A polymorphic microsatellite repeat within the ECE-1c promoter is involved in transcriptional start site determination, human evolution, and Alzheimer's disease

Das Endothelin-Konvertierungsenzym-1 (ECE-1) ist das Schlüsselenzym der Endothelinbiosynthese und damit an zahlreichen (patho-)physiologischen Vorgängen wie Entwicklung des Herz-Kreislauf- und Magen-Darm-Systems, Bluthochdruck oder Kardiomyopathie beteiligt. In mehreren Arbeiten konnte auch gezeigt werden, dass ECE-1c zu den Enzymen gehört, welche in vitro und in vivo Amyloid-Plaques, neben den Tau-Fibrillen das pathologische Hauptkorrelat der Alzheimer-Demenz, abbauen können. Durch alternatives Splicen und Vorliegen alternativer Promotoren existieren mindestens vier ECE-1-Isoformen. Während für ECE-1b bereits ein Einzelnukleotid-Polymorphismus (SNP) mit Einfluss auf die präfrontale ECE-1 mRNA-Expression und einem erhöhten Risiko eine Alzheimer-Demenz zu entwickeln gezeigt werden konnte, ist die Hauptisoform, ECE-1c, diesbezüglich noch weitestgehend unerforscht. In Vorarbeiten konnte ein polymorpher [CpG]m-[CA]n-Mikrosatelliten-repeat im Promotor des humanen ECE-1c mit Nachweis einer in vitro-Funktionalität identifiziert werden. Ferner wurden mittels rapid amplification of cDNA ends (RACE) und genomischem RNase protection assay (RPA) Transkriptionsstartpunkte innerhalb des Mikrosatelliten entdeckt. Darauf basierend lag das Ziel dieser Arbeit zum einen in der molekulargenetischen und funktionellen Charakterisierung des [CpG]m-[CA]n -Mikrosatelliten-repeat sowie Identifikation bindender Transkriptionsfaktoren und zum anderen in der klinischen Assoziation zur Alzheimer-Demenz. Durch Klonierung serieller Deletionsmutanten des ECE-1c-Promotors mit und ohne den [CpG]m-[CA]n-Mikrosatelliten-repeat von 35 verschiedenen humanen Haplotypen, Transfektion und anschließender Aktivitätsmessung im Luziferase-Reportergen- Assay konnte eine Funktionalität in Abhängigkeit des [CpG]m-[CA]n -Mikrosatelliten-repeat nachgewiesen werden. Darüber hinaus wurden mit Hilfe von DNA-Affinitätschromatographie unter Verwendung größenfraktionierter Kernproteine, electromobility shift assay (EMSA) und Massenspektrometrie Poly (ADP-Ribose)-Polymerase-1 (PARP-1) und splicing factor proline- and glutamine- rich (SFPQ) als bindende Transkriptionsfaktoren identifiziert. Für SFPQ konnte die Bindung mittels sogenannter supershift-Analyse und für PARP-1 mittels radioaktiv markiertem Protein im EMSA bestätigt werden. Die Funktionalität beider Faktoren wurde im Luziferase-Reportergen-Assay durch Transfektion oben beschriebener serieller Deletionsmutanten in PARP-knockout-Zellen sowie unter Überexpression von PARP-1 und/ oder SFPQ nachgewiesen. In einer klinischen Assoziationsstudie wurde die [CpG]m-[CA]n-repeat-Länge von 403 Patienten und 444 nicht dementen Kontrollpersonen mittels fluoreszenzmarkierter Polymerasekettenreaktion (PCR) und zum Teil durch Sequenzierung genotypisiert. Statistisch konnte ein signifikanter Unterschied zwischen Patienten- und Kontrollgruppe nachgewiesen werden. Zudem wurde auch der [CpG]m-[CA]n -Mikrosatelliten-repeat von sechs Schimpansen untersucht. Auch bei Schimpansen ist der repeat polymorph, jedoch deutlich kürzer als bei Menschen. Zudem ließ sich im Luziferase-Reportergen-Assay – im Gegensatz zu den menschlichen Haplotypen – keine Aktivitätsänderung durch den repeat nachweisen. Somit zeigt diese Arbeit, dass der [CpG]m-[CA]n-Mikrosatelliten-repeat hochpolymorph und funktionell ist, die Proteine PARP-1 und SFPQ als Transkriptionsfaktoren auf dem repeat binden und es einen signifikanten Unterschied der repeat- Komposition zwischen Alzheimerpatienten und nicht dementen Patienten gibt.Endothelin-converting enzyme -1 (ECE-1) is the key enzyme of endothelin biosynthesis and is involved in various cardiovascular (patho)physiologies. Furthermore, it has been demonstrated that ECE-1c is able to degrade amyloid plaques, one pathologic hallmark of Alzheimer's disease (AD), in vitro and in vivo. At least four isoforms of ECE-1 are generated by alternative splicing and existence of alternative promoters. While a single nucleotide polymorphism in the ECE-1b isoform-specific promoter with influence on the prefrontal ECE-1 mRNA-expression and on the likelihood of developing AD has already been described, a potential link between AD and ECE-1c, the main isoform, is still unexplored. In previous experiments, a polymorphic [CpG]m-[CA]n microsatellite repeat in the human ECE-1c promoter has been identified and its functionality in vitro has been demonstrated. Furthermore, transcriptional start sites within the microsatellite could be detected by rapid amplification of cDNA ends (RACE) and genomic RNase protection assay (RPA). On the one hand, the aim of this work was to characterise the [CpG]m-[CA]n microsatellite repeat as well as to identify binding transcription factors, and on the other hand to look for a clinical association to AD. By subcloning serial deletion mutants of the ECE-1c promoter of 35 different human haplotypes with and without the microsatellite repeat, transfection and subsequent luciferase reporter gene assays, the repeat's influence on the promoter activity was demonstrated. In addition, poly(ADP-ribose) polymerase-1 (PARP-1) and splicing factor proline- and glutamine-rich (SFPQ) were identified as binding transcription factors via DNA affinity chromatography, electromobility shift assay (EMSA) and mass spectometry. The binding of SFPQ was confirmed by so-called supershift analysis, the binding of PARP-1 by EMSA using radioactively labelled proteins. The functionality of both factors was shown in the luciferase reporter gene assay using PARP knockout cells and overexpression of PARP-1 and/ or SFPQ. In a clinical study, the repeat of 403 patients and 444 non demented controls was genotyped via fluorescent-labeled genomic polymerase chain reaction (PCR) and in some cases additionally via sequencing. A statistical significant difference between patients and controls was found. Finally, the [CpG]m-[CA]n microsatellite repeat of six chimpanzees was examined showing that the repeat is shorter than in humans and without effect on promoter activity. In summary, this work depicts that the [CpG]m-[CA]n microsatellite repeat is highly polymorphic and functional, that PARP-1 and SFPQ are binding as transcription factors and that there is a significant difference in repeat composition between AD and controls

Predicate correlation learning for scene graph generation

For a typical Scene Graph Generation (SGG) method, there is often a large gap in the performance of the predicates' head classes and tail classes. This phenomenon is mainly caused by the semantic overlap between different predicates as well as the long-tailed data distribution. In this paper, a Predicate Correlation Learning (PCL) method for SGG is proposed to address the above two problems by taking the correlation between predicates into consideration. To describe the semantic overlap between strong-correlated predicate classes, a Predicate Correlation Matrix (PCM) is defined to quantify the relationship between predicate pairs, which is dynamically updated to remove the matrix's long-tailed bias. In addition, PCM is integrated into a Predicate Correlation Loss function ($L_{PC}$) to reduce discouraging gradients of unannotated classes. The proposed method is evaluated on Visual Genome benchmark, where the performance of the tail classes is significantly improved when built on the existing methods

A time-resolved proteomic and prognostic map of COVID-19

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease

Clinical and virological characteristics of hospitalised COVID-19 patients in a German tertiary care centre during the first wave of the SARS-CoV-2 pandemic: a prospective observational study

Purpose: Adequate patient allocation is pivotal for optimal resource management in strained healthcare systems, and requires detailed knowledge of clinical and virological disease trajectories. The purpose of this work was to identify risk factors associated with need for invasive mechanical ventilation (IMV), to analyse viral kinetics in patients with and without IMV and to provide a comprehensive description of clinical course. Methods: A cohort of 168 hospitalised adult COVID-19 patients enrolled in a prospective observational study at a large European tertiary care centre was analysed. Results: Forty-four per cent (71/161) of patients required invasive mechanical ventilation (IMV). Shorter duration of symptoms before admission (aOR 1.22 per day less, 95% CI 1.10-1.37, p < 0.01) and history of hypertension (aOR 5.55, 95% CI 2.00-16.82, p < 0.01) were associated with need for IMV. Patients on IMV had higher maximal concentrations, slower decline rates, and longer shedding of SARS-CoV-2 than non-IMV patients (33 days, IQR 26-46.75, vs 18 days, IQR 16-46.75, respectively, p < 0.01). Median duration of hospitalisation was 9 days (IQR 6-15.5) for non-IMV and 49.5 days (IQR 36.8-82.5) for IMV patients. Conclusions: Our results indicate a short duration of symptoms before admission as a risk factor for severe disease that merits further investigation and different viral load kinetics in severely affected patients. Median duration of hospitalisation of IMV patients was longer than described for acute respiratory distress syndrome unrelated to COVID-19

Cancer-cell-derived GABA promotes β-catenin-mediated tumour growth and immunosuppression.

Many cancers have an unusual dependence on glutamine. However, most previous studies have focused on the contribution of glutamine to metabolic building blocks and the energy supply. Here, we report that cancer cells with aberrant expression of glutamate decarboxylase 1 (GAD1) rewire glutamine metabolism for the synthesis of γ-aminobutyric acid (GABA)-a prominent neurotransmitter-in non-nervous tissues. An analysis of clinical samples reveals that increased GABA levels predict poor prognosis. Mechanistically, we identify a cancer-intrinsic pathway through which GABA activates the GABAB receptor to inhibit GSK-3β activity, leading to enhanced β-catenin signalling. This GABA-mediated β-catenin activation both stimulates tumour cell proliferation and suppresses CD8+ T cell intratumoural infiltration, such that targeting GAD1 or GABABR in mouse models overcomes resistance to anti-PD-1 immune checkpoint blockade therapy. Our findings uncover a signalling role for tumour-derived GABA beyond its classic function as a neurotransmitter that can be targeted pharmacologically to reverse immunosuppression

A Polymorphic Microsatellite Repeat within the ECE-1c Promoter Is Involved in Transcriptional Start Site Determination, Human Evolution, and Alzheimer's Disease

Genetic factors strongly contribute to the pathogenesis of sporadic Alzheimer's disease (AD). Nevertheless, genome-wide association studies only yielded single nucleotide polymorphism loci of moderate importance. In contrast, microsatellite repeats are functionally less characterized structures within our genomes. Previous work has shown that endothelin-converting enzyme-1 (ECE-1) is able to reduce amyloid {beta} content. Here we demonstrate that a CpG-CA repeat within the human ECE-1c promoter is highly polymorphic, harbors transcriptional start sites, is able to recruit the transcription factors poly(ADP-ribose) polymerase-1 and splicing factor proline and glutamine-rich, and is functional regarding haplotype-specific promoter activity. Furthermore, genotyping of 403 AD patients and 444 controls for CpG-CA repeat length indicated shifted allelic frequency distributions. Sequencing of 245 haplotype clones demonstrated that the overall CpG-CA repeat composition of AD patients and controls is distinct. Finally, we show that human and chimpanzee [CpG](m)-[CA](n) ECE-1c promoter repeats are genetically and functionally distinct. Our data indicate that a short genomic repeat structure constitutes a novel core promoter element, coincides with human evolution, and contributes to the pathogenesis of AD