Sphaeroidkörper der Diatomee Rhopalodia gibba – Obligate Endosymbionten zur molekularen Stickstofffixierung

Die Diatomee Rhopalodia gibba enthält - neben den typischen Organellen von Heterokontophyten, Mitochondrien und einer kompexen Plastide – ungewöhnliche Einschlüsse, welche als Sphaeroidkörper bezeichnet werden. Morphologische Untersuchungen führten zur Vermutung, dass es sich bei diesen Sphaeroidkörpern um Endosymbionten bakterieller Herkunft handeln könnte. Des Weiteren wurde ein Zusammenhang zwischen den Einschlusskörpern und der bei R. gibba beobachteten Fähigkeit zur Fixierung von molekularem Stickstoff diskutiert. Im Rahmen dieser Arbeit sollten die Sphaeroidkörper von R. gibba näher charakterisiert und ihre Funktion innerhalb der Diatomee aufgeklärt werden. Hierzu wurden intakte Sphaeroidkörper aus Diatomeenzellen isoliert und aufgereinigt. Aus diesen Fraktionen konnte DNA präpariert und mittels molekularbiologischer sowie histologischer Analysen den Sphaeroidkörpern zugeordnet werden. Die Identifizierung eines Sphaeroidkörper-Genoms konnte damit die organismische Herkunft der Einschlusskörper und deren endosymbiontische Natur bestätigen. Durch phylogenetische Untersuchungen von Sphaeroidkörper-spezifischen Genen wurde das einzellige, diazotrophe Cyanobakterium Cyanothece sp. ATCC 51142 als nächster freilebender Verwandter der Sphaeroidkörper bestimmt. Durch molekularbiologische Analysen im Rahmen eines Genom-Sequenzierprojekts wurde gezeigt, dass die cyanobakteriellen Endosymbionten von R. gibba alle Strukturgene der Nitrogenase sowie andere Gene für Faktoren der molekularen Stickstofffixierung kodieren. Die Nitrogenase konnte des Weiteren auf Proteinebene durch in-situ Lokalisation an Diatomeenzellen innerhalb der Sphaeroidkörper nachgewiesen werden. Ein Sequenzierprojekt vergleichbarer Sequenzen sowohl der Sphaeroidkörper-DNA als auch von Cyanothece sp. diente dazu, genetische Modifikationen der Endosymbionten zu identifizieren. In diesem Zusammenhang wurden von beiden Organismen über 50 kbp des Genoms sequenziert und analysiert. Die Untersuchungen ergaben, dass das Sphaeroidkörper-Genom zahlreiche Modifikationen aufweist, welche mit der endosymbiontischen Lebensweise einhergehen. Hierzu gehörte vor allem eine reduzierende Evolution des Genoms, die sich durch Mutation, Modifikation sowie Deletion von Genen sowie der Ausbildung großer, nicht-kodierender A/T-reicher DNA-Regionen (NC-Regionen) auszeichnet. Derartige genomische Modifikationen ließen sich zum Teil auf physiologische Veränderungen der endosymbiontischen Bakterien übertragen. Diese Veränderungen – wie der Verlust der photosynthetischen Aktivität – resultieren unter anderem in einer obligaten Abhängigkeit der Endosymbionten von der Wirtszelle. Die Analysen zeigten weiterhin, dass nicht alle Regionen des Sphaeroidkörper-Genoms gleich stark von genetischen Degenerationsprozessen betroffen sind und das Ausmaß der Veränderungen auf ein frühes Stadium der obligat-symbiontischen Interaktion hindeutet. Im Rahmen dieser Arbeit konnte damit gezeigt werden, dass es sich bei den Sphaeroidkörpern von R. gibba um obligate Endosymbionten cyanobakterieller Herkunft handelt, welche innerhalb der Wirtszelle molekularen Stickstoff fixieren. Damit wäre die Sphaeroidkörper-Rhopalodia-Assoziation die erste beschriebene obligate Symbiose zur Stickstofffixierung zwischen einem Bakterium und einem eukaryoten Organismus überhaupt. Die Analyse des Sphaeroidkörper-Genoms sowie die identifizierten genetischen Modifikationen geben einen Einblick in die Genomevolution bakterieller intrazellulärer Symbionten. Dabei konnten mit den NC-Regionen Sequenzbereiche beschrieben werden, welche Hinweise auf genomische Veränderungen in den frühen Stadien symbiontischer Interaktion geben könnten. Die Sphaeroidkörper könnten somit sowohl als Modell für die Evolution bakterieller Endosymbionten als auch für die Etablierung Nitrogenase-abhängiger Stickstofffixierung in eukaryoten Zellen dienen

Nitrogen fixation in eukaryotes – New models for symbiosis

BACKGROUND: Nitrogen, a component of many bio-molecules, is essential for growth and development of all organisms. Most nitrogen exists in the atmosphere, and utilisation of this source is important as a means of avoiding nitrogen starvation. However, the ability to fix atmospheric nitrogen via the nitrogenase enzyme complex is restricted to some bacteria. Eukaryotic organisms are only able to obtain fixed nitrogen through their symbiotic interactions with nitrogen-fixing prokaryotes. These symbioses involve a variety of host organisms, including animals, plants, fungi and protists. RESULTS: We have compared the morphological, physiological and molecular characteristics of nitrogen fixing symbiotic associations of bacteria and their diverse hosts. Special features of the interaction, e.g. vertical transmission of symbionts, grade of dependency of partners and physiological modifications have been considered in terms of extent of co-evolution and adaptation. Our findings are that, despite many adaptations enabling a beneficial partnership, most symbioses for molecular nitrogen fixation involve facultative interactions. However, some interactions, among them endosymbioses between cyanobacteria and diatoms, show characteristics that reveal a more obligate status of co-evolution. CONCLUSION: Our review emphasises that molecular nitrogen fixation, a driving force for interactions and co-evolution of different species, is a widespread phenomenon involving many different organisms and ecosystems. The diverse grades of symbioses, ranging from loose associations to highly specific intracellular interactions, might themselves reflect the range of potential evolutionary fates for symbiotic partnerships. These include the extreme evolutionary modifications and adaptations that have accompanied the formation of organelles in eukaryotic cells: plastids and mitochondria. However, age and extensive adaptation of plastids and mitochondria complicate the investigation of processes involved in the transition of symbionts to organelles. Extant lineages of symbiotic associations for nitrogen fixation show diverse grades of adaptation and co-evolution, thereby representing different stages of symbiont-host interaction. In particular cyanobacterial associations with protists, like the Rhopalodia gibba-spheroid body symbiosis, could serve as important model systems for the investigation of the complex mechanisms underlying organelle evolution

The cyanobacterial endosymbiont of the unicellular algae Rhopalodia gibba shows reductive genome evolution

<p>Abstract</p> <p>Background</p> <p>Bacteria occur in facultative association and intracellular symbiosis with a diversity of eukaryotic hosts. Recently, we have helped to characterise an intracellular nitrogen fixing bacterium, the so-called spheroid body, located within the diatom <it>Rhopalodia gibba</it>. Spheroid bodies are of cyanobacterial origin and exhibit features that suggest physiological adaptation to their intracellular life style. To investigate the genome modifications that have accompanied the process of endosymbiosis, here we compare gene structure, content and organisation in spheroid body and cyanobacterial genomes.</p> <p>Results</p> <p>Comparison of the spheroid body's genome sequence with corresponding regions of near free-living relatives indicates that multiple modifications have occurred in the endosymbiont's genome. These include localised changes that have led to elimination of some genes. This gene loss has been accompanied either by deletion of the respective DNA region or replacement with non-coding DNA that is AT rich in composition. In addition, genome modifications have led to the fusion and truncation of genes. We also report that in the spheroid body's genome there is an accumulation of deleterious mutations in genes for cell wall biosynthesis and processes controlled by transposases. Interestingly, the formation of pseudogenes in the spheroid body has occurred in the presence of intact, and presumably functional, <it>rec</it>A and <it>rec</it>F genes. This is in contrast to the situation in most investigated obligate intracellular bacterium-eukaryote symbioses, where at least either <it>rec</it>A or <it>rec</it>F has been eliminated.</p> <p>Conclusion</p> <p>Our analyses suggest highly specific targeting/loss of individual genes during the process of genome reduction and establishment of a cyanobacterial endosymbiont inside a eukaryotic cell. Our findings confirm, at the genome level, earlier speculation on the obligate intracellular status of the spheroid body in <it>Rhopalodia gibba</it>. This association is the first example of an obligate cyanobacterial symbiosis involving nitrogen fixation for which genomic data are available. It represents a new model system to study molecular adaptations of genome evolution that accompany a switch from free-living to intracellular existence.</p

Nuclear coherent population transfer with x-ray laser pulses

Coherent population transfer between nuclear states using x-ray laser pulses is studied. The laser pulses drive two nuclear transitions between three nuclear states in a setup reminding of stimulated Raman adiabatic passage used for atomic coherent population transfer. To compensate for the lack of $\gamma$-ray laser sources, we envisage accelerated nuclei interacting with two copropagating or crossed x-ray laser pulses. The parameter regime for nuclear coherent population transfer using fully coherent light generated by future X-Ray Free-Electron Laser facilities and moderate or strong acceleration of nuclei is determined. We find that the most promising case requires laser intensities of $10^{17}$-$10^{19}$ W/cm$^{2}$ for complete nuclear population transfer. As relevant application, the controlled pumping or release of energy stored in long-lived nuclear states is discussed.Comment: extended argument about experimental feasibility, added references, results unchanged; v3 updated to the published versio

Prognosis of patients with malignant mesothelioma by expression of programmed cell death 1 ligand 1 and mesothelin in a contemporary cohort in Finland

ObjectivesWe aimed to describe mesothelin (MSLN) and programmed cell death 1 ligand 1 (PD-L1) tumour overexpression amongst patients with malignant mesothelioma (MM), and their associations with survival, amongst a cohort of patients with MM in Finland.MethodsBetween 2004 and 2017, 91 adults with histologically confirmed MM were identified from the Auria Biobank in Finland and followed-up using linked data from electronic health records and national statistics. Biomarker content in tumour cell membranes was determined using automated Immunohistochemistry on histological sections. Stained tumour sections were scored for MSLN and PD-L1 intensity. Adjusted associations between MSLN/PD-L1 co-expression and mortality were evaluated by estimating hazard ratios (HRs) with 95% confidence intervals (CIs) using Cox regression.ResultsBiomarker overexpression occurred in 52 patients for MSLN and 34 patients for PD-L1 and was associated with tumour histology and certain comorbidities. Fifteen per cent of patients had a tumour that overexpressed both biomarkers; r =-0.244, p-value: 0.02. Compared with MSLN+/PD-L1+ patients, HRs (95% CIs) for death were 4.18 (1.71–10.23) for MSLN-/PD-L1+ patients, 3.03 (1.35–6.77) for MSLN-/PD-L1- patients, and 2.13 (0.97–4.67) for MSLN+/PD-L1- patients.ConclusionsBoth MSLN and PD-L1 markers were independent prognostic indicators in patients with MM. Overexpression of MSLN was associated with longer survival; yet their combined expression gave a better indication of survival. The risk of death was four times higher amongst MSLN-/PD-L1+ patients than in MSLN+/PD-L1+ patients.</p

The Electron Capture in 163^{163} Ho Experiment - a Short Update

The definition of the absolute neutrino mass scale is one of the main goals of the Particle Physics today. The study of the end-point regions of the β- and electron capture (EC) spectrum offers a possibility to determine the effective electron (anti-)neutrino mass in a completely model independent way, as it only relies on the energy and momentum conservation. The ECHo (Electron Capture in 163Ho) experiment has been designed in the attempt to measure the effective mass of the electron neutrino by performing high statistics and high energy resolution measurements of the 163 Ho electron capture spectrum. To achieve this goal, large arrays of low temperature metallic magnetic calorimeters (MMCs) implanted with with 163Ho are used. Here we report on the structure and the status of the experiment

Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

<p>Abstract</p> <p>Background</p> <p>DNA methylation in the <it>SHOX2 </it>locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with <it>SHOX2 </it>gene expression and/or copy number alterations. An amplification of the <it>SHOX2 </it>gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples.</p> <p>Methods</p> <p><it>SHOX2 </it>expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect <it>SHOX2 </it>DNA methylation levels. <it>SHOX2 </it>expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH.</p> <p>Results</p> <p>A hypermethylation of the <it>SHOX2 </it>locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the <it>SHOX2 </it>gene showed no difference.</p> <p>Conclusions</p> <p>Frequent gene amplification correlated with hypermethylation of the <it>SHOX2 </it>gene locus. This concerted effect qualifies <it>SHOX2 </it>DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples.</p

SHOX2 DNA Methylation is a Biomarker for the diagnosis of lung cancer based on bronchial aspirates

<p>Abstract</p> <p>Background</p> <p>This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment.</p> <p>Methods</p> <p>Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance.</p> <p>Results</p> <p>Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]).</p> <p>Conclusions</p> <p>Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.</p

Nitrogen fixation in eukaryotes – New models for symbiosis

Abstract Background Nitrogen, a component of many bio-molecules, is essential for growth and development of all organisms. Most nitrogen exists in the atmosphere, and utilisation of this source is important as a means of avoiding nitrogen starvation. However, the ability to fix atmospheric nitrogen via the nitrogenase enzyme complex is restricted to some bacteria. Eukaryotic organisms are only able to obtain fixed nitrogen through their symbiotic interactions with nitrogen-fixing prokaryotes. These symbioses involve a variety of host organisms, including animals, plants, fungi and protists. Results We have compared the morphological, physiological and molecular characteristics of nitrogen fixing symbiotic associations of bacteria and their diverse hosts. Special features of the interaction, e.g. vertical transmission of symbionts, grade of dependency of partners and physiological modifications have been considered in terms of extent of co-evolution and adaptation. Our findings are that, despite many adaptations enabling a beneficial partnership, most symbioses for molecular nitrogen fixation involve facultative interactions. However, some interactions, among them endosymbioses between cyanobacteria and diatoms, show characteristics that reveal a more obligate status of co-evolution. Conclusion Our review emphasises that molecular nitrogen fixation, a driving force for interactions and co-evolution of different species, is a widespread phenomenon involving many different organisms and ecosystems. The diverse grades of symbioses, ranging from loose associations to highly specific intracellular interactions, might themselves reflect the range of potential evolutionary fates for symbiotic partnerships. These include the extreme evolutionary modifications and adaptations that have accompanied the formation of organelles in eukaryotic cells: plastids and mitochondria. However, age and extensive adaptation of plastids and mitochondria complicate the investigation of processes involved in the transition of symbionts to organelles. Extant lineages of symbiotic associations for nitrogen fixation show diverse grades of adaptation and co-evolution, thereby representing different stages of symbiont-host interaction. In particular cyanobacterial associations with protists, like the Rhopalodia gibba-spheroid body symbiosis, could serve as important model systems for the investigation of the complex mechanisms underlying organelle evolution.</p

Robust embedded egomotion estimation

This work presents a method for estimating the egomotion of an aerial vehicle in challenging industrial environments. It combines binocular visual and inertial cues in a tightly-coupled fashion and operates in real time on an embedded platform. An extended Kalman filter fuses measurements and makes motion estimation rely more on inertial data if visual feature constellation is degenerate. Errors in roll and pitch are bounded implicitly by the gravity vector. Inertial sensors are used for efficient outlier detection and enable operation in poorly and repetitively textured environments. We demonstrate robustness and accuracy in an industrial scenario as well as in general indoor environments. The former is accompanied by a detailed performance evaluation supported with ground truth measurements from an external tracking system