Over-optimism in unsupervised microbiome analysis: Insights from network learning and clustering

In recent years, unsupervised analysis of microbiome data, such as microbial network analysis and clustering, has increased in popularity. Many new statistical and computational methods have been proposed for these tasks. This multiplicity of analysis strategies poses a challenge for researchers, who are often unsure which method(s) to use and might be tempted to try different methods on their dataset to look for the “best” ones. However, if only the best results are selectively reported, this may cause over-optimism: the “best” method is overly fitted to the specific dataset, and the results might be non-replicable on validation data. Such effects will ultimately hinder research progress. Yet so far, these topics have been given little attention in the context of unsupervised microbiome analysis. In our illustrative study, we aim to quantify over-optimism effects in this context. We model the approach of a hypothetical microbiome researcher who undertakes four unsupervised research tasks: clustering of bacterial genera, hub detection in microbial networks, differential microbial network analysis, and clustering of samples. While these tasks are unsupervised, the researcher might still have certain expectations as to what constitutes interesting results. We translate these expectations into concrete evaluation criteria that the hypothetical researcher might want to optimize. We then randomly split an exemplary dataset from the American Gut Project into discovery and validation sets multiple times. For each research task, multiple method combinations (e.g., methods for data normalization, network generation, and/or clustering) are tried on the discovery data, and the combination that yields the best result according to the evaluation criterion is chosen. While the hypothetical researcher might only report this result, we also apply the “best” method combination to the validation dataset. The results are then compared between discovery and validation data. In all four research tasks, there are notable over-optimism effects; the results on the validation data set are worse compared to the discovery data, averaged over multiple random splits into discovery/validation data. Our study thus highlights the importance of validation and replication in microbiome analysis to obtain reliable results and demonstrates that the issue of over-optimism goes beyond the context of statistical testing and fishing for significance

Hypoxia evokes increased PDI and PDIA6 expression in the infarcted myocardium of ex-germ-free and conventionally raised mice

The prototypic protein disulfide isomerase (PDI), encoded by the P4HB gene, has been described as a survival factor in ischemic cardiomyopathy. However, the role of protein disulfide isomerase associated 6 (PDIA6) under hypoxic conditions in the myocardium remains enigmatic, and it is unknown whether the gut microbiota influences the expression of PDI and PDIA6 under conditions of acute myocardial infarction. Here, we revealed that, in addition to the prototypic PDI, the PDI family member PDIA6, a regulator of the unfolded protein response, is upregulated in the mouse cardiomyocyte cell line HL-1 when cultured under hypoxia. In vivo, in the left anterior descending artery (LAD) ligation mouse model of acute myocardial infarction, similar to PDI, PDIA6 protein expression was enhanced in the infarcted area (LAD ) relative to uninfarcted sham tissue or the neighbouring area at risk (LAD–) of C57BL/6J mice. Interestingly, we found that ex-germ-free (ex-GF) mice subjected to the LAD ligation model for 24 h had a reduced ejection fraction compared with their conventionally raised (CONV-R) SPF controls. Furthermore, the LAD area in the infarcted heart of ex-GF mice showed reduced PDIA6 expression relative to CONV-R controls, suggesting that the presence of a gut microbiota enhanced LAD ligation-triggered PDIA6 expression. Collectively, our results demonstrate that PDIA6 is upregulated in cardiomyocytes as a consequence of hypoxia. In the LAD mouse model, PDIA6 was also increased in the infarcted area under in vivo conditions, but this increase was suppressed in ex-GF mice relative to CONV-R controls

Adult-Onset Leukoencephalopathy With Axonal Spheroids and Pigmented Glia: Review of Clinical Manifestations as Foundations for Therapeutic Development

A comprehensive review of published literature was conducted to elucidate the genetics, neuropathology, imaging findings, prevalence, clinical course, diagnosis/clinical evaluation, potential biomarkers, and current and proposed treatments for adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), a rare, debilitating, and life-threatening neurodegenerative disorder for which disease-modifying therapies are not currently available. Details on potential efficacy endpoints for future interventional clinical trials in patients with ALSP and data related to the burden of the disease on patients and caregivers were also reviewed. The information in this position paper lays a foundation to establish an effective clinical rationale and address the clinical gaps for creation of a robust strategy to develop therapeutic agents for ALSP, as well as design future clinical trials, that have clinically meaningful and convergent endpoints

VISIR Upgrade Overview and Status

We present an overview of the VISIR upgrade project. VISIR is the mid-infrared imager and spectrograph at ESO’s VLT. The project team is comprised of ESO staff and members of the original VISIR consortium: CEA Saclay and ASTRON. The project plan is based on input from the ESO user community with the goal of enhancing the scientific performance and efficiency of VISIR by a combination of measures: installation of improved hardware, optimization of instrument operations and software support. The cornerstone of the upgrade is the 1k by 1k Si:As AQUARIUS detector array (Raytheon) which has been carefully characterized in ESO’s IR detector test facility (modified TIMMI 2 instrument). A prism spectroscopic mode will cover the N-band in a single observation. New scientific capabilities for high resolution and high-contrast imaging will be offered by sub-aperture mask (SAM) and phase-mask coronagraphic (4QPM/AGPM) modes. In order to make optimal use of favourable atmospheric conditions a water vapour monitor has been deployed on Paranal, allowing for real-time decisions and the introduction of a user-defined constraint on water vapour. During the commissioning in 2012 it was found that the on-sky sensitivity of the AQUARIUS detector was significantly below expectations and that VISIR was not ready to go back to science operations. Extensive testing of the detector arrays in the laboratory and on-sky enabled us to diagnose the cause for the shortcoming of the detector as excess low frequency noise (ELFN). It is inherent to the design chosen for this detector and can’t be remedied by changing the detector set-up. Since this is a form of correlated noise its impact can be limited by modulating the scene recorded by the detector. We have studied several mitigation options and found that faster chopping using the secondary mirror (M2) of the VLT offers the most promising way forward. Faster M2 chopping has been tested and is scheduled for implementation before the end of 2014 after which we plan to re-commission VISIR. In addition an upgrade of the IT infrastructure related to VISIR is planned in order to support burst-mode operations. The upgraded VISIR will be a powerful instrument providing close to background limited performance for diffraction-limited observations at an 8-m telescope. It will offer synergy with facilities such as ALMA, JWST, VLTI and SOFIA, while a wealth of targets is available from survey work (e.g. VISTA, WISE). In addition it will bring confirmation of the technical readiness and scientific value of several aspects of potential mid-IR instrumentation at Extremely Large Telescopes

Human impact on the transport of terrigenous and anthropogenic elements to peri-alpine lakes (Switzerland) over the last decades

Terrigenous (Sc, Fe, K, Mg, Al, Ti) and anthropogenic (Pb and Cu) element fluxes were measured in a new sediment core from Lake Biel (Switzerland) and in previously well-documented cores from two upstream lakes (Lake Brienz and Lake Thun). These three large peri-alpine lakes are connected by the Aare River, which is the main tributary to the High Rhine River. Major and trace element analysis of the sediment cores by inductively coupled plasma mass spectrometry (ICP-MS) shows that the site of Lake Brienz receives three times more terrigenous elements than the two other studied sites, given by the role of Lake Brienz as the first major sediment sink located in the foothills of the Alps. Overall, the terrigenous fluxes reconstructed at the three studied sites suggest that the construction of sediment-trapping reservoirs during the twentieth century noticeably decreased the riverine suspended sediment load at a regional scale. In fact, the extensive river damming that occurred in the upstream watershed catchment (between ca. 1930 and 1950 and up to 2300 m a.s.l.) and that significantly modified seasonal suspended sediment loads and riverine water discharge patterns to downstream lakes noticeably diminished the long-range transport of (fine) terrigenous particles by the Aare River. Concerning the transport of anthropogenic pollutants, the lowest lead enrichment factors (EFs Pb) were measured in the upstream course of the Aare River at the site of Lake Brienz, whereas the metal pollution was highest in downstream Lake Biel, with the maximum values measured between 1940 and 1970 (EF Pb >3). The following recorded regional reduction in aquatic Pb pollution started about 15 years before the actual introduction of unleaded gasoline in 1985. Furthermore, the radiometric dating of the sediment core from Lake Biel identifies three events of hydrological transport of artificial radionuclides released by the nuclear reactor of Mühleberg located at more than 15 km upstream of Lake Biel for the time period 1970 to 200

Collins and Sivers asymmetries in muonproduction of pions and kaons off transversely polarised protons

Measurements of the Collins and Sivers asymmetries for charged pions and charged and neutral kaons produced in semi-inclusive deep-inelastic scattering of high energy muons off transversely polarised protons are presented. The results were obtained using all the available COMPASS proton data, which were taken in the years 2007 and 2010. The Collins asymmetries exhibit in the valence region a non-zero signal for pions and there are hints of non-zero signal also for kaons. The Sivers asymmetries are found to be positive for positive pions and kaons and compatible with zero otherwise. © 2015

Die Rolle von myeloiden Zellen und deren Interaktion mit inflammatorischen Mediatoren bei der Pathogenese des Myokardinfarktes

Myeloide Zellen sind sowohl an der initialen Entzündungsreaktion als auch am anschließenden Heilungsprozess nach Myokardinfarkt (MI) beteiligt. Das Zusammenspiel zwischen diesen Zellen und Inflammationsmediatoren bleibt jedoch bisher unzureichend geklärt. Über Mausmodelle zur gezielten Zelldepletion sowie unter Verwendung systemischer Knock-outs sollte der kardioprotektive oder partiell negative Einfluss von myeloiden Immunzellen (Neutrophile, Monozyten/Makrophagen) und interagierenden Mediatoren (Interferon-gamma, Tumornekrosefaktor-alpha, Cathelicidin) bei der Pathogenese des MI näher untersucht werden. Des Weiteren sollte die Charakterisierung der unterschiedlichen Effekte auf den Inflammationsverlauf und auf den funktionalen Phänotyp - abhängig vom Zeitpunkt nach MI - dazu dienen, neue immunregulatorische Therapieoptionen für die klinische Anwendung zu erschließen. Durchflusszytometrische und molekularbiologische Analysen konnten Hinweise liefern, dass myelomonozytäre Lysozym M positive (LysM+) Zellen essentiell an der Inflammationsantwort nach MI beteiligt sind. Anhand von echokardiografischen sowie histologischen Untersuchungen konnte zudem gezeigt werden, dass die Entzündungs- sowie Reparationsmechanismen, welche über LysM+ Neutrophile und Monozyten/Makrophagen mediiert werden, notwendig für das „cardiac remodeling“ und die kardiale Funktion nach MI sind. Der antikörpervermittelte Eingriff in die Zellmobilisierung sowie genetisch ausgeschaltete Aktivierungsmechanismen nach MI zeigten eine signifikante Reduktion der Neutrophilen- und Monozyten-Chemotaxis sowie eine verminderte Expression an Inflammationsmarkern. Des Weiteren hängt die neutrophilenabhängige Monozyten-Chemotaxis essentiell von neutrophilstämmigen granulären Proteinen (Cathelicidinen) ab - sowohl die „granulocyte differentiation antigen-1“ (Gr-1+) Neutrophilen-Defizienz als auch der über Cathelicidinmangel herbeigeführte Monozytendefizit führten zu einer chemotaktischen sowie systolischen Dysfunktion und war mit einem Anstieg der Mortalität nach MI assoziiert. Weiterhin konnte gezeigt werden, dass proinflammatorische Zytokine (IFN-γ, TNF-α) die myelomonozytäre Zellrekrutierung beim MI vermitteln. Das inflammatorische Verhalten myeloider Zellen nach MI ist hierbei von der Ifngr1 Expression in myelomonozytären Zellen und von einem funktionalen „IFN-γ-signaling“ abhängig. Es konnte zudem ein direkter Zusammenhang zwischen der neutrophilenabhängigen Monozyten-Chemotaxis und dem „IFN-γ-signaling“ nach MI hergestellt werden. Welche Zellpopulation für den zeitigen Anstieg des IFN-γ Level im infarzierten Myokard verantwortlich ist, muss noch geklärt werden. Möglicherweise kommen insbesondere gamma:delta T-Zellen oder „innate lymphocyte cells“ in Frage. Anhand der Resultate dieser Dissertation konnte das für die Infarktheilung wichtige Zusammenspiel von myelomonozytären Zellen, Chemokinen, Zytokinen sowie granulozytären Peptiden weiter analysiert werden. Die gewonnenen Untersuchungsergebnisse tragen zu einem besseren Verständnis der Pathogenese des Myokardinfarktes in Hinblick auf die immunmodulatorische Funktion myeloider Zellen sowie interagierender Mediatoren bei. Ein wichtiger Aspekt bei der Suche nach geeigneten Therapiemöglichkeiten nach MI stellt die gezielte Verminderung der inflammatorischen Beschädigung des Myokards und damit verbundene Reduktion von pathophysiologischen Veränderungen dar. Strategien, welche myelomonozytäre Zellen betreffen, müssen zudem die Zelldiversität, den Zeitpunkt der Zellinfiltration und die Gewebelokalisierung mit einbeziehen. Mit den Erkenntnissen dieser Arbeit sollten zukünftige therapeutische Methoden zur Verhinderung von Herzinsuffizienz nach MI den potentiell positiven Einfluss der zeitigen Neutrophileninfiltration ins infarzierte Myokard mit berücksichtigen.Myeloid cells are involved in the initial injury as well as in the later healing mechanisms of myocardial infarction (MI). However, the exact interactions between these cells and inflammatory mediators remain insufficiently understood. The aim of the study was to investigate the cardio protective or in part adverse role of myeloid immune cells (lysozyme M positive, granulocyte-receptor 1 positive cells) and interacting mediators (interferon gamma, tumor necrosis factor alpha, cathelicidin) on cardiac injury and healing by using mouse models for targeted cell depletion and systemic knock-outs. Depending on the time point after induction of MI different effects on the inflammatory processes and on the functional phenotype should be characterized in order to find new immunoregulatory therapeutic possibilities for clinical application. Flowcytometric and molecular analyses have shown that myeloid LysM+ cells are involved in the inflammatory response after MI. It was also shown by echocardiographic and histological investigations that inflammatory processes and repair mechanisms - mediated by LysM+ neutrophils and monocytes/macrophages - are necessary for cardiac remodeling and cardiac function after MI. The antibody mediated intervention in cell mobilization as well as genetically knocked out activation mechanisms resulted in a significant reduction of the neutrophil and monocyte chemotaxis as well as a reduced expression of inflammatory markers. After MI neutrophils are required for the monocyte chemotaxis. Additionally, the neutrophil dependent monocyte chemotaxis essentially relies on neutrophil-bound granular proteins. Both Gr-1+ neutrophil deficiency and monocyte deficiency caused by the lack of cathelicidin led to chemotactic as well as systolic dysfunction and was associated with an increase in mortality. Moreover, it has been shown that inflammatory cytokines (IFN-γ, TNF-α) mediate the myeloid cell recruitment and play an essential role in survival and cardiac remodeling following MI. The inflammatory behavior of myeloid cells depends on the Ifngr1 expression in myeloid cells and on a functional IFN-γ signaling. In addition, a direct link between the neutrophil dependent monocyte chemotaxis and the IFN-γ signaling according to MI could be established. The cell population which is responsible for the early increase of the IFN-γ level in the infarcted myocardium still has to be detected - possibly γ:δ T-cells or ILCs are in charge of it. The interplay between myeloid cells, chemokines, cytokines and granulocytic peptides, which is important for infarct healing, could be further analyzed. The results of this investigation contribute to a better understanding of the pathogenesis of MI with regard to the immunomodulatory function of myeloid cells as well as interacting mediators. Therapeutic methods to improve post-MI heart failure and targeting myeloid cells must include tissue localization, cell diversity and timing. The data of this study indicate that therapeutic strategies to oppose the inflammatory injury in MI must consider as well the potentially beneficial effect of the early neutrophil influx into the infarcted myocardium

Angiotensin II Infusion Leads to Aortic Dissection in LRP8 Deficient Mice

International audienceMyeloid cells are crucial for the development of vascular inflammation. Low-density lipoprotein receptor-related protein 8 (LRP8) or Apolipoprotein E receptor 2 (ApoER2), is expressed by macrophages, endothelial cells and platelets and has been implicated in the development of cardiovascular diseases. Our aim was to evaluate the role of LRP8, in particular from immune cells, in the development of vascular inflammation. Methods. LRP8+/+ and LRP8−/− mice (on B6;129S background) were infused with angiotensin II (AngII, 1 mg/kg/day for 7 to 28 day) using osmotic minipumps. Blood pressure was recorded using tail cuff measurements. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging. Histological analysis of aortic sections was conducted using sirius red staining. Results. AngII infusion worsened endothelial-dependent vascular relaxation and immune cells rolling and adherence to the carotid artery in both LRP8+/+ as well as LRP8−/− mice. However, only LRP8−/− mice demonstrated a drastically increased mortality rate in response to AngII due to aortic dissection. Bone marrow transplantation revealed that chimeras with LRP8 deficient myeloid cells phenocopied LRP8−/− mice. Conclusion. AngII-infused LRP8 deficient mice could be a useful animal model to study aortic dissection reflecting the lethality of this disease in humans