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A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNAfMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA’s secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.
Research in social insects has shown that hydrocarbons on their cuticle are species-specific. This has also been proven for Diptera and is a promising tool for identifying important fly taxa in Forensic Entomology. Sometimes the empty puparia, in which the metamorphosis to the adult fly has taken place, can be the most useful entomological evidence at the crime scene. However, so far, they are used with little profit in criminal investigations due to the difficulties of reliably discriminate among different species. We analysed the CHC chemical profiles of empty puparia from seven forensically important blow flies Calliphora vicina, Chrysomya albiceps, Lucilia caesar, Lucilia sericata, Lucilia silvarum, Protophormia terraenovae, Phormia regina and the flesh fly Sarcophaga caerulescens. The aim was to use their profiles for identification but also investigate geographical differences by comparing profiles of the same species (here: C. vicina and L. sericata) from different regions. The cuticular hydrocarbons were extracted with hexane and analysed using gas chromatography-mass spectrometry. Our results reveal distinguishing differences within the cuticular hydrocarbon profiles allowing for identification of all analysed species. There were also differences shown in the profiles of C. vicina from Germany, Spain, Norway and England, indicating that geographical locations can be determined from this chemical analysis. Differences in L. sericata, sampled from England and two locations in Germany, were less pronounced, but there was even some indication that it may be possible to distinguish populations within Germany that are about 70 km apart from one another.
Mining is one of the major pollution sources worldwide, causing huge disturbances to the environment. Industrial and artisanal mining activities are widespread in Mexico, a major global producer of various metals. This study aimed to assess the ecological impairments resulting from mining activities using aquatic macroinvertebrates assemblages (MA). A multiple co-inertia analysis was applied to determine the relationships between environmental factors, habitat quality, heavy metals, and aquatic macroinvertebrates in 15 study sites in two different seasons (dry and wet) along two rivers running across the Central Plateau of Mexico. The results revealed three contrasting environmental conditions associated with different MAs. High concentrations of heavy metals, nutrients, and salinity limit the presence of several families of seemingly sensitive macroinvertebrates. These factors were found to influence structural changes in MAs, showing that not only mining activities, but also agriculture and presence of villages in the basin, exert adverse effects on macroinvertebrate assemblages. Diversity indices showed that the lowest diversity matched both the most polluted and the most saline rivers. The rivers studied displayed high alkalinity and hardness levels, which can reduce the availability of metals and cause adverse effects on periphyton by inhibiting photosynthesis and damaging MAs. Aquatic biomonitoring in rivers, impacted by mining and other human activities, is critical for detecting the effect of metals and other pollutants to improve management and conservation strategies. This study supports the design of cost-effective and accurate water quality biomonitoring protocols in developing countries.
Dynamic imaging of landmark organelles, such as nuclei, cell membrane, nuclear envelope, and lipid droplets enables image-based phenotyping of functional states of cells. Multispectral fluorescent imaging of landmark organelles requires labor-intensive labeling, limits throughput, and compromises cell health. Virtual staining of label-free images with deep neural networks is an emerging solution for this problem. Multiplexed imaging of cellular landmarks from scattered light and subsequent demultiplexing with virtual staining saves the light spectrum for imaging additional molecular reporters, photomanipulation, or other tasks. Published approaches for virtual staining of landmark organelles are fragile in the presence of nuisance variations in imaging, culture conditions, and cell types. This paper reports model training protocols for virtual staining of nuclei and membranes robust to label-free imaging parameters, cell states, and cell types. We developed a flexible and scalable convolutional architecture, named UNeXt2, for supervised training and self-supervised pre-training. The strategies we report here enable robust virtual staining of nuclei and cell membranes in multiple cell types, including neuromasts of zebrafish, across a range of imaging conditions. We assess the models by comparing the intensity, segmentations, and application-specific measurements obtained from virtually stained and experimentally stained nuclei and membranes. The models rescue the missing label, non-uniform expression of labels, and photobleaching. We share three pre-trained models, named VSCyto3D, VSCyto2D, and VSNeuromast, as well as VisCy, a PyTorch-based pipeline for training, inference, and deployment that leverages the modern OME-Zarr format.
This work aimed to investigate the regulation and activity of 5-lipoxygenase (5-LO), the central enzyme in leukotriene biosynthesis, in two colorectal cancer cell lines. The leukotriene pathway is positively correlated with the progression of several solid malignancies; however, factors regulating 5-LO expression and activity in tumors are poorly understood.
Cancer development, as well as cancer progression, are strongly dependent on the tumor microenvironment. In the conventional monolayer culture of cancer cell lines, cell-matrix and cell-cell interactions present in native tumors are absent. Furthermore, it is already known that various colon cancer cell lines dysregulate several important signaling pathways due to 3D growth. Therefore, the expression of the leukotriene cascade in HT-29 and HCT-116 colorectal cancer cells was investigated within a three-dimensional context using multicellular tumor spheroids to mimic a more physiological environment compared to conventional cell culture. Especially the expression of 5-LO, cPLA2α, and LTA4 hydrolase was altered due to threedimensional (3D) cell growth, which was investigated by qPCR and Western blot analysis. High cellular density in monolayer cultures led to similar results. The observed 5-LO upregulation was found inversely correlated with cell proliferation, determined by cell cycle analysis, and activation of PI3K/mTORC-2- and MEK-1/ERK-dependent pathways, determined using pharmacological pathway inhibition, stable shRNA knockdown cell lines, and analysis via qPCR and Western blot analysis. Following, the transcription factor E2F1 and its target gene MYBL2 were identified to play a role in the repression of 5-LO during cell proliferation. For this purpose, several stable MYBL2 over-expression and ALOX5 reporter cell lines were prepared and analyzed. Since 5-LO was already identified as a direct p53 target gene, the influence of p53, which is variably expressed in the cell lines (HT-29, p53 R273H mut; HCT-116 p53 wt; HCT-116 p53 KO), was investigated as well. Furthermore, HCT-116 cells carrying a p53 knockout were investigated. The PI3K/mTORC-2- and MEK-1/ERK-dependent suppression of 5-LO was also found in tumor cells from other origins (Capan-2, Caco-2, MCF-7), which was determined using pharmacological pathway inhibition and following analysis via qPCR. This suggests that the identified mechanism might apply to other tumor entities as well.
5-LO activity was previously described as attenuated in HT-29 and HCT-116 cells compared to polymorphonuclear leukocytes, which express a highly active 5-LO. However, the present study showed that the enzyme activity is indeed low but inducible in HT-29 and HCT-116 cells. Of note, the general lipid mediator profile and the mediator concentrations were comparable to those of M2 macrophages. Finally, the analysis of substrate availability in HT-29 and HCT-116 cells revealed a vast difference between formed metabolite concentrations and supplemented fatty acid concentrations, indicating that the substrates are either transformed into lipoxygenase-independent metabolites or are esterified into the cellular membrane.
In summary, the data presented in this work demonstrate that 5-LO expression and activity are tightly regulated in HT-29 and HCT-116 cells and fine-tuned due to environmental conditions. The cells suppress 5-LO during proliferation but upregulate the expression and activity of the enzyme under cellular stress-triggering conditions. This implies a possible role of 5-LO in manipulating the tumor stroma to support a tumor-promoting microenvironment.
Echolocating bats exhibit remarkable auditory behaviors, enabled by adaptations within and outside their auditory system. Yet, research in echolocating bats has focused mostly on brain areas that belong to the classic ascending auditory pathway. This study provides direct evidence linking the cerebellum, an evolutionarily ancient and non-classic auditory structure, to vocalization and hearing. We report that in the fruit-eating bat Carollia perspicillata, external sounds can evoke cerebellar responses with latencies below 20 ms. Such fast responses are indicative of early inputs to the bat cerebellum. In vocalizing bats, distinct spike train patterns allow the prediction with over 85% accuracy of the sound they are about to produce, or have just produced, i.e., communication calls or echolocation pulses. Taken together, our findings provide evidence of specializations for vocalization and hearing in the cerebellum of an auditory specialist.
On the potential for GWAS with phenotypic population means and allele-frequency data (popGWAS)
(2024)
This study explores the potential of a novel genome-wide association study (GWAS) approach for identifying loci underlying quantitative polygenic traits in natural populations. Extensive population genetic forward simulations demonstrate that the approach is generally effective for oligogenic and moderately polygenic traits and relatively insensitive to low heritability, but applicability is limited for highly polygenic architectures and pronounced population structure. The required sample size is moderate with very good results being obtained already for a few dozen populations scored. The method performs well in predicting population means even with a moderate false positive rate. When combined with machine learning for feature selection, this rate can be further reduced. The data efficiency of the method, particularly when using pooled sequencing, makes GWAS studies more accessible for research in biodiversity genomics. Overall, this study highlights the promise of this popGWAS approach for dissecting the genetic basis of complex traits in natural populations.
Abstract
Seed harvesting from wild plant populations is key for ecological restoration, but may threaten the persistence of source populations. Consequently, several countries have set guidelines limiting the proportions of harvestable seeds. However, these guidelines are so far inconsistent, and they lack a solid empirical basis. Here, we use high-resolution data from 298 plant species to model the demographic consequences of seed harvesting. We find that the current guidelines do not protect populations of annuals and short-lived perennials, while they are overly restrictive for long-lived plants. We show that the maximum possible fraction of seed production – what can be harvested without compromising the long-term persistence of populations – is strongly related to the generation time of the target species. When harvesting every year, this safe seed fraction ranges from 80% in long-lived species to 2% in most annuals. Less frequent seed harvesting substantially increases the safe seed fraction: In the most vulnerable annual species, it is safe to harvest 5%, 10% or 30% of population seed production when harvesting every two, five or ten years, respectively. Our results provide a quantitative basis for seed harvesting legislations worldwide, based on species’ generation time and harvesting regime.
Significance The UN Decade on Ecosystem Restoration, 2021-2030, foresees upscaling restoration, and the demand for native seed is skyrocketing. Seeds for restoring native vegetation are often harvested in wild, but too intensive harvest can threaten the donor populations. Existing guidelines that set limits to wild seed harvest are mostly based on expert opinions, yet they commonly lack empirical basis and vary among regions in one order of magnitude. We show that the current guidelines urgently need to be reformulated, because they are overly restrictive in long-lived species, while they do not protect annual plants from extinction. Using matrix population models of nearly 300 plant species, we provide a quantitative basis for a new seed harvesting legislation world-wide.
Cyclin CLB2 mRNA localization and protein synthesis link cell cycle progression to bud growth
(2024)
Clb2 is a conserved mitotic B-type cyclin, the levels of which are finely controlled to drive progression through the cell cycle. While it is known that CLB2 transcription and Clb2 protein degradation are important for precise control of its expression, it remains unclear whether the synthesis of Clb2 is also regulated. To address whether and how Clb2 expression levels respond to cell growth changes and adapt cell cycle progression, we combined single-cell and single-molecule imaging methods to measure CLB2 mRNA and protein expression throughout the Saccharomyces cerevisiae cell cycle. We found that the CLB2 mRNA was efficiently localized to the yeast bud as soon as this compartment was formed, but strikingly the Clb2 protein accumulated in the mother nucleus. The CLB2 mRNA localization in the yeast bud by the She2-3 complex did not control protein localization but rather promoted CLB2 translation. Moreover, CLB2 mRNA bud localization and protein synthesis were coupled and dependent on a single secondary structure -a ZIP code-located in the coding sequence. In a CLB2 ZIP code mutant, mRNA localization was impaired and Clb2 protein synthesis decreased, resulting in changes in cell cycle distribution and increased size of daughter cells at birth. Finally, while in WT cells the Clb2 protein concentration followed bud growth, this relationship was impaired in the ZIP code mutant. We propose that S. cerevisiae couples the control of CLB2 mRNA bud localization and protein synthesis to coordinate cell growth and cell cycle progression. This mechanism extends our knowledge of CLB2 expression regulation, and constitutes a novel function for mRNA localization.
Research on the human and animal microbiome has become increasingly important in recent years. It is now widely accepted the gut microbiome is of crucial importance to health, as it is involved in a large number of physiological processes. The term ‘microbiome’ refers to the all living microorganisms including their genes and metabolites in a defined environment, while the specific composition of microorganisms consisting of bacteria, archaea and protozoa is referred to as the ‘microbiota’ (Lane-Petter, 1962; Lederberg and McCray, 2001).
In recent years, research has focused on various of these communities in the soil (Fierer, 2017), water (Sunagawa et al., 2015), air (Leung et al., 2014) and especially in the human gut. However, this topic is also becoming increasingly relevant for the conservation of endangered species. In the face of global mass extinctions and the listing of over 42,000 animal species as ‘critically endangered’, conservation breeding programmes are more important than ever (Díaz et al., 2019; IUCN, 2022). The responsibility for these tasks lies with zoological institutions, which are dedicated to animal conservation and the continuous monitoring of animal welfare. Microbiome research offers a non-invasive method to support species conservation. By analysing faecal samples, microbial markers can be identified that provide important information about the health status and reproductive cycle of animals (Weingrill et al., 2004; Antwis et al., 2019). Zoological facilities also provide an ideal research environment for comparing individuals from different habitats. In addition, all necessary metadata such as age, sex, kinship or medical treatment are documented and can be used for the analysis.
This is the starting point for this thesis. In order to identify such microbial markers, it is necessary to understand the microbiome of a variety of animal species. The first aim is therefore to characterise the faecal microbiota of 31 mammalian species, focusing on herbivores and carnivores. It could be shown that they differ significantly in terms of both microbial diversity and microbiota composition. Herbivorous species express a very diverse microbial composition, consisting mainly of cellulose-degrading taxa of the families Fibrobacteraceae or Spirochaetaceae. In contrast, the microbiota of carnivorous species is less diverse and is dominated by protein-degrading Fusobacteriaceae and Clostridiaceae. In addition, this thesis proves that the microbiota of herbivorous species is highly consistent, whereas the microbiota of carnivorous species is highly variable. The results of this study provide important insights for the sampling scheme of future projects. Especially when analysing carnivorous species, single samples are not sufficient to capture the full variability of the microbiome.
These results lead to the question of whether this variability can be explained by daily fluctuations in the individual microbiome and whether this can be used to distinguish between species or individuals. Using individual longitudinal data and a combined approach of clustering algorithms and dynamic time warping, it is shown that such a distinction is possible at the species and individual level. This was confirmed for both a carnivorous (Panthera tigris) and a herbivorous (Connochaetes taurinus) species. These results confirm the influence of the host individual on the faecal microbiota, in addition to the often described influence of diet (Ley et al., 2008a; Kartzinel et al., 2019).
Based on the knowledge gained from these studies, a methodology has been developed that will enable the conservation of species in the field to be supported by microbiome research in the future. The focus here lays on the identification of host-specific metadata based on the faecal microbiota. The developed regression model is able to distinguish between carnivorous, herbivorous and omnivorous hosts with up to 99% accuracy. In addition, a more accurate phylogenetic classification of the family (Canidae, Felidae, Ursidae, Herpestidae) can be made for carnivorous hosts. For herbivorous hosts, the model can predict the respective digestive system with up to 100% accuracy, distinguishing between ruminants, hindgut fermenters and a simple digestive system. The acquisition of host-specific metadata from an unknown faecal sample is an important step towards establishing microbiome research in species conservation. Field studies in particular will benefit from such new methods. Usually, costly microsatellite analysis and high-quality host DNA are required to obtain host-specific information from faecal samples. The newly developed method offers a less costly and labour-intensive alternative to conventional techniques and opens up a more accessible field for microbiome research in the field.
Aryl hydrocarbon receptor-dependent and -independent pathways mediate curcumin anti-aging effects
(2022)
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor whose activity can be modulated by polyphenols, such as curcumin. AhR and curcumin have evolutionarily conserved effects on aging. Here, we investigated whether and how the AhR mediates the anti-aging effects of curcumin across species. Using a combination of in vivo, in vitro, and in silico analyses, we demonstrated that curcumin has AhR-dependent or -independent effects in a context-specific manner. We found that in Caenorhabditis elegans, AhR mediates curcumin-induced lifespan extension, most likely through a ligand-independent inhibitory mechanism related to its antioxidant activity. Curcumin also showed AhR-independent anti-aging activities, such as protection against aggregation-prone proteins and oxidative stress in C. elegans and promotion of the migratory capacity of human primary endothelial cells. These AhR-independent effects are largely mediated by the Nrf2/SKN-1 pathway.
Mucormycosis is an invasive fungal infection associated with high mortality, partly due to delayed diagnosis and inadequate empiric therapy. As fungal cultures often fail to grow Mucorales, identification of respective hyphae in tissue is frequently needed for diagnosis but may be challenging. We studied fluorescence in situ hybridization (FISH) targeting specific regions of the fungal ribosomal RNA (rRNA) of Mucorales to improve diagnosis of mucormycosis from tissue samples. We generated a probe combination specifically targeting Mucorales. Probe specificity was verified in silico and using cultivated fungi. Mucorales hyphae in tissue of a mouse model demonstrated a bright cytoplasmatic hybridization signal. In tissue samples of patients with mucormycosis, a positive signal was seen in 7 of 12 (58.3%) samples. However, autofluorescence in 3 of 7 (42.9%) samples impaired the diagnostic yield. Subsequent experiments suggested that availability of nutrients and antifungal therapy may impact on the FISH signal obtained with Mucorales hyphae. Diagnosis of mucormycosis from tissue might be improved by rRNA FISH in a limited number of cases only. FISH signals may reflect different wphysiological states of fungi in tissue. Further studies are needed to define the value of FISH to diagnose mucormycosis from other clinical samples.
Chronische Entzündungen und die daraus resultierenden Morbiditäten gehören zu den häufigsten Ursachen für einen frühen Tod beim Menschen. Einer der Hauptfaktoren für die Verschlechterung des Gesundheitszustands bei Patienten mit chronischen-entzündlichen Erkrankungen ist die pathologische Infiltration von Leukozyten in gesundes Gewebe, die zu Gewebeschäden und dem Fortschreiten der Krankheit führt. Das vaskuläre Endothel, das die Innenseite der Blutgefäße auskleidet, spielt eine entscheidende Rolle bei der Entzündungsreaktion, da es als Schnittstelle für die Interaktion mit Leukozyten fungiert, um die Extravasation von Leukozyten aus dem Blutstrom in das Gewebe zu ermöglichen. Die Adhäsion von Leukozyten an die Zellen des Endothels wird dabei hauptsächlich durch die von Zytokinen ausgelösten pro-inflammatorischen NFκB- und AP-1-Signalkaskaden ermöglicht, die die Hochregulierung der wichtigsten endothelialen Adhäsionsmoleküle – ICAM-1, VCAM-1 und E-Selektin – bewirken. Eine Klasse von Wirkstoffen, die für ihre entzündungshemmenden Eigenschaften und ihren Nutzen bei der Behandlung chronischer Entzündungskrankheiten bekannt sind, sind die Mikrotubuli-bindenden-Substanzen (microtubule-targeting-agents; MTAs), die nachweislich auch den Entzündungszustand in den Zellen des Endothels und die Leukozyten-Adhäsionskaskade beeinflussen können. MTAs lassen sich in Mikrotubuli-Destabilisatoren, die eine Depolymerisation des Mikrotubuli-Zytoskeletts bewirken, und Mikrotubuli-Stabilisatoren, die die Depolymerisation der Mikrotubuli verhindern, unterteilen. Die zugrundeliegenden biomolekularen Vorgänge und Wirkungen, die die MTAs auf die Zellen des Gefäßendothels haben, und wie sie die Adhäsionskaskade der Leukozyten beeinflussen, sind jedoch weitgehend unbekannt.
Ziel dieser Studie war es, die Auswirkungen des neuartigen Mikrotubuli-Destabilisators Prätubulysin, eines Vorläufers der Tubulysine, die ursprünglich in Stämmen des Myxobakteriums Angiococcus disciformis entdeckt wurden, auf die entzündlichen Prozesse zu untersuchen, die die Leukozyten-adhäsion in TNF-aktivierten primären Endothelzellen aus der menschlichen Nabelschnurvene (HUVECs) ermöglichen. Zusätzlich wurden auch die Auswirkungen der bereits klinisch etablierten Mikrotubuli-Destabilisatoren Colchicin und Vincristin sowie des Mikrotubuli-Stabilisators Paclitaxel untersucht.
Das entzündungshemmende Potenzial von Prätubulysin wurde daher zunächst in vivo in einem Imiquimod-induzierten psoriasiformen Dermatitis-Mausmodell getestet, wobei sich zeigte, dass Prätubulysin den Entzündungszustand deutlich verringert. Um zu beweisen, dass der entzündungshemmende Effekt mit einer verringerten Interaktion von Leukozyten mit dem Endothel zusammenhängt, wurde die Wirkung von Prätubulysin in vivo mittels Intravitalmikroskopie des TNF-aktivierten Kremaster-Muskels der Maus untersucht. Dabei zeigte sich, dass die Behandlung mit Prätubulysin zu einer signifikant verringerten Adhäsion von Leukozyten an die Zellen des Gefäßendothels führte. Die verringerte Adhäsion von Leukozyten an Endothelzellen wurde auch in der in vitro Umgebung bestätigt, indem die Adhäsion von Leukozyten unter Flussbedingungen getestet wurde. Mittels Durchflusszytometrie, Western-Blot-Analyse, sowie qRT-PCR-Analyse der jeweiligen mRNA-Level konnte gezeigt werden, dass die verringerten Leukozyten-Interaktionen auf der verringerten Expression der Zelladhäsionsmoleküle ICAM-1 und VCAM-1 sowie teilweise von E-Selektin nach Behandlung mit Prätubulysin, Vincristin und Colchicin beruhen, wobei Paclitaxel keine signifikanten hemmenden Auswirkungen hatte. Weitere Untersuchungen des Einflusses von Prätubulysin auf die NFκB- und AP-1-Signalübertragung zeigten, dass diese intrazellulären Signalkaskaden durch Prätubulysin nicht behindert werden, wobei NFκB und AP-1 weitgehend in den Promotoren der Zelladhäsionsmoleküle angereichert waren, wie durch Chromatin-Immunpräzipitation nachgewiesen wurde. Darüber hinaus induzierte die Behandlung mit Prätubulysin die Aktivität der NFκB-induzierenden Kinase IKK und führte zu einem signifikanten Anstieg der Aktivität der AP-1 Upstream-Kinase JNK, wie eine Western Blot Analyse ergab. Die Prüfung der Transkriptionsaktivität von NFκB und AP-1 in Reportergen Assays zeigte, dass insbesondere die Mikrotubuli-Destabilisatoren die Promotoraktivität dieser Transkriptionsfaktoren in einer konzentrationsabhängigen Weise verringerten. Weitere Tests zur Abhängigkeit der durch Prätubulysin induzierten Hemmung der Zelladhäsionsmoleküle von der Aktivität der JNK zeigten, dass die Hemmung empfindlich auf die Aktivität dieser Kinase reagiert. Es konnte gezeigt werden, dass die Inhibition der Aktivität der JNK die Expression der Zelladhäsionsmoleküle durch die Behandlung mit Prätubulysin auf mRNA und Proteinebene wiederherstellt. Mit Hilfe der Chromatin-Immunpräzipitation konnte weiterhin gezeigt werden, dass die Behandlung mit Prätubulysin zunächst die Assoziation des Bromodomänen-enthaltenden Proteins 4 mit den Promotoren/Genen von ICAM-1 und VCAM-1 erhöhte, aber zu einem behandlungszeitabhängigen Rückgang der Anreicherung führte. Darüber hinaus wurde durch die Behandlung mit Prätubulysin auch der Abbau dieses Proteins leicht erhöht. Durch den Einsatz eines JNK Inhibitors konnte gezeigt werden, dass die Verdrängung des Bromodomänen-enthaltenden Proteins 4 von icam-1 und vcam-1, sowie der erhöhte Abbau dieses Faktors auch von der Aktivität der JNK abhängig sind. Die Verdrängung des Bromodomänen-enthaltenden Proteins 4 induzierte auch das Vorhandensein von repressiven Chromatinmarkierungen in den Genen von ICAM-1 und VCAM-1. Die Prüfung der Anreicherung der RNA-Polymerase II an den Promotoren/Genen von ICAM-1 und VCAM-1 zeigte jedoch auch eine behandlungszeitabhängige differentielle Anreicherung dieser Polymerase, wobei die Anreicherung nach kurzen Behandlungszeiten reduziert war, sich nach mittleren Behandlungszeiten erholte und nach längeren Behandlungszeiten wieder stark reduziert war. Die anschließende Prüfung der Bedeutung des Bromodomänen-enthaltenden Proteins 4 für die Expression von ICAM-1 und VCAM-1 durch Knock-down-Experimente ergab, dass das vcam-1 Gen durch Knock-down dieses Proteins unterdrückt, das icam-1 Gen jedoch induziert wird. Dies deutet auf das Vorhandensein zusätzlicher Faktoren hin, die auch auf die Aktivität der JNK reagieren und neben dem Bromodomänen-enthaltenden Proteins 4 die Transkriptionsverlängerung des icam-1 Gens bewirken.
The negative effect of fossil-based industrial processes on the environment, especially the contribution to global warming by emitting greenhouse gases such as CO2 causes a global threat to mankind. Therefore, technologies are demanded by the society for a sustainable and environmentally friendly economy. The biotechnological use of sugar-based feedstocks to produce valuable products are in conflict with, for example, food production. In order to overcome this issue, waste products such as syngas (H2, CO and CO2) or CO2 taken from the atmosphere are of increasing interest for biotechnological applications. Acetogenic bacteria are already used at industrial scale to produce sustainable and environmentally friendly biofuels from syngas. A promising candidate due to its physiological flexibility is the thermophilic acetogen Moorella thermoacetica. In contrast to most acetogens M. thermoacetica is not restricted to one energy conserving system. In addition to the Ech complex, cytochromes and quinones may be involved in energy conservation by, for example, DMSO respiration. The extra energy conserved can be used to form highly valuable but energy demanding products. In this review we give insights into the physiology of this acetogen, the current state of the art of M. thermoacetica as a platform for biotechnological applications and discuss future perspectives.
The ability of wild animals to navigate and survive in complex and dynamic environments depends on their ability to store relevant information and place it in a spatial context. Despite the centrality of spatial memory, and given our increasing ability to observe animal movements in the wild, it is perhaps surprising how difficult it is to demonstrate spatial memory empirically. We present a cognitive analysis of movements of several wolves (Canis lupus) in Finland during a summer period of intensive hunting and den-centered pup-rearing. We tracked several wolves in the field by visiting nearly all GPS locations outside the den, allowing us to identify the species, location and timing of nearly all prey killed. We then developed a model that assigns a spatially explicit value based on memory of predation success and territorial marking. The framework allows for estimation of multiple cognitive parameters, including temporal and spatial scales of memory. For most wolves, fitted memory-based models outperformed null models by 20 to 50% at predicting locations where wolves chose to forage. However, there was a high amount of individual variability among wolves in strength and even direction of responses to experiences. Some wolves tended to return to locations with recent predation success—following a strategy of foraging site fidelity—while others appeared to prefer a site switching strategy. These differences are possibly explained by variability in pack sizes, numbers of pups, and features of the territories. Our analysis points toward concrete strategies for incorporating spatial memory in the study of animal movements while providing nuanced insights into the behavioral strategies of individual predators.
Dynamic imaging of landmark organelles, such as nuclei, cell membrane, nuclear envelope, and lipid droplets enables image-based phenotyping of functional states of cells. Multispectral fluorescent imaging of landmark organelles requires labor-intensive labeling, limits throughput, and compromises cell health. Virtual staining of label-free images with deep neural networks is an emerging solution for this problem. Multiplexed imaging of cellular landmarks from scattered light and subsequent demultiplexing with virtual staining saves the light spectrum for imaging additional molecular reporters, photomanipulation, or other tasks. Published approaches for virtual staining of landmark organelles are fragile in the presence of nuisance variations in imaging, culture conditions, and cell types. This paper reports model training protocols for virtual staining of nuclei and membranes robust to cell types, cell states, and imaging parameters. We developed a flexible and scalable convolutional architecture, named UNeXt2, for supervised training and self-supervised pre-training. The strategies we report here enable robust virtual staining of nuclei and cell membranes in multiple cell types, including neuromasts of zebrafish, across a range of imaging conditions. We assess the models by comparing the intensity, segmentations, and application-specific measurements obtained from virtually stained and experimentally stained nuclei and membranes. The models rescue the missing label, non-uniform expression of labels, and photobleaching. We share three pre-trained models, named VSCyto3D, VSCyto2D, and VSNeuromast, as well as VisCy, a PyTorch-based pipeline for training, inference, and deployment that leverages the modern OME-Zarr format.
Argonaute 2 (AGO2) is an indispensable component of the RNA-induced silencing complex, operating at the translational or posttranscriptional level. It is compartmentalized into structures such as GW- and P-bodies, stress granules and adherens junctions as well as the midbody. Here we show using immunofluorescence, image and bioinformatic analysis and cytogenetics that AGO2 also resides in membrane protrusions such as open- and close-ended tubes. The latter are cytokinetic bridges where AGO2 colocalizes at the midbody arms with cytoskeletal components such as α-Τubulin and Aurora B, and various kinases. AGO2, phosphorylated on serine 387, is located together with Dicer at the midbody ring in a manner dependent on p38 MAPK activity. We further show that AGO2 is stress sensitive and important to ensure the proper chromosome segregation and cytokinetic fidelity. We suggest that AGO2 is part of a regulatory mechanism triggered by cytokinetic stress to generate the appropriate micro-environment for local transcript homeostasis.
Oncogenic transformation of lung epithelial cells is a multi-step process, frequently starting with the inactivation of tumor suppressors and subsequent activating mutations in proto-oncogenes, such as members of the PI3K or MAPK family. Cells undergoing transformation have to adjust to changes, such as metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors, which manifest these adjustments. Here, we report that the deubiquitylase USP28 enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes, such as c-JUN, c-MYC, NOTCH and ΔNP63, at early stages of malignant transformation. USP28 is increased in cancer compared to normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors, such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small molecule inhibitor reset the proteome of transformed cells towards a ‘pre-malignant’ state, and its inhibition cooperated with clinically established compounds used to target EGFRL858R, BRAFV600E or PI3KH1047R driven tumor cells. Targeting USP28 protein abundance already at an early stage via inhibition of its activity therefore is a feasible strategy for the treatment of early stage lung tumours and the observed synergism with current standard of care inhibitors holds the potential for improved targeting of established tumors.
Neurodevelopmental psychiatric disorders (NPDs) like attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and schizophrenia, affect millions of people worldwide. Despite recent progress in NPD research, much remains to be discovered about their underpinnings, therapeutic targets, effects of biological sex and age. Risk factors influencing brain development and signalling include prenatal inflammation and genetic variation. This dissertation aimed to build upon these findings by combining behavioural, molecular, and neuromorphological investigations in mouse models of such risk factors, i.e. maternal immune activation (MIA), neuron-specific overexpression (OE) of the cytoplasmatic isoforms of the RNA-binding protein RBFOX1, and neuronal deletion of the small Ras GTPase DIRAS2.
Maternal infections during pregnancy pose an increased risk for NPDs in the offspring. While viral-like MIA has been previously established elsewhere, this study was the first in our institution to implement the model. I validated NPD-relevant deficits in anxiety- and depression-like behaviours, as well as dose- and sex-specific social deficits in mouse offspring following MIA in early gestation. Proteomic analyses in embryonic and adult hippocampal (HPC) synaptoneurosomes highlighted novel and known targets affected by MIA. Analysis of the embryonic dataset implicated neurodevelopmental disruptions of the lipid, polysaccharide, and glycoprotein metabolism, important for proper membrane function, signalling, and myelination, for NPD-pertinent sequelae. In adulthood, the observed changes encompassed transmembrane trafficking and intracellular signalling, apoptosis, and cytoskeletal organisation pathways. Importantly, 50 proteins altered by MIA in embryonic and adult HPC were enriched in the NPD-relevant synaptic vesicle cycle. A persistently upregulated protein cluster formed a functional network involved in presynaptic signalling and proteins downregulated in embryos but upregulated in adults by MIA were correlated with observed social deficits. 49/50 genes encoding these proteins were significantly associated with NPD- and comorbidity-relevant traits in human phenome-wise association study data for psychiatric phenotypes. These findings highlight NPD-relevant targets for future study and early intervention in at-risk individuals. MIA-evoked changes in the neuroarchitecture of the NPD-relevant HPC and prefrontal cortex (PFC) of male and female mice highlighted sex- and region-specific alterations in dendritic and spine morphology, possibly underlining behavioural phenotypes.
To further investigate genetic risk factors of NPDs, I performed a study based on the implications of RBFOX1’s pleiotropic role in neuropsychiatric disorders and previous preclinical findings. Cytoplasmatic OE of RBFOX1, which affects the stability and translation of thousands of targets, was used to disseminate its role in morphology and behaviour. RBFOX1 OE affected dendritic length and branching in the male PFC and led to spine alterations in both PFC and HPC. Due to previously observed ASD-like endophenotypes in our Rbfox1 KO mice and the importance of gene × environment effects on NPD susceptibility, I probed the interaction of cytoplasmatic OE and a low-dose MIA on offspring. Both RBFOX1 OE alone and with MIA led to increased offspring loss during the perinatal period. Preliminary data suggested that RBFOX1 OE × MIA might increase anxiety- and anhedonia-like behaviours. Morphological changes in the adult male OE HPC and PFC suggested increased spine density and reduced dendritic complexity. A small post-mortem study in human dorsolateral PFC of older adults did not reveal significant effects of a common risk variant on RBFOX1 abundance.
To expand upon NPD genetic risks, I evaluated the effects of a homo- (KO) or heterozygous (HET) Diras2 deletion in a novel, neuron-specific mouse model. DIRAS2’s function is largely unknown, but it has been associated with ADHD in humans and neurodevelopment in vitro. In adult mice, there were subtle sex-specific effects on behaviour, i.e. more pronounced NPD-relevant deficits in males, in keeping with human data. KO mice had subtly improved cognitive performance, while HET mice exhibited behaviours in line with core ADHD symptoms, e.g. earning difficulties (females), response inhibition deficits and hyperactivity (males), suggesting Diras2 dose-sensitivity and sex-specificity. The morphological findings revealed multiple aberrations in dendritic and spine morphology in the adult PFC, HPC, and amygdala of HET males. KOs changes in spine and dendritic morphology were exclusively in the PFC and largely opposite to those in HETs and NPD-like phenotypes. Region- and genotype-specific expression changes in Diras2 and Diras1 were observed in six relevant brain regions of adult HET and KO females, also revealing differences in the survival and morphology regulator mTOR, which might underlie observed differences.
In conclusion, the effects of MIA and partial Diras2 knockdown resembled each other in core, NPD-associated behavioural and morphological phenotypes, while cytoplasmatic RBFOX1 OE and full Diras2 KO differed from those. My findings suggest complex dose- and sex-dependent relationships between these prenatal and genetic interventions, whose NPD-relevant influences might converge onto neurodevelopmental molecular pathways. An assessment of such putative overlap, based on available data from the MIA proteomic analyses of embryonic and adult HPC, suggested the three models might be linked via downstream targets, interactions, and upstream regulators. Future studies should disseminate both distinct and shared aspects of MIA, RBFOX1, and DIRAS2 relevant to NPDs and build upon these findings.
Mitochondria are dynamic organelles exhibiting diverse shapes. While the variation of shapes, ranging from spheres to elongated tubules, and the transition between them, are clearly seen in many cell types, the molecular mechanisms governing this morphological variability remain poorly understood. Here, we propose a novel shaping mechanism based on the interplay between the inner and outer mitochondrial membranes. Our biophysical model suggests that the difference in surface area, arising from the pulling of the inner membrane into cristae, correlates with mitochondrial elongation. Analysis of live cell super-resolution microscopy data supports this correlation, linking elongated shapes to the extent of cristae in the inner membrane. Knocking down cristae shaping proteins further confirms the impact on mitochondrial shape, demonstrating that defects in cristae formation correlate with mitochondrial sphericity. Our results suggest that the dynamics of the inner mitochondrial membrane are important not only for simply creating surface area required for respiratory capacity, but go beyond that to affect the whole organelle morphology. This work explores the biophysical foundations of individual mitochondrial shape, suggesting potential links between mitochondrial structure and function. This should be of profound significance, particularly in the context of disrupted cristae shaping proteins and their implications in mitochondrial diseases.