene was cloned into a pBAD directional TOPO vector containing an N-terminal His-tag and a gene for kanamycin resistance. The recombinant vector was then transformed into One Shot TOP10 competent Escherichia coli cells. Transformants were selected for by plating on LB medium supplemented with kanamycin. Transformed colonies were analyzed using PCR and restriction digestion. Positive transformants were selected and expression was induced with arabinose. SDS-PAGE assay with His-tag In-gel stain revealed TolC expression. Furthermore, analysis of TolC-null mutations using high performance liquid chromatography (HPLC) reveals that the TolC mutant secretes little, if any, enterobactin. However, some levels of breakdown products 2,3-dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer are observed. These data establish that TolC may be a critical component of the E. coli enterobactin secretion machinery and may represent a type of siderophore export mechanism previously undescribed. TolC family proteins are ubiquitous among gram-negative bacteria, and the conserved apertures present a possible chemotherapeutic target in multidrug-resistant pathogens.Molecular Biology Progra

Momentum-resolved electron-phonon interaction in lead determined by neutron resonance spin-echo spectroscopy

Neutron resonance spin-echo spectroscopy was used to monitor the temperature evolution of the linewidths of transverse acoustic phonons in lead across the superconducting transition temperature, $T_c$, over an extended range of the Brillouin zone. For phonons with energies below the superconducting energy gap, a linewidth reduction of maximum amplitude $\sim 6 \mu$eV was observed below $T_c$. The electron-phonon contribution to the phonon lifetime extracted from these data is in satisfactory overall agreement with {\it ab-initio} lattice-dynamical calculations, but significant deviations are found

Präoperative Nüchternzeiten: Sicht der Patienten

Zusammenfassung: Hintergrund: Mit dem Ziel der subjektiven perioperativen Qualitätsverbesserung scheint es wünschenswert, die präoperativen Nüchternzeiten im Rahmen der als sicher geltenden Grenzen so kurz als möglich zu halten. Diese Maßnahmen sollten mit einer messbaren Verminderung von präoperativem Hunger und Durst einhergehen und v.a. in einer Verbesserung der präoperativen Befindlichkeit resultieren. Welchen Einfluss Durst und Hunger aus Patientensicht auf den präoperativen Komfort haben, ist jedoch weit gehend unbekannt. Ziel dieser Studie war es, das Ausmaß der Beeinträchtigung der Patienten durch eine traditionelle Nüchternheitsregelung abzuschätzen. Patienten und Methoden: Ein Kollektiv von 412Patienten der "American-Society-of-Anesthesiologists"- (ASA-)RisikoklassenI und II, das sich einem kleineren chirurgischen Eingriff unterzog, wurde mithilfe eines Fragebogens zum Ausmaß und Stellenwert von präoperativem Durst und Hunger befragt. Ergebnisse: Es hatten 33% der Patienten mäßigen oder starken Durst, 19% mäßigen bis starken Hunger. Von den Befragten möchten 47% vor der Operation noch trinken, 72% hätten gern noch ein leichtes Frühstück eingenommen. Die mittlere Nüchternzeit war 12,8±3,4h für Flüssigkeiten und 15,5±4,4h für Essen. Durst wurde von 3,3% und Hunger von 0,8% der Patienten als Hauptgrund für die Beeinträchtigung des präoperativen Wohlbefindens genannt. Das lange Warten (8,5%), Nervosität (6,5%) und Angst (4,8%) wurden am häufigsten genannt. Die Antworten waren unabhängig von der Zeitdauer der präoperativen Nüchternheit. Schlussfolgerung: Der Patientenkomfort ist durch eine traditionelle Nüchternheitsregelung beeinträchtigt, und Minimierung der präoperativen Nüchternzeiten wird von den Patienten gewünscht. Anstrengungen mit dem Ziel der Reduktion von präoperativer Angst und Nervosität bergen jedoch zusätzliches großes Potenzial für eine Steigerung der perioperativen Behandlungsqualität aus Sicht der Patiente

Q-dependence of the inelastic neutron scattering cross section for molecular spin clusters with high molecular symmetry

For powder samples of polynuclear metal complexes the dependence of the inelastic neutron scattering intensity on the momentum transfer Q is known to be described by a combination of so called interference terms. They reflect the interplay between the geometrical structure of the compound and the spatial properties of the wave functions involved in the transition. In this work, it is shown that the Q-dependence is strongly interrelated with the molecular symmetry of molecular nanomagnets, and, if the molecular symmetry is high enough, is actually completely determined by it. A general formalism connecting spatial symmetry and interference terms is developed. The arguments are detailed for cyclic spin clusters, as experimentally realized by e.g. the octanuclear molecular wheel Cr8, and the star like tetranuclear cluster Fe4.Comment: 8 pages, 1 figures, REVTEX

Analysis of animal-to-human translation shows that only 5% of animal-tested therapeutic interventions obtain regulatory approval for human applications

There is an ongoing debate about the value of animal experiments to inform medical practice, yet there are limited data on how well therapies developed in animal studies translate to humans. We aimed to assess 2 measures of translation across various biomedical fields: (1) The proportion of therapies which transition from animal studies to human application, including involved timeframes; and (2) the consistency between animal and human study results. Thus, we conducted an umbrella review, including English systematic reviews that evaluated the translation of therapies from animals to humans. Medline, Embase, and Web of Science Core Collection were searched from inception until August 1, 2023. We assessed the proportion of therapeutic interventions advancing to any human study, a randomized controlled trial (RCT), and regulatory approval. We meta-analyzed the concordance between animal and human studies. The risk of bias was probed using a 10-item checklist for systematic reviews. We included 122 articles, describing 54 distinct human diseases and 367 therapeutic interventions. Neurological diseases were the focus of 32% of reviews. The overall proportion of therapies progressing from animal studies was 50% to human studies, 40% to RCTs, and 5% to regulatory approval. Notably, our meta-analysis showed an 86% concordance between positive results in animal and clinical studies. The median transition times from animal studies were 5, 7, and 10 years to reach any human study, an RCT, and regulatory approval, respectively. We conclude that, contrary to widespread assertions, the rate of successful animal-to-human translation may be higher than previously reported. Nonetheless, the low rate of final approval indicates potential deficiencies in the design of both animal studies and early clinical trials. To ameliorate the efficacy of translating therapies from bench to bedside, we advocate for enhanced study design robustness and the reinforcement of generalizability.</p

Generation and application of river network analogues for use in ecology and evolution

Several key processes in freshwater ecology are governed by the connectivity inherent to dendritic river networks. These have extensively been analyzed from a geomorphological and hydrological viewpoint, yet structures classically used in ecological modeling have been poorly representative of the structure of real river basins, often failing to capture well-known scaling features of natural rivers. Pioneering work identified optimal channel networks (OCNs) as spanning trees reproducing all scaling features characteristic of natural stream networks worldwide. While OCNs have been used to create landscapes for studies on metapopulations, biodiversity, and epidemiology, their generation has not been generally accessible. Given the increasing interest in dendritic riverine networks by ecologists and evolutionary biologists, we here present a method to generate OCNs and, to facilitate its application, we provide the R-package OCNet. Owing to the stochastic process generating OCNs, multiple network replicas spanning the same surface can be built; this allows performing computational experiments whose results are irrespective of the particular shape of a single river network. The OCN construct also enables the generation of elevational gradients derived from the optimal network configuration, which can constitute three-dimensional landscapes for spatial studies in both terrestrial and freshwater realms. Moreover, the package provides functions that aggregate OCNs into an arbitrary number of nodes, calculate several descriptors of river networks, and draw relevant network features. We describe the main functionalities of the package and its integration with other R-packages commonly used in spatial ecology. Moreover, we exemplify the generation of OCNs and discuss an application to a metapopulation model for an invasive riverine species. In conclusion, OCNet provides a powerful tool to generate realistic river network analogues for various applications. It thereby allows the design of spatially realistic studies in increasingly impacted ecosystems and enhances our knowledge on spatial processes in freshwater ecology in general

Volumetric preload measurement by thermodilution: a comparison with transoesophageal echocardiography

Background. End-diastolic volume indices determined by transpulmonary thermodilution and pulmonary artery thermodilution may give a better estimate of left ventricular preload than pulmonary capillary wedge pressure monitoring. The aim of this study was to compare volume preload monitoring using the two different thermodilution techniques with left ventricular preload assessment by transoesophageal echocardiography (TOE). Methods. Twenty patients undergoing elective cardiac surgery with preserved left-right ventricular function were studied after induction of anaesthesia. Conventional haemodynamic variables, global end-diastolic volume index using the pulse contour cardiac output (PiCCO) system (GEDVIPiCCO), continuous end-diastolic volume index (CEDVIPAC) measured by a modified pulmonary artery catheter (PAC), left ventricular end-diastolic area index (LVEDAI) using TOE and stroke volume indices (SVI) were recorded before and 20 and 40 min after fluid replacement therapy. Analysis of variance (Bonferroni-Dunn), Bland-Altman analysis and linear regression were performed. Results. GEDVIPiCCO, CEDVIPAC, LVEDAI and SVIPiCCO/PAC increased significantly after fluid load (P10% for GEDVIPiCCO and LVEDAI was observed in 85% and 90% of the patients compared with 45% for CEDVIPAC. Mean bias (2 sd) between percentage changes (Δ) in GEDVIPiCCO and ΔLVEDAI was −3.2 (17.6)% and between ΔCEDVIPAC and ΔLVEDAI −8.7 (30.0)%. The correlation coefficient (r2) for ΔGEDVIPiCCO vs ΔLVEDAI was 0.658 and for ΔCEDVIPAC vs ΔLVEDAI 0.161. The relationship between ΔGEDVIPiCCO and ΔSVIPiCCO was stronger (r2=0.576) than that between ΔCEDVIPAC and ΔSVIPAC (r2=0.267). Conclusion. GEDVI assessed by the PiCCO system gives a better reflection of echocardiographic changes in left ventricular preload, in response to fluid replacement therapy, than CEDVI measured by a modified PA