Excision and transfer of an integrating and conjugative element in a bacterial species with high recombination efficiency

Horizontal transfer of mobile genetic elements, such as integrating and conjugative elements (ICEs), plays an important role in generating diversity and maintaining comprehensive pan-genomes in bacterial populations. The human gastric pathogen Helicobacter pylori, which is known for its extreme genetic diversity, possesses highly efficient transformation and recombination systems to achieve this diversity, but it is unclear to what extent these systems influence ICE physiology. In this study, we have examined the excision/integration and horizontal transfer characteristics of an ICE (termed ICEHptfs4) in these bacteria. We show that transfer of ICEHptfs4 DNA during mating between donor and recipient strains is independent of its conjugation genes, and that homologous recombination is much more efficient than site-specific integration into the recipient chromosome. Nevertheless, ICEHptfs4 excision by site-specific recombination occurs permanently in a subpopulation of cells and involves relocation of a circularization-dependent promoter. Selection experiments for excision indicate that the circular form of ICEHptfs4 is not replicative, but readily reintegrates by site-specific recombination. Thus, although ICEHptfs4 harbours all essential transfer genes, and typical ICE functions such as site-specific integration are active in H. pylori, canonical ICE transfer is subordinate to the more efficient general DNA uptake and homologous recombination machineries in these bacteria

A comprehensive analysis of Helicobacter pylori plasticity zones reveals that they are integrating conjugative elements with intermediate integration specificity

Background: The human gastric pathogen Helicobacter pylori is a paradigm for chronic bacterial infections. Its persistence in the stomach mucosa is facilitated by several mechanisms of immune evasion and immune modulation, but also by an unusual genetic variability which might account for the capability to adapt to changing environmental conditions during long-term colonization. This variability is reflected by the fact that almost each infected individual is colonized by a genetically unique strain. Strain-specific genes are dispersed throughout the genome, but clusters of genes organized as genomic islands may also collectively be present or absent. Results: We have comparatively analysed such clusters, which are commonly termed plasticity zones, in a high number of H. pylori strains of varying geographical origin. We show that these regions contain fixed gene sets, rather than being true regions of genome plasticity, but two different types and several subtypes with partly diverging gene content can be distinguished. Their genetic diversity is incongruent with variations in the rest of the genome, suggesting that they are subject to horizontal gene transfer within H. pylori populations. We identified 40 distinct integration sites in 45 genome sequences, with a conserved heptanucleotide motif that seems to be the minimal requirement for integration. Conclusions: The significant number of possible integration sites, together with the requirement for a short conserved integration motif and the high level of gene conservation, indicates that these elements are best described as integrating conjugative elements (ICEs) with an intermediate integration site specificity

Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate.

AIM: To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles. MATERIAL & METHODS: The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. RESULTS: Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. CONCLUSION: In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes

Prognostic impact of pretransplant measurable residual disease assessed by peripheral blood WT1-mRNA expression in patients with AML and MDS

OBJECTIVE As peripheral blood (PB) Wilm's Tumor 1 (WT1)-mRNA expression is established as MRD-marker during conventional AML chemotherapy, impact of pretransplant WT1 expression remains unclear. Therefore, we aimed to assess prognostic impact of pretransplant WT1 expression on post-transplant outcome in patients with AML/MDS. METHODS In 64 AML/MDS patients, pretransplant WT1 expression was retrospectively analyzed using a standardized assay offering high sensitivity, specificity, and a validated cut-off. Patients were divided into three groups determined by pretransplant remission and WT1 expression. Post-transplant outcome of these groups was compared regarding cumulative incidence of relapse (CIR), relapse-free (RFS), and overall survival (OS). RESULTS Pretransplant forty-six patients (72%) showed hematologic remission, including 21 (46%) MRD-negative and 25 (54%) MRD-positive patients indicated by WT1 expression, while 18 refractory patients (28%) showed active disease. Two-year estimates of post-transplant CIR, RFS, and OS were similar in MRD-positive (61%, 37%, 54%) and refractory patients (70%, 26%, 56%), but significantly inferior compared with MRD-negative patients (10%, 89%, 90%). After multivariable adjustment, pretransplant MRD negativity measured by WT1 expression retained its prognostic impact on CIR (P~=~.008), RFS (P~=~.005), and OS (P~=~.049). CONCLUSIONS PB WT1 expression represents a useful method to estimate pretransplant MRD, which is highly predictable for post-transplant outcome and may help improving peri-transplant management in AML/MDS patients

Yale Global Tic Severity Scale (YGTSS): Psychometric Quality of the Gold Standard for Tic Assessment Based on the Large-Scale EMTICS Study.

The Yale Global Tic Severity Scale (YGTSS) is a clinician-rated instrument considered as the gold standard for assessing tics in patients with Tourette's Syndrome and other tic disorders. Previous psychometric investigations of the YGTSS exhibit different limitations such as small sample sizes and insufficient methods. To overcome these shortcomings, we used a subsample of the large-scale "European Multicentre Tics in Children Study" (EMTICS) including 706 children and adolescents with a chronic tic disorder and investigated convergent, discriminant and factorial validity, as well as internal consistency of the YGTSS. Our results confirm acceptable convergent and good to very good discriminant validity, respectively, indicated by a sufficiently high correlation of the YGTSS total tic score with the Clinical Global Impression Scale for tics (r <sub>s</sub> = 0.65) and only low to medium correlations with clinical severity ratings of attention deficit/hyperactivity symptoms (r <sub>s</sub> = 0.24), obsessive-compulsive symptoms (r <sub>s</sub> = 27) as well as internalizing symptoms (r <sub>s</sub> = 0.27). Internal consistency was found to be acceptable (Ω = 0.58 for YGTSS total tic score). A confirmatory factor analysis supports the concept of the two factors "motor tics" and "phonic tics," but still demonstrated just a marginal model fit (root mean square error of approximation = 0.09 [0.08; 0.10], comparative fit index = 0.90, and Tucker Lewis index = 0.87). A subsequent analysis of local misspecifications revealed correlated measurement errors, suggesting opportunities for improvement regarding the item wording. In conclusion, our results indicate acceptable psychometric quality of the YGTSS. However, taking the wide use and importance of the YGTSS into account, our results suggest the need for further investigations and improvements of the YGTSS. In addition, our results show limitations of the global severity score as a sum score indicating that the separate use of the total tic score and the impairment rating is more beneficial

Intestinal APCs of the endogenous nanomineral pathway fail to express PD-L1 in Crohn's disease.

Crohn's disease is a chronic inflammatory condition most commonly affecting the ileum and colon. The aetiology of Crohn's disease is complex and may include defects in peptidoglycan recognition, and/or failures in the establishment of intestinal tolerance. We have recently described a novel constitutive endogenous delivery system for the translocation of nanomineral-antigen-peptidoglycan (NAP) conjugates to antigen presenting cells (APCs) in intestinal lymphoid patches. In mice NAP conjugate delivery to APCs results in high surface expression of the immuno-modulatory molecule programmed death receptor ligand 1 (PD-L1). Here we report that NAP conjugate positive APCs in human ileal tissues from individuals with ulcerative colitis and intestinal carcinomas, also have high expression of PD-L1. However, NAP-conjugate positive APCs in intestinal tissue from patients with Crohn's disease show selective failure in PD-L1 expression. Therefore, in Crohn's disease intestinal antigen taken up by lymphoid patch APCs will be presented without PD-L1 induced tolerogenic signalling, perhaps initiating disease

Reduction of T-Helper Cell Responses to Recall Antigen Mediated by Codelivery with Peptidoglycan via the Intestinal Nanomineral-Antigen Pathway.

Naturally occurring intestinal nanomineral particles constituently form in the mammalian gut and trap luminal protein and microbial components. These cargo loaded nanominerals are actively scavenged by M cells of intestinal immune follicles, such as Peyer's patches and are passed to antigen-presenting cells. Using peripheral blood mononuclear cell populations as an in vitro model of nanomineral uptake and antigen presentation, we show that monocytes avidly phagocytose nanomineral particles bearing antigen and peptidoglycan (PGN), and that the presence of PGN within particles downregulates their cell surface MHC class II and upregulates programmed death receptor ligand 1. Nanomineral delivery of antigen suppresses antigen-specific CD4+ T cell responses, an effect that is enhanced in the presence of PGN. Blocking the interleukin-10 receptor restores CD4+ T cell responses to antigen codelivered with PGN in nanomineral form. Using human intestinal specimens, we have shown that the in vivo nanomineral pathway operates in an interleukin-10 rich environment. Consequently, the delivery of a dual antigen-PGN cargo by endogenous nanomineral in vivo is likely to be important in the establishment of intestinal tolerance, while their synthetic mimetics present a potential delivery system for therapeutic applications targeting the modulation of Peyer's patch T cell responses

Synthetic mimetics of the endogenous gastrointestinal nanomineral: Silent constructs that trap macromolecules for intracellular delivery.

Amorphous magnesium-substituted calcium phosphate (AMCP) nanoparticles (75-150nm) form constitutively in large numbers in the mammalian gut. Collective evidence indicates that they trap and deliver luminal macromolecules to mucosal antigen presenting cells (APCs) and facilitate gut immune homeostasis. Here, we report on a synthetic mimetic of the endogenous AMCP and show that it has marked capacity to trap macromolecules during formation. Macromolecular capture into AMCP involved incorporation as shown by STEM tomography of the synthetic AMCP particle with 5nm ultra-fine iron (III) oxohydroxide. In vitro, organic cargo-loaded synthetic AMCP was taken up by APCs and tracked to lysosomal compartments. The AMCP itself did not regulate any gene, or modify any gene regulation by its cargo, based upon whole genome transcriptomic analyses. We conclude that synthetic AMCP can efficiently trap macromolecules and deliver them to APCs in a silent fashion, and may thus represent a new platform for antigen delivery

The CANNA-TICS Study Protocol: A Randomized Multi-Center Double-Blind Placebo Controlled Trial to Demonstrate the Efficacy and Safety of Nabiximols in the Treatment of Adults With Chronic Tic Disorders

Background: Gilles de la Tourette syndrome (TS) is a chronic neuropsychiatric disorder characterized by motor and vocal tics. First-line treatments for tics are antipsychotics and tic-specific behavioral therapies. However, due to a lack of trained therapists and adverse events of antipsychotic medication many patients seek alternative treatment options including cannabis. Based on the favorable results obtained from case studies on different cannabis-based medicines as well as two small randomized controlled trials using delta-9-tetrahydrocannabinol (THC), we hypothesize that the cannabis extract nabiximols can be regarded as a promising new and safe treatment strategy in TS. Objective: To test in a double blind randomized clinical trial, whether treatment with the cannabis extract nabiximols is superior to placebo in patients with chronic tic disorders. Patients and Methods: This is a multicenter, randomized, double-blind, placebo controlled, parallel-group, phase IIIb trial, which aims to enroll 96 adult patients with chronic tic disorders (TS or chronic motor tic disorder) across 6 centers throughout Germany. Patients will be randomized with a 2:1 ratio into a nabiximols and a placebo arm. The primary efficacy endpoint is defined as tic reduction of at least 30% (compared to baseline) according to the Total Tic Score of the Yale Global Tic Severity Scale (YGTSS-TTS) after 13 weeks of treatment. In addition, several secondary endpoints will be assessed including changes in different psychiatric comorbidities, quality of life, driving ability, and safety assessments. Discussion: This will be the first large, controlled study investigating efficacy and safety of a cannabis-based medicine in patients with TS. Based on available data using different cannabis-based medicines, we expect not only a reduction of tics, but also an improvement of psychiatric comorbidities. If the cannabis extract nabiximols is proven to be safe and effective, it will be a valuable alternative treatment option. The results of this study will be of high health-economic relevance, because a substantial number of patients uses cannabis (illegally) as self-medication. Conclusion: The CANNA-TICS trial will clarify whether nabiximols is efficacious and safe in the treatment of patients with chronic tic disorders

Auskunft ist Jazz : Projektbericht zur Erstellung eines Lehrfilms zum Auskunftsinterview in Bibliotheken ; Lehrveranstaltung BBPD1 Projekt Auskunftsinterview des Bachelorstudiengangs Bibliothekswesen im Wintersemester 2013/14 und Sommersemester 2014

„Auskunft ist Jazz – Das bibliothekarische Auskunftsinterview“ wurde als Lehrfilm im Rahmen eines Studienprojekts des Studiengangs Bibliothekswesen der FH Köln im Wintersemester 2013/14 und im Sommersemester 2014 durch eine studentische Arbeitsgruppe unter der Leitung von Prof. Dr. Hermann Rösch konzipiert und hergestellt. Der hier vorliegende Bericht bietet detaillierte Informationen zur Entstehung und Durchführung des Projekts von der ersten Idee bis zum fertigen Film. Im Anhang werden darüber hinaus unter anderem das Drehbuch und das Storyboard des Films zur Verfügung gestellt. „Auskunft ist Jazz – Das bibliothekarische Auskunftsinterview“ zeigt sämtliche Aspekte des Auskunftsinterviews anhand eines beispielhaften Szenarios, in dem die Schülerin Clarissa auf der Suche nach Informationen für ihre Facharbeit an der Auskunftstheke der Bibliothekarin Nadine begegnet. In jeweils negativen und positiven Varianten werden anhand des folgenden Auskunftsgesprächs die Erzeugung der Grundstimmung zu Anfang, die Ermittlung des Informationsbedarfs mit Hilfe geeigneter Fragetechniken, die gemeinsame Recherche und die Qualitätskontrolle durch das Follow-up zum Abschluss gezeigt. Zwischen den Szenen sorgt ein Moderator erklärend für Orientierung und es werden weitere unterhaltsame und lehrreiche Szenen aus dem bibliothekarischen Alltag gezeigt. Darüber hinaus geht der Film auch auf schwierige Phänomene wie großer Andrang und ungeduldige Nutzer an der Auskunftstheke ein. Sämtliche Aufgaben bei der Planung und Herstellung des Films wurden arbeitsteilig durch ein Drehbuchteam, ein Technikteam und ein Organisationsteam erfüllt. Die Herausforderung für die Mitglieder der verschiedenen Teams bestand neben der Aneignung spezialisierter Fähigkeiten in den jeweiligen Arbeitsbereichen besonders in der Kooperation und Koordination des komplexen Gesamtprojekts. Das Zusammenspiel bei gleichzeitiger Spezialisierung machte den für alle Beteiligten hochinteressanten und lehrreichen Charakter des Projekts aus. Nachdem der Film am 12. Juni 2014 im Rahmen einer Premierenfeier am Institut für Informationswissenschaften der Fachhochschule Köln vorgestellt wurde, steht er nun auf Youtube der Öffentlichkeit zur Verfügung. Zusätzlich zum eigentlichen Lehrfilm wurde noch ein Zusammenschnitt des positiven Interviews im Zusammenhang und eine Checkliste mit den wichtigsten Stichworten zum Auskunftsinterview veröffentlicht. Eingesetzt werden soll der Film in universitärer Lehre sowie bibliothekarischer Aus- und Fortbildung. Alle drei Variationen sind unter einer Creative Commons-Namensnennung-Nicht kommerziell-4.0-International-Lizenz veröffentlicht und stehen somit für die freie und nichtkommerziellen Verwendung zur Verfügung