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Clonal hematopoiesis of indeterminate potential (CHIP) is caused by recurrent somatic mutations leading to clonal blood cell expansion. However, direct evidence of the fitness of CHIP-mutated human hematopoietic stem cells (HSCs) in blood reconstitution is lacking. Because myeloablative treatment and transplantation enforce stress on HSCs, we followed 81 patients with solid tumors or lymphoid diseases undergoing autologous stem cell transplantation (ASCT) for the development of CHIP. We found a high incidence of CHIP (22%) after ASCT with a high mean variant allele frequency (VAF) of 10.7%. Most mutations were already present in the graft, albeit at lower VAFs, demonstrating a selective reconstitution advantage of mutated HSCs after ASCT. Thus, CHIP-mutated stem and progenitor cells largely gain on clone size upon ASCT-related blood reconstitution, leading to an increased future risk of CHIP-associated complications. CHIP increase with age and is also associated with atherosclerosis and inflammation. Age and inflammation are the major risk factors for heart failure, yet the association of CHIP with chronic ischemic heart failure (CHF) in humans is unknown. Therefore, we analyzed bone marrow-derived mononuclear cells from 200 patients with CHF by NGS to detect the presence of CHIP and associated such with long-term prognosis in patients with CHF. Forty-seven mutations with a VAF of at least 2% were found in 18.5% of 200 patients with CHF. The mutations most commonly occurred in the genes DNMT3A and TET2. During a median follow-up of 4.4 years, a significantly worse clinical outcome for patients with either DNMT3A or TET2 mutations compared with non-CHIP carriers was notable. Importantly, there was a significant dose-response association between VAF and clinical outcome. Our data suggest that somatic mutations in hematopoietic cells, may be significantly associated with the progression and poor prognosis of CHF.
Chloroplasts are difficult to assemble because of the presence of large inverted repeats. At the same time, correct assemblies are important, as chloroplast loci are frequently used for biogeography and population genetics studies. In an attempt to elucidate the orientation of the single-copy regions and to find suitable loci for chloroplast single nucleotide polymorphism (SNP)-based studies, circular chloroplast sequences for the ultra-centenary reference individual of European Beech (Fagus sylvatica), Bhaga, and an additional Polish individual (named Jamy) was obtained based on hybrid assemblies. The chloroplast genome of Bhaga was 158,458 bp, and that of Jamy was 158,462 bp long. Using long-read mapping on the configuration inferred in this study and the one suggested in a previous study, we found an inverted orientation of the small single-copy region. The chloroplast genome of Bhaga and of the individual from Poland both have only two mismatches as well as three and two indels as compared to the previously published genome, respectively. The low divergence suggests low seed dispersal but high pollen dispersal. However, once chloroplast genomes become available from Pleistocene refugia, where a high degree of variation has been reported, they might prove useful for tracing the migration history of Fagus sylvatica in the Holocene.
Genetic and genomic tools have provided researchers with the opportunity to address fundamental questions regarding the reintroduction of species into their historical range with greater precision than ever before. Reintroduction has been employed as a conservation method to return locally extinct species to their native range for decades. However, it remains unknown how genetic factors may impact population establishment and persistence at the population and metapopulation level in the short- and long-term. Genetic methods are capable of producing datasets from many individuals, even when only low quality DNA can be collected. These methods offer an avenue to investigate unanswered questions in reintroduction biology, which is vital to provide evidence based management strategies for future projects. The Eurasian lynx (Lynx lynx) and European wildcat (Felis silvestris) are elusive carnivores native to Eurasia and have been the subject of multiple reintroduction attempts into their native range. During the 19th and 20th century, the Eurasian lynx was extirpated from West and Central Europe due to increasing habitat fragmentation and persecution. Similarly, the European wildcat was the subject of human persecution, residing in a few refugia in West and Central Europe. After legal protection in the 1950s, subsequent reintroduction projects of both species began in the 1970s and 1980s and continue to the present. Despite this large focus on species conservation, little attention has been given to the consequences these reintroductions have on the genetic composition of the reintroduced populations and if the populations have a chance of persisting in the long term. These species have not yet benefited from the large range of genetic and genomic techniques currently available to non-model organisms, leaving many fundamental aspects of their reintroduction poorly understood. In my dissertation, I investigate demography, population structure, genetic diversity and inbreeding at the population and metapopulation level in both species. In the introduction, which lays the foundation for the subsequent chapters of this PHD, I provide background on reintroduction, its role in conservation and the genetic consequences on populations, especially populations of apex and mesocarnivores. In Publication I, I investigated the reemergence of the European wildcat in a low mountain region in Germany using fine-scale spatial analysis. I found that the reintroduced population has persisted and merged with an expanding natural population. The reintroduced population showed no genetic differentiation from the natural population suggesting there is a good chance this population has retained sufficient genetic diversity despite reintroduction. In Publication II, I tracked population development and genetic diversity over 15 years in a reintroduced lynx population to determine the genetic ramifications on a temporal scale. I found slow genetic erosion after a period of outbreeding, which fits in line with other reintroduced taxa sharing similar demographic histories. I also found the number of genetic founders to be a fraction of the total released individuals, indicating that reintroduced populations of elusive carnivores may have fewer founder individuals than previously thought. In Publication III, I sampled all surviving lynx reintroductions in West and Central Europe as well as 11 natural populations to compare levels of genetic diversity and inbreeding across the species distribution. I found that all reintroduced populations have lower genetic variability and higher inbreeding than natural populations, which urgently requires further translocations to mitigate possible negative consequences. These translocations could stem from other reintroduced populations or from surrounding natural populations. The results contribute to a growing body of evidence indicating that inbreeding is likely to be more prevalent in wild populations than previously understood. Finally, in the discussion I explore how genetic methods can be applied to post-reintroduction monitoring of felid species to illuminate questions relating to genetic composition after release. The methods employed in these studies and in future work will be highly dependent on the research questions posed. Additionally, I investigate the drivers of the observed genetic patterns including founder size, source population, environmental factors, and population growth. I found that genetic diversity loss patterns across these two felid species are not clearly defined, however, management actions can be taken to mitigate the negative effects of reintroductions. These management actions include further translocation, introducing a sufficient number of released individuals and situating reintroductions adjacent to natural populations. All of these actions can minimize genetic drift and inbreeding, two factors which negatively impact small populations. This thesis further supports mounting evidence that genetic considerations should be assessed before releasing individuals, which allows for incorporation of scientific evidence into the planning process thereby increasing the overall success of reintroduction projects. Ultimately, the resources developed during this dissertation provide a solid baseline and foundation for future work regarding the consequences of reintroductions. This is especially important as an increasing number of species are at risk of extinction and reintroductions of both the European wildcat and Eurasian lynx, as well as many others, are planned in the coming years.
Morbus Parkinson (abgekürzt als PD vom Englischen Parkinson’s disease) ist nach Alzheimer die zweithäufigste neurodegenerative Erkrankung. Die Hauptmerkmale sind Rigidität und Bradykinesie, sowie Tremor und posturale Instabilität. Im Gehirn lässt sich bei Parkinsonpatienten post mortem ein Verlust an Neuronen in der Substantia nigra feststellen, was zu den ersten beiden Anzeichen führt. Zudem gibt es intrazelluläre Einschlüsse in den betroffenen Nervenzellen – Lewy-Körperchen genannt – die aus Alpha-Synuklein und anderen Proteinen wie Ubiquitin zusammengesetzt sind. Außerdem ist der Eisenmetabolismus in Gehirnen von Parkinsonpatienten gestört und man findet Eisen-Ablagerungen, vor allem im Mittelhirn. Die Ursachen für PD sind bislang nicht abschließend geklärt. Der Großteil der Fälle ist sporadischer Natur mit unbekannter Ursache und nur bei einem geringen Anteil liegt eine Mutation in einem einzelnen Gen zugrunde. Die häufigsten Mutationen tritt in den Genen für Alpha-Synuklein (SNCA), PINK1 und PARKIN auf.
Die Serin-Threonin-Kinase PINK1 und die E3-Ubiquitin-Protein-Ligase PARKIN sind zwei Proteine, die in Stresssituationen an der Mitochondrien-Außenmembran am Abbau von alten oder nicht richtig funktionierenden Mitochondrien beteiligt sind. Dieser Vorgang nennt sich Mitophagie.
Die dieser Arbeit zugrunde liegenden Publikationen gehen den Zusammenhängen zwischen mitochondrialen Fehlfunktionen und der Pathogenese von PD nach. Da die Krankheit meist erst im hohen Alter auftritt, davon größtenteils ohne direkte Ursache, liegt der Schluss nahe, dass neben genetischen Ursachen auch Umweltfaktoren eine größere Rolle spielen könnten. Um dies näher zu analysieren, wurden experimentell verschiedene Stressoren eingesetzt.
Insgesamt wurden folgende Aspekte untersucht:
I. Welche Auswirkungen hat das Fehlen von PINK1 auf die Zelle? Gibt es einen Biomarker, der mit höherem Alter immer stärker verändert ist?
II. Welchen Einfluss haben Umweltfaktoren wie veränderte Eisen-Exposition auf die Zelle und was verändert sich beim Fehlen von PINK1?
III. Wie können mitochondriale Fehlfunktionen präferentiell das Nervensystem betreffen, wenn es nicht um respiratorische Insuffizienz geht?
Die einzelnen Studien zeigten folgende Ergebnisse:
Torres-Odio/Key et al. 2017 widmete sich der Suche nach molekularen Biomarkern, wodurch PD präsymptomatisch erkannt und die Progression der Erkrankung eingeschätzt werden kann. Die Transkriptom-Analyse der Kleinhirne von Mäusen mit Pink1-/--Mutation in drei verschiedenen Altersstufen zeigte eindrücklich, dass nicht ein einzelner Faktor immer stärker verändert war, sondern, dass immer mehr Faktoren und daher auch eine steigende Zahl an
Signalwegen mit höherem Alter beteiligt waren. Diese Veränderungen betrafen inflammatorische Signalwege, insbesondere Faktoren, die mit der Erkennung und Verarbeitung von zellfremden Nukleinsäuren assoziiert sind. Aufgrund der evolutionären Herkunft von Mitochondrien als frühere Protobakterien haben mitochondriale Nukleinsäuren und Proteine zum Teil bakterielle Ähnlichkeiten, und könnten bei Fehlfunktionen ins Zytosol gelangen. Vor diesem Hintergrund lassen die Ergebnisse der Studie den Schluss zu, dass das angeborene Immunsystem in Neuronen durch eine PINK1-assoziierte mitochondriale Störung aktiviert wird.
In der Publikation Key et al. 2020 wurde Eisen als ein im täglichen Leben vorkommender Stressor eingesetzt und es wurden systematisch Faktoren des Eisenstoffwechsels bei hohen und niedrigen Eisenspiegeln im Zusammenhang mit Parkinson-Mutationen untersucht. Da Eisen für die Gesundheit von Mitochondrien eine große Rolle spielt und Eisen-Chelatoren als Therapie bei PD Patienten bereits diskutiert werden, haben die molekularen Befunde große Relevanz. Die Ergebnisse zeigen, dass unter niedrigen Eisenspiegeln Proteine reduziert waren, die am Nukleotid-Stoffwechsel beteiligt sind, sowie Faktoren, die Eisen-Schwefel-Cluster als Cofaktoren haben und wichtig für die Nukleotid-Qualitätskontrolle sind. Das Fehlen von Eisen führte zu einer Induktion von Pink1 und Prkn, was auf verstärkte Mitophagie hindeutet. Insgesamt konnte gezeigt werden, dass die mitochondriale Eisen-Schwefel-Cluster Biogenese und die post-transkriptionelle Eisenregulation entscheidend für die Pathogenese von PD, bzw. das gesunde Fortbestehen einer Zelle und letztlich auch eines Organismus sind.
In Key et al. 2019 wurde erstmalig das Gesamt-Ubiquitylom aus Gehirnen von gealterten Parkin-knockout (KO) Mäusen erhoben und analysiert, um Ubiquitylierungs-Substrate von PARKIN zu identifizieren. Hierbei zeigte sich eine veränderte Ubiquitylierung von mehreren Faktoren, die an der zellulären Calcium-Homöostase beteiligt sind. Weitere elektrophysiologische Experimente in Gehirnen von gealterten Parkin-/--KO Mäusen ergaben, dass in Nervenzellen im Locus coeruleus die Geschwindigkeit der spontanen Taktgeber erhöht, dass die langsame Nachhyperpolarisation reduziert und, dass die Dauer der Aktionspotentiale erniedrigt war, ohne Veränderung der Kaliumkanal-Ströme.
Insgesamt geht aus den drei Studien hervor, dass mitochondriale Fehlfunktionen bei dauerhaftem Bestehen weitreichende Folgen für die Gesundheit des Nervensystems haben können, denn auch kleine Veränderungen, seien es durch Mutationen oder Umweltfaktoren wie Eisen, können in einer so großen Lebensspanne wie der des Menschen über Krankheit oder Gesundheit entscheiden!
Nematodes represent a diverse and ubiquitous group of metazoans in terrestrial environments. They feed on bacteria, fungi, plants, other nematodes or parasitize a variety of animals and hence may be considered as active members of many food webs. Deadwood is a structural component of forest ecosystems which harbors many niches for diverse biota. As fungi and bacteria are among the most prominent decomposing colonizers of deadwood, we anticipated frequent and diverse nematode populations to co-occur in such ecosystems. However, knowledge about their ability to colonize this habitat is still limited. We applied DNA-based amplicon sequencing (metabarcoding) of the 18S rRNA gene to analyze nematode communities in sapwood and heartwood of decaying logs from 13 different tree species. We identified 247 nematode ASVs (amplicon sequence variants) from 27 families. Most of these identified families represent bacterial and fungal feeders. Their composition strongly depended on tree species identity in both wood compartments. While pH and water content were the only wood properties that contributed to nematodes’ distribution, co-occurring fungal and prokaryotic (bacteria and archaea) α- and β-diversities were significantly related to nematode communities. By exploring thirteen different tree species, which exhibit a broad range of wood characteristics, this study provides first and comprehensive insights into nematode diversity in deadwood of temperate forests and indicates connectivity to other wood-inhabiting organisms.
Downy mildews caused by obligate biotrophic oomycetes result in severe crop losses worldwide. Among these pathogens, Pseudoperonospora cubensis and P. humuli, two closely related oomycetes, adversely affect cucurbits and hop, respectively. Discordant hypotheses concerning their taxonomic relationships have been proposed based on host–pathogen interactions and specificity evidence and gene sequences of a few individuals, but population genetics evidence supporting these scenarios is missing. Furthermore, nuclear and mitochondrial regions of both pathogens have been analyzed using microsatellites and phylogenetically informative molecular markers, but extensive comparative population genetics research has not been done. Here, we genotyped 138 current and historical herbarium specimens of those two taxa using microsatellites (SSRs). Our goals were to assess genetic diversity and spatial distribution, to infer the evolutionary history of P. cubensis and P. humuli, and to visualize genome-scale organizational relationship between both pathogens. High genetic diversity, modest gene flow, and presence of population structure, particularly in P. cubensis, were observed. When tested for cross-amplification, 20 out of 27 P. cubensis-derived gSSRs cross-amplified DNA of P. humuli individuals, but few amplified DNA of downy mildew pathogens from related genera. Collectively, our analyses provided a definite argument for the hypothesis that both pathogens are distinct species, and suggested further speciation in the P. cubensis complex.
Fatty acids in oomycetes
(2021)
In the published article, there was an error regarding the affiliation for Diana Abondano Almeida. As well as having affiliation 2, they should also have Department of Wildlife-/Zoo-Animal-Biology and Systematics, Faculty of Biological Sciences, Goethe Universität, Frankfurt, Germany.
The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.
Cancer is a multifactorial disease with increasing incidence. There are more than 100 different cancer types, defined by location, cell of origin, and genomic alterations that influence oncogenesis and therapeutic response. This heterogeneity between tumors of different patients and also the heterogeneity within the same patient’s tumor pose an enormous challenge to cancer treatment. In this review, we explore tumor heterogeneity on the longitudinal and the latitudinal axis, reviewing current and future approaches to study this heterogeneity and their potential to support oncologists in tailoring a patient’s treatment regimen. We highlight how the ideal of precision oncology is reaching far beyond the knowledge of genetic variants to inform clinical practice and discuss the technologies and strategies already available to improve our understanding and management of heterogeneity in cancer treatment. We will focus on integrating multi-omics technologies with suitable in vitro models and their proficiency in mimicking endogenous tumor heterogeneity.
The central dogma of biology is based on the concatenated transfer of information from DNA, via transcribed mRNA, to the translated protein. In eukaryotes, transcription and translation are separated locally as well as temporally by cellular compartmentalization. Prior to active export factor-dependent transport from the nucleus to the cytosol, the newly formed pre-mRNA must mature. This involves 5'capping, splicing, and endonucleolytic cleavage and polyadenylation (CPA).
Transcription of a new pre-mRNA is terminated by hydrolytic cleavage in the 3'-UTR, and the newly formed 3'-end is protected from premature degradation by synthesis of a poly(A) tail. These processes are catalyzed by four multi-protein complexes (CFIm, CFIIm, CPSF, and CsTF) and poly(A) polymerase (PAP). CPA is sequence-specific and dependent on RNA-binding proteins (RBPs). APA-specific sequences include the poly(A) motif ('AAUAAA' and certain motif variants), the UGUA motif, and U/GU-rich sequences upstream and downstream of the poly(A) signal, respectively. About 70% of mammalian genes have more than one polyadenylation site (PAS) and express transcripts of different lengths by a mechanism called alternative polyadenylation (APA). This can affect the length of the 3'UTR (3'UTR-APA) or the coding sequence of the transcript (CDS-APA) if the alternative PAS is upstream of the STOP codon. The length of the 3'UTR affects the stability, export efficiency, subcellular localization, translation rate, and local translation of the nascent transcript. 3'UTR-APA is regulated in the interplay of the cis-elements (poly(A) motif, UGUA and U/GU) and trans-elements (expression of CPA factors). In this context, the functions of the individual cis and trans elements have been extensively studied, yet the regulation of alternative polyadenylation-the decision whether to use the proximal or distal PAS-is less deciphered and requires additional study.
In murine P19 cells, we were able to demonstrate for the first time a direct link between 3'UTR-APA and nuclear export of mature mRNA by the splicing factors SRSF3 and SRSF7 and decipher the mechanism. At the core here is the direct recruitment of the export factor NXF1 by SRSF3 and SRSF7 to transcripts with 3'UTRs of different lengths.
The primary goal of the thesis presented here was to decipher the function of SRSF3 and SRSF7 in the regulation of 3'UTR-APA and to determine the basic mechanism. For this purpose, various genome-wide methods, such as RNA-Seq, MACE-Seq, and iCLIP-Seq, were integrated and the findings were supported by reporter gene and mutation studies.
Initial determination of the poly(A)-tome in P19 cells by MACE-Seq yielded approximately 16,000 PAS and showed that slightly less than 50% of all genes used two or more PAS and expressed alternative 3'UTR isoforms. Further DaPARS analyses after knockdown of Srsf3 or Srsf7 confirmed that SRSF3 affected more transcripts than SRSF7 and led primarily to the expression of long 3'UTRs, whereas SRSF7 promoted the expression of short 3'UTRs. Integration of SRSF3- and SRSF7-specific iCLIP data suggested a possible competition between SRSF3 and SRSF7 at the proximal PAS (pPAS), which could thus act as a hotspot of 3'UTR regulation.
Experiments with intron-free reporter genes revealed that SRSF3- and SRSF7-dependent regulation of 3'UTR-APA is independent of splicing. With respect to SRSF7, a concentration dependence was demonstrated. Mutation experiments involving the SRSF3- and SRSF7-specific binding motifs in the 3'UTR also confirmed the hypothesis of competition between the two SR proteins.
Extensive Co-IP experiments clearly demonstrated that only SRSF7, but not SRSF3, can interact with CFIm and FIP1 (a subunit from the CPSF complex) in an RNA-independent manner. In addition, we showed that these interactions exhibited some phosphorylation dependence, such that the interaction to FIP1 arose primarily in the semi- to hypophosphorylated state of SRSF7. Whereas the interaction to CFIm was mainly detected in the hyperphosphorylated state. The differential affinity between SRSF3 and SRSF7 for polyadenylation factors could be attributed to two SRSF7-specific domains in subsequent mutation experiments: A CCHC-type Zn finger between the RRM and the RS domain, and a hydrophobic 27 amino acid long region in the middle of the RS domain. Together, this suggested that SRSF3 could block the utilization of pPAS, whereas SRSF7 could activate it by directly recruiting polyadenylation factors.
Interestingly, we showed that knockdown of Srsf3 also negatively regulates the expression of Cpsf6 (a subunit of CFIm) through alternative splicing, which subsequently leads to decreased expression of CPSF6 and of CFIm. Reduction of CFIm led to increased expression of transcripts with short 3'UTR, analogous to knockdown of Srsf3. This mirrors the results of previous studies. A direct comparison between SRSF3- and CPSF6-specific transcripts revealed that not all targets were congruent. In addition, we found preliminary evidence for CFIm-related masking of essential cis-pPAS elements by bimodal UGUA motifs at the pPAS. In summary, we present a novel mechanism of indirect 3'UTR-APA regulation through SRSF3-conditional expression of the CFIm subunit CPSF6.
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