Characterization of a type I-B CRISPR-Cas system of Clostridium thermocellum

CRISPR-Cas systems are adaptive immune systems, found in bacteria and archaea that provide inheritable resistance against mobile genetic elements, e.g. viruses and plasmids. CRISPR-Cas systems comprise one or more CRISPR loci that contain virus-derived DNA sequences (spacers) that are interspaced by identical repeat sequences (repeats), and a set of cas genes. The degradation of nucleic acid targets is mediated by ribonucleoprotein (RNP) complexes, formed by Cas proteins, that are guided by small CRISPR RNA molecules (crRNAs). A Cas protein classification has been established which reflects the diversification of CRISPR systems during the co-evolution of phages and their respective hosts. In this study, the type I-B CRISPR-Cas system of the thermophilic bacterium Clostridium thermocellum was investigated. CRISPR loci are transcribed into precursor-crRNAs and individual crRNAs are produced by Cas6 endonucleases. C. thermocellum contains two Cas6 proteins and the recombinant enzymes exclusively process their respective precursor transcripts in vitro. RNA-Seq analyses confirmed crRNA maturation and highlighted crRNA abundance differences in vivo. These analyses identified internal promotion of precursor-crRNA transcription and reverse crRNA transcripts (anti-crRNAs). Anti-crRNAs display a distinct processing pattern and the abundance of the complementary crRNA is often reduced in vivo. Cleavage assays with crRNAs and double-stranded crRNA/anti-crRNA hybrids identified RNase III to be capable of anti-crRNA processing. RNase III cleavage is mediated by recognition motifs within the repeat RNA duplexes. In type I-B systems, CRISPR interference is mediated by a dsDNA targeting crRNP complex, termed Cascade, which consists of the Cas proteins Cas3, Cas5, Cas6, Cas7 and the subtype-specific Cas8b subunit. All five recombinant Cascade subunits were produced in Escherichia coli. Cascade assembly studies revealed that a stable core-complex is formed by Cas5, Cas7, Cas8b and crRNA in vitro. Cas3 does not assemble with the complex. Cas6 is a temporarily associated subunit. Mass-spectrometric analyses confirmed protein interactions between Cas5, Cas7 and Cas8b and determined an uneven complex stoichiometry of 1:1:6:2.5 for Cas5:Cas6:Cas7:Cas8b. The large subunit Cas8b forms an additional small C-terminal protein fragment that is also observed in C. thermocellum cell extracts and assembles with the complex. This thesis provides details for the in vitro assembly of individual Cas proteins into type I-B Cascade. Furthermore, RNA-Seq analyses of the CRISPR arrays highlight the impact of individual spacer and repeat sequences on the functionality of CRISPR-Cas systems

Altered Ca2+ Homeostasis in Immune Cells during Aging: Role of Ion Channels

Aging is an unstoppable process and begins shortly after birth. Each cell of the organism is affected by the irreversible process, not only with equal density but also at varying ages and with different speed. Therefore, aging can also be understood as an adaptation to a continually changing cellular environment. One of these very prominent changes in age affects Ca2+ signaling. Especially immune cells highly rely on Ca2+-dependent processes and a strictly regulated Ca2+ homeostasis. The intricate patterns of impaired immune cell function may represent a deficit or compensatory mechanisms. Besides, altered immune function through Ca2+ signaling can profoundly affect the development of age-related disease. This review attempts to summarize changes in Ca2+ signaling due to channels and receptors in T cells and beyond in the context of aging

Einfluss der R-CHOP Therapie auf die Verteilung und die Funktion von Immunzellen bei Patienten mit aggressivem Non-Hodgkin-Lymphom

Das häufig auftretende diffus großzellige B-Zell-Lymphom (DLBCL) wird als Standardtherapie mit einer Kombination aus Chemotherapie und dem zusätzlichen Einsatz des therapeutischen anti-CD20 Antikörpers Rituximab behandelt. Trotzdem kommt es bei ca. 30-50 % der Patienten zur Bildung eines Rezidivs oder die Patienten werden refraktär. Daher stellt eine bessere prognostische Beurteilung der Patienten einen wichtigen Schritt in der Entwicklung einer individualisierten Therapie bei Non Hodgkin Lymphomen dar. Innerhalb dieser Arbeit wurden jeweils 27 ml EDTA-Blut (3 x 9 ml) von insgesamt 33 DLBCL Patienten zu unterschiedlichen Zeitpunkten (Diagnosestellung, Verlauf der Therapie und Nachsorge) analysiert, um einen Überblick über quantitative und qualitative Veränderungen der im Blut zirkulierenden Immunzellen zu erhalten. Als Referenzpunkt diente das Blut, das zum Zeitpunkt der „Diagnosestellung“ abgenommen wurde, das zudem mit Blut immunologisch gesunder Spender verglichen wurde. Im weiteren Therapieverlauf, wenn klinisch möglich, wurde das Blut jedes Patienten jeweils vor Rituximab-Gabe zu allen 6 8 Zyklen R CHOP untersucht. Nach Therapie folgte eine Blutabnahme alle drei Monate zum Nachsorgetermin, sodass der erste in diese Studie eingegangene Patient bereits über einen Zeitraum von drei Jahren untersucht wurde. Aus allen Blutproben wurden PBMC (periphere mononukleäre Zellen) aufgereinigt und durchflusszytometrisch untersucht. Folgende Subpopulationen wurden durch die Verwendung unterschiedlicher Antikörper und einer entsprechenden Gatingstrategie identifiziert: Leukozyten (Singlets, CD45+), Granulozyten (FSC A/SSC-A), Lymphozyten (CD45+/CD14-) und Monozyten (CD45+/CD14+). Die Lymphozytenpopulation wurde dann weiter unterteilt in T-Zellen (CD3+, mögliche Auftrennung in CD4+/CD8+), B-Zellen (CD19+) und NK Zellen (CD56+, CD3-, CD19-). Da die Bindung des CD20 Antikörpers Rituximab über den Fcγ-Rezeptor CD16 vermittelt wird, wurden die NK Zellen, T Zellen und Monozyten zusätzlich hinsichtlich ihrer CD16 Expression analysiert. Zudem wird die CD56 Expression in T-Zellen und Monozyten mit Tumorerkrankungen assoziiert, sodass diese Zellpopulationen auch auf ihre CD56-Expression hin untersucht wurden. Zum Zeitpunkt der Diagnosestellung ergaben sich für den prozentualen Anteil der NK und B Zellen (gemessen an den CD45+ Leukozyten) keine Unterschiede im Vergleich zu gesunden Spendern. Der Anteil an Granulozyten und T-Zellen war überraschenderweise verringert und der Anteil an Monozyten erhöht. Hier war insbesondere auch der Anteil der CD16+ Monozyten im Vergleich zu gesunden Spendern erhöht und korrelierte negativ mit der Prognose der Patienten. Zudem zeigte sich ein zusätzlicher Anstieg der CD16+ Monozyten über den Therapieverlauf. Funktional wurde in dieser Studie das Tötungsverhalten von NK und T-Zellen im Populationskillingassay untersucht. Als Zielzelllinien wurden MHC I-defiziente K562-Zellen (natürliche Zytotoxizität) und die CD20+ DLBCL-Zelllinie U-2932 verwendet. Das Killing der U 2932 wurde über Rituximab vermittelt, wobei der Fc-Teil an CD16+ NK bzw. T Zellen bindet, um eine Antikörper-abhängige zellvermittelte Zytotoxizität (ADCC) zu induzieren. Hierbei zeigte sich keine veränderte Zytotoxizität der NK Zellen sowohl im Therapieverlauf als auch im Vergleich zu gesunden Kontrollspendern. Das T-Zellkilling war hingegen sowohl Rituximab spezifisch gegen U 2932 als auch gegen K562 Zellen zum Zeitpunkt der Diagnosestellung im Vergleich zur Kontrollgruppe signifikant erhöht. Die für diese Zytotoxizität verantwortlichen Zellen sind wahrscheinlich unkonventionelle T-Zellen, wie NKT-Zellen oder T-Zellen. Diese Zellen lysieren ihre Zielzelllinien Antigen-unspezifisch und werden mit wichtigen Funktionen bei der Tumorimmunität in Zusammenhang gebracht. Die genaue Bestimmung der verantwortlichen Zellen ging über den Rahmen dieser Studie hinaus, wird aber innerhalb unserer Arbeitsgruppe weiter untersucht, da das Vorhandensein der Zellen im Blut bei DLBCL Patienten ein wichtiger immunologischer Parameter sein könnte. Mit dieser Studie wurde die Aufreinigung, die durchflusszytometrische und funktionelle Analyse von Blutproben von DLBCL Patienten in unserem Labor grundlegend etabliert. Daraus haben sich Folgearbeiten, wie die genetische Untersuchung unterschiedlicher Polymorphismen, Zytokin- und Vitamin D-Spiegel Analysen im Plasma und die Bestimmung weiterer Oberflächenrezeptoren, z.B. mittels CyTOF, ergeben. Damit erhoffen wir uns weitere immunologisch relevante Unterschiede zu finden bzw. die in dieser Studie bereits gefundenen Unterschiede genauer zu charakterisieren, um eine diagnostische oder prognostische Relevanz bestimmen zu können.The standard therapy for the common diffuse large B-cell lymphoma (DLBCL) is a combination of chemotherapy (CHOP) with the additional use of the therapeutic anti CD20 antibody Rituximab. Nevertheless, approximately 30-50 % of patients develop a relapse or become refractory. Therefore, a better prognostic assessment of patients represents an important step in the development of individualized therapy for non-Hodgkin lymphoma. Within this work, 27 ml EDTA blood (3 x 9 ml) each from a total of 33 DLBCL patients were analysed at different time points (diagnosis, course of therapy and follow-up) to obtain an overview of quantitative and qualitative changes of the immune cells circulating in the blood. The blood samples taken at the time of "diagnosis" served as the reference point and was compared with blood samples from healthy controls. In the further course, when clinically possible, the blood of each patient was analysed every 6-8 cycles of R-CHOP before rituximab administration. After therapy, blood sampling followed every three months at the follow-up appointment, so that the first patient included in this study had already been examined over a period of three years. PBMC (peripheral blood mononuclear cells) were purified from all blood samples and analysed by flow cytometry. The following subpopulations were identified by using different antibodies and an appropriate gating strategy: leukocytes (singlets, CD45+), granulocytes (FSC A/SSC-A), lymphocytes (CD45+/CD14-) and monocytes (CD45+/CD14+). The lymphocyte population was then further subdivided into T cells (CD3+, possible separation into CD4+/CD8+), B cells (CD19+) and NK cells (CD56+, CD3-, CD19-). Since the binding of the CD20 antibody rituximab is mediated by the Fcγ receptor CD16, the NK cells, T cells and monocytes were additionally analysed for their CD16 expression. In addition, CD56 expression in T cells and monocytes is associated with tumour disease, consequently these cell populations were also analysed for CD56 expression. At the time of diagnosis, there were no differences in the percentage of NK and B cells (measured by CD45+ leukocytes) compared with healthy donors. Surprisingly, the percentage of granulocytes and T cells was decreased and the percentage of monocytes was increased. In particular, the proportion of CD16+ monocytes was also increased compared to healthy controls and correlated negatively with the prognosis of the patients. Furthermore, an additional increase of CD16+ monocytes was shown over the course of therapy. Functionally, this study investigated the killing behaviour of NK and T cells in the population killing assay. MHC I-deficient K562 cells (natural cytotoxicity) and the CD20+ DLBCL cell line U-2932 were used as target cell lines. Killing of U 2932 was mediated via rituximab, with the Fc portion binding to CD16+ NK or T cells to induce antibody-dependent cell mediated cytotoxicity (ADCC). Here, there was no altered cytotoxicity of NK cells both during the course of therapy and compared to healthy controls. In contrast, T cell killing was significantly increased, both rituximab-specific against U-2932 and against K562 cells at the time of diagnosis compared to the control group. These cells lyse their target cell lines in an antigen-unspecific manner and are associated with important functions in tumour immunity. The exact determination of the responsible cells was outside the frame of this study, but will be further investigated within our group, as the presence of these cells in the blood might be an important immunological parameter in DLBCL patients. This study fundamentally established the purification, flow cytometric and functional analysis of blood samples from DLBCL patients in our laboratory. This has resulted in follow-up work, such as the genetic investigation of different polymorphisms, cytokine and vitamin D level analyses in plasma, and the determination of additional surface receptors, e.g., by CyTOF. Thus, we hope to find further immunologically relevant differences or rather to characterize the differences already found in this study more precisely in order to be able to determine a diagnostic or prognostic relevance.DFG; SFB89

Fledermausschutz in Sachsen

Zahlreiche Aktivitäten begleiten die Aktion zum Schutz der Fledermäuse in Sachsen. Der Bevölkerung sollen mit diesem Faltblatt Informationen darüber vermittelt werden, wie jeder Einzelne am Erhalt und Ausbau von Fledermausquartieren mitwirken kann

Heterozygous OT-I mice reveal that antigen-specific CD8+ T cells shift from apoptotic to necrotic killers in the elderly

Numerous alterations in CD8+ T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell-intrinsic dysfunctions and microenvironmental changes is challenging. TCR transgenic OT-I mice are utilized to investigate CD8+ T-cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT-I model minimizes the current limitations and provides a valuable tool to assess antigen-specific T-cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8+ T cells from OT-I +/+ and OT-I +/− mice to prove the applicability of the heterozygous system. Our data reveal that OVA-activated CD8+ T cells from adult OT-I +/− mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age-related alterations in CD8+ T cells, including naive T-cell deterioration and decreased proliferative capacity, also occur in elderly OT-I +/− mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT-I +/− model to investigate cell-intrinsic alterations affecting the cytotoxic behavior of aged CD8+ T cells after antigen-specific in vitro activation. Time-resolved analysis of antigen-directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8+ T cells increases with age. Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8+ T cells shifts the mode of target cell death from granzymemediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation

Faster cytotoxicity with age : Increased perforin and granzyme levels in cytotoxic CD8+ T cells boost cancer cell elimination

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8+ T cells responses in the elderly has come back into focus since the COVID-19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8+ T cells in aging. We focused on the different subpopulations and time-resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8+ T cells from elderly mice are much faster than those of CD8+ T cells from adult mice. This is true not only in the total CD8+ T cell population but also for their effector (TEM) and central memory (TCM) T cell subpopulations. TIRF experiments reveal that CD8+ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8+ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell-intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked

A comparative study of machine learning methods for time-to-event survival data for radiomics risk modelling

Radiomics applies machine learning algorithms to quantitative imaging data to characterise the tumour phenotype and predict clinical outcome. For the development of radiomics risk models, a variety of different algorithms is available and it is not clear which one gives optimal results. Therefore, we assessed the performance of 11 machine learning algorithms combined with 12 feature selection methods by the concordance index (C-Index), to predict loco- regional tumour control (LRC) and overall survival for patients with head and neck squamous cell carcinoma. The considered algorithms are able to deal with continuous time-to-event survival data. Feature selection and model building were performed on a multicentre cohort (213 patients) and validated using an independent cohort (80 patients). We found several combinations of machine learning algorithms and feature selection methods which achieve similar results, e.g., MSR-RF: C-Index = 0.71 and BT-COX: C-Index = 0.70 in combination with Spearman feature selection. Using the best performing models, patients were stratified into groups of low and high risk of recurrence. Significant differences in LRC were obtained between both groups on the validation cohort. Based on the presented analysis, we identified a subset of algorithms which should be considered in future radiomics studies to develop stable and clinically relevant predictive models for time-to-event endpoints

Kurzgutachten zur regionalen Ungleichverteilung der Netznutzungsentgelte: Bestandsaufnahme und pragmatische Lösungsansätze; im Auftrag der 50Hertz Transmission GmbH

Der zur Umsetzung der Energiewende notwendige Netzausbau fällt regional sehr unterschiedlich hoch aus. Durch die bestehende Entgeltsystematik ergeben sich hierbei potentielle Mehrbelastungen für Stromkunden in Regionen mit einem hohen Anteil an Erneuerbaren Energien. Aktuell sind vor allem in den neuen Bundesländern höhere Entgelte zu verzeichnen. Im Rahmen dieses Kurzgutachtens werden mittels eines detaillierten Modells der Netzkosten auf den unterschiedlichen Spannungsebenen nach Landkreisen aufgeschlüsselte Netznutzungsentgelte bis zum Jahr 2024 prognostiziert. Darüber hinaus werden fünf Anpassungsvarianten der bestehenden Entgeltsystematik quantitativ analysiert und diskutiert: Einheitliches Übertragungsnetzentgelt Streichung der vermiedenen Netznutzungsentgelte für dargebotsabhängige Erzeuger Preiskorridore für Endkundenentgelte Bundeseinheitliche Entgelte für Endkunden Wälzen der durch Erneuerbare Energien (EE) bedingten Verteilernetzkosten Aus den Analysen ergeben sich vor allem für die Varianten Entgeltkorridore, bundeseinheitliche Entgelte sowie für das Wälzen der EE-bedingten Verteilernetzkosten signifikante Entlastungseffekte für Kunden mit sehr hohen Entgelten bei moderater Mehrbelastung der übrigen Stromkunden. Obwohl die letzte Variante zu einer verursachungsgerechteren Kostenverteilung führen würde, ist deren Umsetzbarkeit äußerst fraglich. Erste Maßnahmen um ein Auseinanderdriften der Entgelte abzuschwächen, die deutlich einfacher umzusetzen sind, wären die Einführung eines einheitlichen Übertragungsnetzentgelts sowie die Streichung vermiedener Netznutzungsentgelte für dargebotsabhängige Erzeuger.:Inhaltsverzeichnis III Abbildungsverzeichnis V Tabellenverzeichnis VI Abkürzungsverzeichnis VII Länderkürzel VIII 1 Zusammenfassung und Schlussfolgerungen 1 2 Einleitung und Gegenstand der Analyse 5 3 Allgemeine Herausforderungen bei der Entwicklung der Netznutzungsentgelte 7 4 Herangehensweise und Modellbeschreibung 12 5 Ergebnisse unter der heutigen Systematik und verschiedener Anpassungsvarianten 23 6 Sensitivitätsanalyse und Differenzierung ausgewählter Modellaspekte 48 7 Umsetzung der Anpassungsvarianten 54 8 Bewertung der Anpassungsvarianten 60 9 Literaturverzeichnis 64 10 Anhang 6

Avifaunistische Beobachtungen im Westchentej

This paper reports the results of bird observations at four study sites with many different habitats in the western Chentej-mountains (forest steppe, taiga) at the end of the spring-migration and the beginning of the breeding time. Altogether 134 bird species were observed, about 107 of which are probably breeding birds. Additional birds were caught using 6 mist-nets. In this way 98 birds of 21 species were caught, measured, and ringed. Furthermore feathers have been collected (molting feathers and near two breeding places of Accipiter nisus). The bird fauna of the larch forests shows a high species richness (108 species), but the abundances are often very low (for instance for woodpeckers, thrushes, tits). Muscicapa griseisticta is a new record (possibly irregularly) as a migrating bird for Mongolia (11-6-1990 [i.e. June 11, 1990], Davaany Sörlög - three individuals in a willow-shrub near a railway). We suppose that Gallinago megala, Perisoreus infaustus, Losucstella lanceolata, Turdus sibiricus, and Pyrrhula pyrrhula are regular breeders in the forest areas of the western Chentej. Aquila clanga, Grus grus lilfordi, Hirundapus caudacutus, Corvus frugilegus*, Cinclus cinclus, Prunella collaris*, Regulus regulus, Luscinia sibilans, Luscinia cuyne, Pinicula enucleator, Pyrrhula cineracea, and Emberiza rutila are possible breeders in this area, or have been observed breeding (*). Furthermore, Haliaeetus albicilla, Falco peregrinus, Prunella montanella, and Turdus naumanni were recorded. We propose to organize a registration-catching-program for birds in the western Chentej. We recommend nature protection for the flood-plains of some rivers for example Onon and Eröö

Functionalization of Pyrene To Prepare Luminescent Materials—Typical Examples of Synthetic Methodology

Pyrene-based π-conjugated materials are considered to be an ideal organic electro-luminescence material for application in semiconductor devices, such as organic light-emitting diodes (OLEDs), organic field-effect transistors (OFETs) and organic photovoltaics (OPVs), and so forth. However, the great drawback of employing pyrene as an organic luminescence material is the formation of excimer emission, which quenches the efficiency at high concentration or in the solid-state. Thus, in order to obtain highly efficient optical devices, scientists have devoted much effort to tuning the structure of pyrene derivatives in order to realize exploitable properties by employing two strategies, 1) introducing a variety of moieties at the pyrene core, and 2) exploring effective and convenient synthetic strategies to functionalize the pyrene core. Over the past decades, our group has mainly focused on synthetic methodologies for functionalization of the pyrene core; we have found that formylation/acetylation or bromination of pyrene can selectly lead to functionalization at K-region by Lewis acid catalysis. Herein, this Minireview highlights the direct synthetic approaches (such as formylation, bromination, oxidation, and de-tert-butylation reactions, etc.) to functionalize the pyrene in order to advance research on luminescent materials for organic electronic applications. Further, this article demonstrates that the future direction of pyrene chemistry is asymmetric functionalization of pyrene for organic semiconductor applications and highlights some of the classical asymmetric pyrenes, as well as the latest breakthroughs. In addition, the photophysical properties of pyrene-based molecules are briefly reviewed. To give a current overview of the development of pyrene chemistry, the review selectively covers some of the latest reports and concepts from the period covering late 2011 to the present day