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Nowak, Larissa ; Schleuning, Matthias ; Bender, Irene M. A. ; Kissling, W. Daniel ; Fritz, Susanne A.
Aim
How species respond to climate change is influenced by their sensitivity to climatic conditions (i.e. their climatic niche) and aspects of their adaptive capacity (e.g. their dispersal ability and ecological niche). To date, it is largely unknown whether and how species’ sensitivity to climate change and their adaptive capacity covary. However, understanding this relationship is important to predict the potential consequences of a changing climate for species assemblages. Here, we test how species’ sensitivity to climate change and trait-based measures of their ecological adaptive capacity (i) vary along a broad elevational gradient and (ii) covary across a large number of bird species.
Location
A Neotropical elevational gradient (300–3600 m.a.s.l.) in the Manú Biosphere Reserve, south-east Peru.
Methods
We focus on 215 frugivorous bird species along a Neotropical elevational gradient. We approximate species’ sensitivity to climate change by their climatic niche breadth, based on species occurrences across South America and bioclimatic variables. In addition, we use a trait-based approach to estimate the dispersal ability of species (approximated by their wing pointedness), their dietary niche breadth (approximated by bill width) and their habitat niche breadth (the number of used habitat classes).
Results
We found that (i) species’ climatic niche breadth increased with elevation, while their trait-based dispersal ability and dietary niche breadth decreased with elevation, and (ii) sensitivity to climate change and trait-based adaptive capacity were not related across species.
Main conclusions
These results suggest different mechanisms of how species in lowland and highland assemblages might respond to climate change. The independent variation of species’ sensitivity to climate change and their trait-based adaptive capacity suggests that accounting for both dimensions will improve assessments of species’ susceptibility to climate change and potential impacts of climate change on diverse species assemblages.
Exploration of macrofungi in the Jammu and Kashmir regions, India, led to the discovery of two novel species, described herein as Lactarius indohirtipes sp. nov. and L. sharmai sp. nov. This paper presents detailed morphological descriptions and illustrations, as well as a phylogenetic analysis based on nrITS sequences. A comparative analysis with related species is also included.
The evolution of cell-free protein synthesis (CFPS) over recent decades has made it a widely used system for expressing membrane proteins (MPs). Unlike traditional methods, CFPS allows direct and translocon-independent expression of MPs within lipid membranes, such as liposomes or nanodiscs (NDs), without the need for detergent solubilization. This open nature of CF systems enables customization of the experimental environment, including expression conditions, choice of nanoparticles (NPs), lipid composition, and addition of stabilizing molecules.
Membrane scaffold protein (MSP)-based NDs emerged as a gold standard for cotranslational solubilization of MPs using the CF-system. This approach allowed not only biochemical characterization, but also structural studies of MPs and even GPCRs. However, to solubilize MPs inside nanoparticles via the traditional reconstitution route, apart from MSPs other scaffolds were successfully implemented, e.g. the saposin A (commercially known as Salipro) scaffold system or the synthetic styrene maleic acid lipid particles (SMALPs). In this study the potential of saposin A-based nanoparticles (SapNPs) was explored for cotranslational MP solubilization.
Three strategies for applying SapNPs in CF systems were investigated: preassembly, (i) coassembly (ii), and coexpression (iii). (i) Preassembly involved forming SapNPs before CF expression and adding them to the CF reaction. In coassembly mode SapA and lipids were mixed in the CF reaction for spontaneous assembly with the synthesized MP. In coexpression mode lipids were added to the CF reaction while coexpressing SapA with the MP target. Proteorhodopsin (PR) served as a model protein to evaluate these strategies due to its ability to oligomerize and straightforward quantification using the cofactor retinal. Preassembled SapNPs provided homogeneous, aggregate-free particles yielding up to 200 µM solubilized PR inside in the CF reaction. Coassembly was also successfully applied to produce PR/SapNP complexes at slightly lower yields, however the system was prone to produce soluble aggregates at too high PR template concentrations and overall needed more adjustments. Coexpression resulted in PR yields below 20 µM and was not considered viable for MP production. Finally, the preassembled SapNPs were used to produce functional G-protein coupled receptor probes. Despite lower overall performance compared to MSP-based systems, SapNPs showed potential as an alternative in CF systems for specific MPs.
The second optimization approach was directed at the CF lysate itself. CF synthesis for NMR analysis benefits from selective labeling schemes enabled by truncated amino acid (AA) metabolic pathways in lysates, reducing spectral ambiguity. However, residual enzymatic AA conversions persist, leading to label dilution and ambiguous NMR spectra. This study aimed to eliminate these residual activities in the E. coli A19 strain, generating optimized CF lysates for NMR applications.
The approach involved cumulative gene deletions of the most problematic scrambling enzymes. The new strain, “Stablelabel,” included deletions and modifications in genes asnA, ansA, ansB, glnA, aspC, and ilvE, effectively eliminating background activities of L-Asn, L-Asp, and conversions of L-Glu to L-Asp and L-Gln. However, residual conversion of L-Gln to L-Glu persisted due to glutaminase activity of several glutaminases using the inhibitor 6 diazo-5-oxo-L-norleucine (DON). Stablelabel showed a slightly slower growth than A19, and an overall good performance with 2.7 mg/mL GFP expressed in the reaction mixture (RM) compared to the parental A19 strain with 3.5 mg/mL. Furthermore, the strain was successfully applied to demonstrate methyl group labeling of MPs using preconverted L-val and L-leu from their respective precursors 2-ketoisovalerate and 4-methyl-2-oxovalerate.
In this study, lipid nanoparticle particle-and strain engineering vividly demonstrated the potential of CFPS systems and their versatility. While the SapNP system requires further engineering to potentially reach the efficiency of the well-studied MSP NDs, this study provides an example of nanoparticle characterization allowing new insights into NP behavior in CF systems. Furthermore, it was shown that strain engineering is a straightforward solution to tailor CF lysates to the individual requirements. After this thesis was submitted, Stablelabel in fact was successfully applied for backbone assignment of casein kinase 1, thereby demonstrating its suitability to express complex targets for NMR studies.
Schmidt, Florian ; Marx, Alexander ; Baumgarten, Nina ; Hebel, Marie ; Wegner, Martin ; Kaulich, Manuel ; Leisegang, Matthias ; Brandes, Ralf ; Göke, Jonathan ; Vreeken, Jilles ; Schulz, Marcel Holger
Understanding how epigenetic variation in non-coding regions is involved in distal gene-expression regulation is an important problem. Regulatory regions can be associated to genes using large-scale datasets of epigenetic and expression data. However, for regions of complex epigenomic signals and enhancers that regulate many genes, it is difficult to understand these associations. We present StitchIt, an approach to dissect epigenetic variation in a gene-specific manner for the detection of regulatory elements (REMs) without relying on peak calls in individual samples. StitchIt segments epigenetic signal tracks over many samples to generate the location and the target genes of a REM simultaneously. We show that this approach leads to a more accurate and refined REM detection compared to standard methods even on heterogeneous datasets, which are challenging to model. Also, StitchIt REMs are highly enriched in experimentally determined chromatin interactions and expression quantitative trait loci. We validated several newly predicted REMs using CRISPR-Cas9 experiments, thereby demonstrating the reliability of StitchIt. StitchIt is able to dissect regulation in superenhancers and predicts thousands of putative REMs that go unnoticed using peak-based approaches suggesting that a large part of the regulome might be uncharted water.
Kreiß, Marius ; Oberlis, Julia H. ; Seuter, Sabine ; Bischoff-Kont, Iris ; Sürün, Duran ; Thomas, Dominique Jeanette ; Göbel, Tamara ; Schmid, Tobias ; Rådmark, Olof ; Brandes, Ralf ; Fürst, Robert ; Häfner, Ann-Kathrin ; Steinhilber, Dieter
Human 5-lipoxygenase (5-LO) is the key enzyme of leukotriene biosynthesis, mostly expressed in leukocytes and thus a crucial component of the innate immune system.
In this study, we show that 5-LO, besides its canonical function as an arachidonic acid metabolizing enzyme, is a regulator of gene expression associated with euchromatin. By Crispr-Cas9-mediated 5-LO knockout (KO) in MonoMac6 (MM6) cells and subsequent RNA-Seq analysis, we identified 5-LO regulated genes which could be clustered to immune/defense response, cell adhesion, transcription and growth/developmental processes. Analysis of differentially expressed genes identified cyclooxygenase-2 (COX2, PTGS2) and kynureninase (KYNU) as strongly regulated 5-LO target genes. 5-LO knockout affected MM6 cell adhesion and tryptophan metabolism via inhibition of the degradation of the immunoregulator kynurenine. By subsequent FAIRE-Seq and 5-LO ChIP-Seq analyses, we found an association of 5-LO with euchromatin, with prominent 5-LO binding to promoter regions in actively transcribed genes. By enrichment analysis of the ChIP-Seq results, we identified potential 5-LO interaction partners. Furthermore, 5-LO ChIP-Seq peaks resemble patterns of H3K27ac histone marks, suggesting that 5-LO recruitment mainly takes place at acetylated histones.>
In summary, we demonstrate a noncanonical function of 5-LO as transcriptional regulator in monocytic cells.
In the deep-sea, the interaction between benthic fauna and substrate mainly occurs through bioturbational processes which can be preserved as traces (i.e., lebensspuren). Lebensspuren are common features of deep seafloor landscapes and usually more abundant than the organism that produce them (i.e., tracemakers), rendering them promising proxies to infer biodiversity. The density and diversity relationships between lebensspuren and benthic fauna are to the present day unclear and contradicting hypotheses have been proposed suggesting negative, positive, or even null correlations. To test these hypotheses, in this study lebensspuren, tracemakers (specific epibenthic fauna that produce these traces), degrading fauna (benthic fauna that can erase lebensspuren), and fauna in general were characterized taxonomically at eight deep-sea stations in the Kuril Kamchatka Trench area. No general correlation (over-all study area) could be observed between diversities of lebensspuren, tracemakers, degrading fauna and fauna. However, a diversity correlation was observed between specific stations, showing both negative and positive correlations depending on: 1) the number of unknown tracemakers (especially significant for dwelling lebensspuren); and 2) the lebensspuren with multiple origins; and 3) tracemakers that can produce different lebensspuren. Lebensspuren and faunal density were not correlated. However, lebensspuren density was either positively or negatively correlated with tracemaker densities, depending on the lebensspuren morphotypes. A positive correlation was observed for resting lebensspuren (e.g., ophiuroid impressions, Actinaria circular impressions), while negative correlations were observed for locomotion-feeding lebensspuren (e.g., echinoid trails). In conclusion, lebensspuren diversity may be a good proxy for tracemaker biodiversity when the lebensspuren-tracemaker tandem can be reliable characterized; and lebensspuren-density correlations vary depending the specific lebensspuren residence time, tracemaker density and associated behaviour (rate of movement), but on a global scale abiotic and other biotic 42 factors may also play an important role.
In the deep sea, interactions between benthic fauna and seafloor sediment primarily occur through bioturbation that can be preserved as traces (i.e. lebensspuren). Lebensspuren are common features of deep-sea landscapes and are more abundant than the organisms that produce them (i.e. tracemakers), rendering lebensspuren promising proxies for inferring biodiversity. The density and diversity relationships between lebensspuren and benthic fauna remain unclear, and contradicting correlations have been proposed (i.e. negative, positive, or even null correlations). To approach these variable correlations, lebensspuren and benthic fauna were characterized taxonomically at eight deep-sea stations in the Kuril-Kamchatka Trench area, together with two novel categories: tracemakers (specific epibenthic fauna that produce these traces) and degrading fauna (benthic fauna that can erase lebensspuren). No general correlation (overall study area) was observed between diversities of lebensspuren, tracemakers, degrading fauna, and fauna. However, a diversity correlation was observed at specific stations, showing both negative and positive correlations depending on: (1) the number of unknown tracemakers (especially significant for dwelling lebensspuren); (2) the lebensspuren with multiple origins; and (3) tracemakers that can produce different lebensspuren. Lebensspuren and faunal density were not correlated. However, lebensspuren density was either positively or negatively correlated with tracemaker densities, depending on the lebensspuren morphotypes. A positive correlation was observed for resting lebensspuren (e.g. ophiuroid impressions, Actiniaria circular impressions), while negative correlations were observed for locomotion-feeding lebensspuren (e.g. echinoid trails). In conclusion, lebensspuren diversity may be a good proxy for tracemaker biodiversity when the lebensspuren–tracemaker relationship can be reliable characterized. Lebensspuren–density correlations vary depending on the specific lebensspuren residence time, tracemaker density, and associated behaviour (rate of movement). Overall, we suggest that lebensspuren density and diversity correlations should be studied with tracemakers rather than with general benthic fauna. On a global scale, abiotic (e.g. hydrodynamics, substrate consistency) and other biotic factors (e.g. microbial degradation) may also play an important role.
The attention on the protein PURA has increased recently following the discovery of the rare PURA Syndrome. This neurodevelopmental disorder is caused by de novo mutations in the PURA gene. Notably, our collaborators could show that the protein PURA can bind DNA and RNA in vitro. As a result, I was motivated to explore PURA's cellular RNAbinding activity. Furthermore, I inquired on the connection of PURA-RNA binding to the cellular effect of a reduction of functional PURA as present in PURA Syndrome patients.
To investigate the binding of PURA and the impact of PURA de ciency on cellular RNA and protein expression, I performed an integrative computational analysis of multimodal data from complementary high-throughput experiments. An essential component was the examination of UV Crosslinking and immunoprecipitation (CLIP) experiments, which can query the global RNA-binding behaviour of a given protein in a cellular context. As the processing and analysis of CLIP data are rather complex, I introduce an automated command line tool for the processing of CLIP data named racoon_clip as part of this dissertation. Therefore, this dissertation comprises two major segments. Firstly, I describe the implementation and usage of racoon clip for CLIP data analysis. Secondly, I discuss my research on the protein PURA, demonstrating its global RNA-binding properties, the effects of PURA depletion and its association with neuronal functions and P-bodies, among others.
racoon_clip is a command line application that I have developed for processing of individualnucleotide resolution CLIP (iCLIP) and enhanced CLIP (eCLIP) experiments - two of the most commonly used types of CLIP experiments - in a comparable and user-friendly way.
For this, I built racoon_clip as an automated work how that encompasses all CLIP processing steps from raw data to single-nucleotide resolution crosslink events. racoon_clip is available as a command line tool that users can run with a single command. The work how is implemented with Snakemake work how management providing computational advantage tages including parallelisation, scalability and portability of the work how. The main task of racoon_clip is to extract single-nucleotide crosslink events from iCLIP, iCLIP2, eCLIP and similar data types. To strike a balance between being highly customisable and easy to use, racoon_clip supplies pre-set options for the most common types of experiments.
Additionally, it is possible for users to create a custom setup of barcode and adapter architectures, which allows them to use the software for other types of CLIP data. While accounting for the different architectures in the reads, the performed central processing steps remain the same. This leads to a high degree of comparability between the different experiment types, which I demonstrate in the exemplary processing of U2AF2 iCLIP and eCLIP data. Taken together, I am confident that racoon_clip will be beneficial to numerous researchers interested in RNA-Protein interactions as it offers easily accessible processing for CLIP data and enhances the comparability of multiple CLIP datasets across di erent experiment types.
In the second part of this dissertation, I focus on the cellular function of the RNAbinding protein PURA. Through in-depth computational analysis of one iCLIP data set of endogenous PURA and two iCLIP data sets of overexpressed PURA in HeLa cells, I establish that PURA is a global RNA-binding protein. It preferentially binds RNAs in either the coding sequence (CDS) or the 3' untranslated region (3'UTR) of mature protein-coding transcripts by recognising a Purine-rich degenerated sequence motif. Even though overexpression of PURA results in less specific binding behaviour, the same overall binding patterns as from endogenous PURA persist. Overall characteristics of PURA binding remain similar in three distinct PURA iCLIP data sets with and without PURA overexpression.
To learn about the molecular consequences of a depletion of functional PURA in a cellular context, I used a 50% reduction of PURA in HeLa cells as a model for the heterozygous loss of PURA in PURA Syndrome and evaluated its impact on global RNA and protein expression. The results demonstrate that PURA depletion globally a ects RNA and protein expression. Additionally, I integrate PURA RNA binding with the changes in expression of RNAs and proteins in the context of PURA depletion. This reveals 234 targets of PURA that are bound by PURA and are impacted at both RNA and protein levels by the PURA protein. RNAs that are bound by PURA or change in abundance upon PURA depletion are enriched in neuronal development factors, RNA lifecycle regulators, and mitochondrial factors, among others. Consistent with a possible role of PURA in neuronal transport, there is considerable overlap between PURA bound transcripts and transcripts, that are transported to the dendritic end of neurons.
Notably, there is a link between PURA and P-bodies, as documented by the enrichment of PURA-bound RNAs in both the P-body and stress granule transcriptome. Further, PURA was found by our collaborators to be localised within P-bodies and P-body numbers were strongly reduced in cells that are depleted of PURA. This absence might be attributed to the downregulation of the proteins encoded by the PURA targets LSM14A and DDX6 as both of them were previously identified as essential for P-body formation.
Overall, the reduction of P-body numbers in PURA depletion, the neuronal function of PURA, and its association with mitochondria and RNA lifecycle regulation may indicate the cellular foundation of both PURA Syndrome and related neuronal diseases.
In summary, I present a versatile and user-friendly computational tool for the analysis of CLIP data. Subsequently, I conduct a thorough computational analysis of CLIP and other high-throughput data in the context of the RNA-binding protein PURA, which offers valuable insights into the cellular functions of PURA. These insights advance our understanding of the impact of PURA loss in PURA Syndrome and other disease contexts.
Rahm, Johanna Viola ; Balakrishnan, Ashwin ; Wehrheim, Maren ; Kaminer, Alexandra ; Glogger, Marius ; Kessler, Laurell F. ; Kaschube, Matthias ; Barth, Hans-Dieter ; Heilemann, Mike
Stimulated emission depletion (STED) microscopy is a super-resolution technique that surpasses the diffraction limit and has contributed to the study of dynamic processes in living cells. However, high laser intensities induce fluorophore photobleaching and sample phototoxicity, limiting the number of fluorescence images obtainable from a living cell. Here, we address these challenges by using ultra-low irradiation intensities and a neural network for image restoration, enabling extensive imaging of single living cells. The endoplasmic reticulum (ER) was chosen as the target structure due to its dynamic nature over short and long timescales. The reduced irradiation intensity combined with denoising permitted continuous ER dynamics observation in living cells for up to 7 hours with a temporal resolution of seconds. This allowed for quantitative analysis of ER structural features over short (seconds) and long (hours) timescales within the same cell, and enabled fast 3D live-cell STED microscopy. Overall, the combination of ultra-low irradiation with image restoration enables comprehensive analysis of organelle dynamics over extended periods in living cells.
Liu, Ziwen ; Hirata Miyasaki, Eduardo ; Pradeep, Soorya ; Rahm, Johanna Viola ; Foley, Christian ; Chandler, Talon ; Ivanov, Ivan E. ; Woosley, Hunter O. ; Lao, Tiger ; Balasubramanian, Akilandeswari ; Marreiros, Rita ; Liu, Chad ; Leonetti, Manuel D. ; Aviner, Ranen ; Arias, Carolina ; Jacobo, Adrian ; Mehta, Shalin B.
Correlative dynamic imaging of cellular landmarks, such as nuclei and nucleoli, cell membranes, nuclear envelope and lipid droplets is critical for systems cell biology and drug discovery, but challenging to achieve with molecular labels. Virtual staining of label-free images with deep neural networks is an emerging solution for correlative dynamic imaging. Multiplexed imaging of cellular landmarks from scattered light and subsequent demultiplexing with virtual staining leaves the light spectrum for imaging additional molecular reporters, photomanipulation, or other tasks. Current approaches for virtual staining of landmark organelles are fragile in the presence of nuisance variations in imaging, culture conditions, and cell types. We report training protocols for virtual staining of nuclei and membranes robust to variations in imaging parameters, cell states, and cell types. We describe a flexible and scalable convolutional architecture, 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 human cell lines, neuromasts of zebrafish and stem cell (iPSC)-derived neurons, 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 cell membranes. The models rescue missing labels, non-uniform expression of labels, and photobleaching. We share three pre-trained models (VSCyto3D, VSNeuromast, and VSCyto2D) and a PyTorch-based pipeline (VisCy) for training, inference, and deployment that leverages current community standards for image data and metadata.
Snapshots of acetyl-CoA synthesis, the final step of CO₂ fixation in the Wood-Ljungdahl pathway
(2024)
Yin, Max Dongsheng ; Lemaire, Olivier N. ; Rosas-Jiménez, José Guadalupe ; Belhamri, Mélissa ; Shevchenko, Anna ; Hummer, Gerhard ; Wagner, Tristan ; Murphy, Bonnie J.
In the ancient microbial Wood-Ljungdahl pathway, CO2 is fixed in a multi-step process with acetyl-CoA synthesis at the bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS). Here, we present catalytic snapshots of the CODH/ACS from the gas-converting acetogen Clostridium autoethanogenum, characterizing the molecular choreography of the overall reaction including electron transfer to the CODH for CO2 reduction, methyl transfer from the corrinoid iron-sulfur protein (CoFeSP) partner to the ACS active site and acetyl-CoA production. Unlike CODH, the multidomain ACS undergoes large conformational changes to form an internal connection to the CODH active site, accommodate the CoFeSP for methyl transfer and protect the reaction intermediates. Altogether, the structures allow us to draw a detailed reaction mechanism of this enzyme crucial for CO2 fixation in anaerobic organisms.
Members of the arginine–serine-rich protein family (SR proteins) are multifunctional RNA-binding proteins that have emerged as key determinants for mRNP formation, identity and fate. They bind to pre-mRNAs early during transcription in the nucleus and accompany bound transcripts until they are translated or degraded in the cytoplasm. SR proteins are mostly known for their essential roles in constitutive splicing and as regulators of alternative splicing. However, many additional activities of individual SR proteins, beyond splicing, have been reported in recent years. We will summarize the different functions of SR proteins and discuss how multifunctionality can be achieved. We will also highlight the difficulties of studying highly versatile SR proteins and propose approaches to disentangle their activities, which is transferrable to other multifunctional RBPs.
Levin, Roman ; Löhr, Frank ; Karakoc, Betül ; Lichtenecker, Roman ; Dötsch, Volker ; Bernhard, Frank
Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of 15N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative “Stablelabel” containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in “Stablelabel” lysates. By taking advantage of ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.
Molecular concepts for pandemic viruses : membrane fusion assays and targeting of reservoir cells
(2024)
In den letzten Jahren haben verschiedene pandemische Viren zu beträchtlichen Krankheits- und Todesfällen geführt. Um dieser ständigen Bedrohung entgegenzuwirken, ist es wichtig diagnostische Testsysteme und Therapien anzupassen oder neu zu etablieren. Diese Arbeit konzentriert sich auf die pandemischen Viren SARS-CoV-2 und HIV.
Der Zelleintritt von SARS-CoV-2 wird durch das Spike-Protein (S) ausgelöst, welches die Fusion der Virushülle mit der zellulären Membran bewirkt. Erste Studien haben gezeigt, dass das S-Protein eine hohe Fusionsaktivität aufweist. Aus diesem Grund sollten in dieser Arbeit neue Fusionstests etabliert werden, um potenzielle Inhibitoren der Zellfusion zu evaluieren. Im ersten Teil dieser Thesis wird die Etablierung von quantitativen Tests zur Evaluierung der Zell-Zell und Partikel-Zell Fusionsaktivität, welche durch S bewirkt wird, demonstriert.
Trotz jahrelanger Forschung können HIV-Patienten nicht geheilt werden und Virusinfektionen treten weiterhin weltweit auf. Das größte Problem bei der Entwicklung eines Heilmittels ist die frühe Bildung von Reservoirzellen während einer Infektion. Um diese Reservoirzellen zu identifizieren, wurde der Oberflächenmarker CD32a vorgeschlagen. Die Nutzung von Cas9-Nukleasen zur Inaktivierung von HIV ist in vitro erfolgreich, aber der effiziente Transfer in Reservoirzellen bleibt weiterhin herausfordernd. Im zweiten Teil dieser Thesis werden Rezeptor-gerichtete Adeno-assoziierte Vektoren (AAVs) für die HIV-Gentherapie präsentiert, die CD4 und CD32a für den Zelleintritt nutzen.
Zur Charakterisierung der Fusionsaktivität von SARS-CoV-2 wurden drei quantitative Fusionstests etabliert, welche Partikel- und Zell-Zell Fusionen berücksichtigen. Für den Partikel-Zell Fusionstest wurden lentivirale Vektoren (LV) verwendet, welche das S-Protein auf ihrer Oberfläche präsentierten. Die Transduktionseffizienz von S-LV erreichte auf Zellen, die den SARS-CoV-2 Rezeptor ACE2 exprimieren, ein Signal-zu-Hintergrund Verhältnis von über 2000. Durch die Präsentation von S auf leeren LV-Partikeln konnte die Fusion von benachbarten Zellen detektiert und quantifiziert werden („fusion-from-without“ (FFWO)). Für die Quantifizierung wurde ein Reporter-Komplementationstest etabliert. Hierbei wurden die Alpha- und Omega-Fragmente der β-Galaktosidase getrennt in zwei Zielzellpopulationen exprimiert, die beide ACE2 exprimierten. Durch die Zugabe von S-Partikeln kam es zur Fusion der Zielzellen und zur Komplementation der Alpha- und Omega-Fragmente. Die resultierende β-Galaktosidase-Aktivität konnte anschließend quantifiziert werden. Unter optimalen Versuchsbedingungen erreichte dieser Assay ein Signal-zu-Hintergrund Verhältnis von 2,7 Größenordnungen. Anschließend wurde der Komplementationstest für die Messung der Zell-Zell Fusion verwendet. In diesem Test exprimierten Effektorzellen S und das Alpha-Fragment, Zielzellen ACE2 und das Omega-Fragment. Obwohl die S-Expression auf den Effektorzellen sehr gering war, konnte dennoch eine signifikante Fusion nachgewiesen werden. Auch hier konnte unter optimalen Versuchsbedingungen ein hohes Signal-zu-Hintergrund Verhältnis von 2,9 Größenordnungen festgestellt werden. Nach der Etablierung der Testsysteme wurden S-spezifische Inhibitoren verwendet. Im Gegensatz zu Partikel-Zell-Fusionen wurde die Fusionsaktivität von S auf Zellen nur mäßig inhibiert. Dies deutet daraufhin, dass das Eindringen von Partikeln in Zellen wirksamer verhindert werden kann als die Ausbreitung durch Zell-Zell Fusionen.
Um AAVs spezifisch an HIV-Reservoirzellen zu binden, wurden CD4- und CD32a-spezifische DARPins („designed ankyrin repeat proteins“) in Rezeptor-verblindete AAVs eingebaut. Ebenso wurden beide DARPins gleichzeitig auf dem Kapsid präsentiert, um eine höhere Spezifität für doppelt-positive Zellen zu erreichen. Wenn diese Partikel einer Zellmischung aus CD4-, CD32a- und CD4/CD32a-exprimierenden Zellen zugesetzt wurden, transduzierten die bispezifischen Vektoren vorzugsweise doppelt-positive Zellen. Diese Präferenz war am höchsten in Zellkulturen, die stark unterrepräsentierte CD4/CD32a-exprimierende Zellen enthielten. Unter diesen Voraussetzungen erreichten bispezifische Vektoren eine bis zu 66-fach höhere Transduktionseffizienz auf CD4/CD32a-positive Zellen im Vergleich zu CD32a-exprimierenden Zellen. Darüber hinaus zeigten bispezifische AAV eine präferentielle Bindung und Transduktion von isolierten Primärzellen und Zellen in Vollblut. Selbst nach systemischer Injektion in humanisierte Mäuse wurden doppelt-positive Zellen effizienter von bispezifischen als von monospezifischen AAVs transduziert. Schließlich zeigten die generierten Vektoren, welche die Cas9 Nuklease transferierten, eine effiziente Inhibition der HIV-Replikation.
In almost all parts of the world the industrialisation grows continuously and thus, the chemical pollution of natural waters has become a major public concern. A major consequence and one of the key environmental problems we are facing today is the increasing contamination of freshwater systems with chemicals. The chemicals are detected in wastewater, surface (river) water, ground water and drinking water ubiquitously in natural waters and not only in industrialised areas. The main point sources for water pollution and the release of these synthetic organic substances of human origin, so called micropollutants (MPs), are wastewater treatment plants (WWTPs). These MPs such as pharmaceuticals, personal care products, disinfectant chemicals, chemicals used in the industry and in households, contraceptives, hormones, food additives, artificial sweeteners, pesticides, biocides, and many emerging contaminants are only incompletely removed by the existing conventional wastewater treatment technologies. The MPs end up in the water cycle and have adverse effects on wildlife aquatic ecosystems and human health even at very low concentrations. Therefore, advanced wastewater treatment (AWWT) technologies, such as ozonation, treatment with activated carbon, biofiltration, membrane bioreactors (MBRs) or exposure to ultraviolet light are investigated as options to upgrade conventional WWTPs. However, several studies show that especially the ozonation of wastewater generates diverse transformation products (TPs) with unknown properties. These TPs could be more toxic than the mother compound. Thus, a post-treatment after the ozonation process is required.
The present thesis was part of the BMBF-funded TransRisk project dealing with “the characterisation, communication, and minimisation of risks of emerging pollutants and pathogens in the water cycle”. One main objective was the investigation of conventional treated wastewater after a full-scale ozonation with four post-treatments (each non-aerated and aerated granular activated carbon (GAC) filtration and biofiltration) in comparison to a MBR treatment of raw (untreated) wastewater separately and in combination with an additional ozonation on a pilot WWTP. For this purpose, the wastewater samples were characterised with a comprehensive battery of in vitro and in vivo bioassays. The in vitro bioassays were performed to detect endocrine activities (such as (anti)estrogenic and (anti)androgenic activities), genotoxicity, and mutagenicity. The results showed a decreased estrogenic activity due to the conventional wastewater treatment as well as the ozonation, but a distinct increase of the anti-estrogenic activity and the mutagenicity in the ozonated wastewater, possibly caused by new formed TPs, that were reduced after the post-treatments whereas the GAC filtration performed better than the biofiltration. The in vivo bioassays included for example the impact of the wastewater on mortality, reproduction, development, and energy reserves of the test organisms. The in vivo on-site tests with the mudsnail Potamopyrgus antipodarum and with the amphipod Gammarus fossarum indicated a major impact of conventional treated wastewater, ozonated wastewater, and MBR treated wastewater. The flow channel experiments in the laboratory with Gammarus pulex pointed to a serious impact of an estrogenic effluent on life-history traits of the amphipod. Finally, an ozonation of the wastewater with subsequent GAC filtration represented the most promising option. In addition, chemical analyses of 40 selected MPs, so called tracer substances, performed in parallel to the in vitro and in vivo bioassays underlined this assumption.
A second main objective was the optimisation of the preparation of water and wastewater samples for ecotoxicological in vitro bioassays because common sample preparation techniques are predominantly adapted for chemical analyses. Therefore, the impact of sample filtration, long-term acidification with following neutralisation as well as the enrichment with solid phase extraction (SPE) in combination with short-term acidification were investigated using amongst others raw (untreated) wastewater, hospital wastewater, conventional treated and ozonated wastewater, surface water, and ground water. Overall, eleven in vitro bioassays were performed for the detection of endocrine activities, genotoxicity, and mutagenicity. The results show that sample filtration and acidification/neutralisation significantly affected the outcome of the bioassays especially the anti-estrogenic activity and the mutagenicity whereas the sample filtration had a minor impact than the acidification. Thus, the testing of untreated (waste)water samples is advisable because the sample is minimally processed. Furthermore, the SPE extracts showed in parts high cytotoxic effects whereby no conclusions on the results of the bioassays were possible. However, the enrichment of endocrine activity and mutagenicity was predominantly effective but depended on the used SPE cartridge and the pH value of the (waste)water samples. Based on the results the use of a Telos C18/ENV cartridge and an acidified sample is recommendable. In the end, there is a need to optimise the sample preparation for in vitro bioassays to reach their maximum outcome for the best possible assessment of the water quality.
The archaeal ATP synthase is a multisubunit complex that consists of a catalytic A(1) part and a transmembrane, ion translocation domain A(0). The A(1)A(0) complex from the hyperthermophile Pyrococcus furiosus was isolated. Mass analysis of the complex by laser-induced liquid bead ion desorption (LILBID) indicated a size of 730 +/- 10 kDa. A three-dimensional map was generated by electron microscopy from negatively stained images. The map at a resolution of 2.3 nm shows the A(1) and A(0) domain, connected by a central stalk and two peripheral stalks, one of which is connected to A(0), and both connected to A(1) via prominent knobs. X-ray structures of subunits from related proteins were fitted to the map. On the basis of the fitting and the LILBID analysis, a structural model is presented with the stoichiometry A(3)B(3)CDE(2)FH(2)ac(10).
One like all? Behavioral response range of native and invasive amphipods to neonicotinoid exposure
(2024)
Soose, Laura J. ; Rex, Tobias ; Oehlmann, Jörg ; Schiwy, Andreas Herbert ; Krauss, Martin ; Brack, Werner ; Klimpel, Sven ; Hollert, Henner ; Jourdan, Jonas
Highlights
• Short-time neonicotinoid exposure causes behavioral responses in non-target species.
• Environmentally relevant concentrations can induce changes in invertebrate behavior.
• Different baseline activity of ecological similar crustacean amphipods.
• Species respond specifically to thiacloprid exposure.
• Acantocephalan infection affects locomotion of intermediate host Gammarus roeselii.
Abstract
Native and invasive species often occupy similar ecological niches and environments where they face comparable risks from chemical exposure. Sometimes, invasive species are phylogenetically related to native species, e.g. they may come from the same family and have potentially similar sensitivities to environmental stressors due to phylogenetic conservatism and ecological similarity. However, empirical studies that aim to understand the nuanced impacts of chemicals on the full range of closely related species are rare, yet they would help to comprehend patterns of current biodiversity loss and species turnover. Behavioral sublethal endpoints are of increasing ecotoxicological interest. Therefore, we investigated behavioral responses (i.e., change in movement behavior) of the four dominant amphipod species in the Rhine-Main area (central Germany) when exposed to the neonicotinoid thiacloprid. Moreover, beyond species-specific behavioral responses, ecological interactions (e.g. parasitation with Acanthocephala) play a crucial role in shaping behavior, and we have considered these infections in our analysis. Our findings revealed distinct baseline behaviors and species-specific responses to thiacloprid exposure. Notably, Gammarus fossarum exhibited biphasic behavioral changes with hyperactivity at low concentrations that decreased at higher concentrations. Whereas Gammarus pulex, Gammarus roeselii and the invasive species Dikerogammarus villosus, showed no or weaker behavioral responses. This may partly explain why G. fossarum disappears in chemically polluted regions while the other species persist there to a certain degree. But it also shows that potential pre-exposure in the habitat may influence behavioral responses of the other amphipod species, because habituation occurs, and potential hyperactivity would be harmful to individuals in the habitat. The observed responses were further influenced by acanthocephalan parasites, which altered baseline behavior in G. roeselii and enhanced the behavioral response to thiacloprid exposure. Our results underscore the intricate and diverse nature of responses among closely related amphipod species, highlighting their unique vulnerabilities in anthropogenically impacted freshwater ecosystems.
Highlights
• The higher the extinction risk, the fewer exposure-effect data are available.
• Lack of studies in the Southern Hemisphere shows a spatial bias in the literature.
• Commonly studied pollutants are persistent organic pollutants, metals, pesticides.
• Pollution-effect studies focus on molecular and cellular levels.
• In silico and in vitro approaches aid in assessing in vivo effects.
Abstract
Marine mammals, due to their long life span, key position in the food web, and large lipid deposits, often face significant health risks from accumulating contaminants. This systematic review examines published literature on pollutant-induced adverse health effects in the International Union for Conservation of Nature (IUCN) red-listed marine mammal species. Thereby, identifying gaps in literature across different extinction risk categories, spatial distribution and climatic zones of studied habitats, commonly used methodologies, researched pollutants, and mechanisms from cellular to population levels. Our findings reveal a lower availability of exposure-effect data for higher extinction risk species (critically endangered 16%, endangered 15%, vulnerable 66%), highlighting the need for more research. For many threatened species in the Southern Hemisphere pollutant-effect relationships are not established. Non-destructively sampled tissues, like blood or skin, are commonly measured for exposure assessment. The most studied pollutants are POPs (31%), metals (30%), and pesticides (17%). Research on mixture toxicity is scarce while pollution-effect studies primarily focus on molecular and cellular levels. Bridging the gap between molecular data and higher-level effects is crucial, with computational approaches offering a high potential through in vitro to in vivo extrapolation using (toxico-)kinetic modelling. This could aid in population-level risk assessment for threatened marine mammals.
Beside mosquitoes, ticks are well-known vectors of different human pathogens. In the Northern Hemisphere, Lyme borreliosis (Eurasia, LB) or Lyme disease (North America, LD) is the most commonly occurring vector-borne infectious disease caused by bacteria of the genus Borrelia which are transmitted by hard ticks of the genus Ixodes. The reported incidence of LB in Europe is about 22.6 cases per 100,000 inhabitants annually with a broad range depending on the geographical area analyzed. However, the epidemiological data are largely incomplete, because LB is not notifiable in all European countries. Furthermore, not only differ reporting procedures between countries, there is also variation in case definitions and diagnostic procedures. Lyme borreliosis is caused by several species of the Borrelia (B.) burgdorferi sensu lato (s.l.) complex which are maintained in complex networks including ixodid ticks and different reservoir hosts. Vector and host influence each other and are affected by multiple factors including climate that have a major impact on their habitats and ecology. To classify factors that influence the risk of transmission of B. burgdorferi s.l. to their different vertebrate hosts as well as to humans, we briefly summarize the current knowledge about the pathogens including their astonishing ability to overcome various host immune responses, regarding the main vector in Europe Ixodes ricinus, and the disease caused by borreliae. The research shows, that a higher standardization of case definition, diagnostic procedures, and standardized, long-term surveillance systems across Europe is necessary to improve clinical and epidemiological data.
Autophagy is an important degradation pathway mediating the engulfment of cellular material (cargo) into autophagosomes followed by degradation in autophagosomes.
Different stress stimuli, e.g. nutrient deprivation, oxidative stress or organelle damage, engage autophagy to maintain cellular homeostasis, recycle nutrients or remove damaged cell organelles. Autophagy not only degrades bulk cytoplasmic material but also selective autophagic cargo, for example lysosomes (lysophagy), mitochondria (mitophagy), ER (ER-phagy), lipid droplets (lipophagy), protein aggregates (aggrephagy) or pathogens (xenophagy). Selective autophagy pathways are regulated by selective autophagy receptors which bind to ubiquitinated cargo proteins and link them to LC3 on the autophagosomal membrane.
Ubiquitination is an essential post-translational modification controlling different cellular processes such as proteasomal and lysosomal degradation or innate immune signaling.
M1-linked (linear) poly-Ubiquitin (poly-Ub) chains are exclusively assembled by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and removed by the M1 poly-Ub-specific OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN). In addition to key functions in innate immune signaling and nuclear factor-κB (NF-κB) activation, M1 ubiquitination is also implicated in the regulation of autophagy.
LUBAC and OTULIN control autophagy initiation and maturation and the autophagic clearance of invading bacteria via xenophagy. However, additional functions of LUBAC- and OTULIN-regulated M1 ubiquitination in autophagy are largely unknown and it also remains unexplored if LUBAC and OTULIN control other selective autophagy pathways in addition to xenophagy. This study aimed to unravel the role of LUBAC- and OTULIN-controlled M1 ubiquitination in bulk and selective autophagy in more detail.
In this study, characterization of OTULIN-depleted MZ-54 glioblastoma (GBM) cells revealed that OTULIN deficiency results in enhanced LC3 lipidation in response to autophagy induction and upon blockade of late stage autophagy with Bafilomycin A1 (BafA1). Furthermore, electron microscopy analysis showed that OTULIN-deficient cells have an increased number of degradative compartments (DGCs), confirming enhanced autophagy activity upon loss of OTULIN. APEX2-based autophagosome content profiling identified various OTULIN-dependent autophagy cargo proteins. Among these were the autophagy receptor TAX1BP1 which regulates different forms of selective autophagy (e.g. lysophagy, aggrephagy) and the glycan-binding protein galectin-3 which serves key functions in lysophagy, suggesting a role of OTULIN and M1 poly-Ub in the regulation of aggrephagy and lysophagy.
Abstract 2
To study aggrephagy, protein aggregation was induced with puromycin which causes premature termination of translation and accumulation of defective ribosomal products (DRiPs). Loss of OTULIN increased the number of M1 poly-Ub-positive foci and insoluble proteins and reduced the levels of soluble TAX1BP1 and p62 in response to puromycin-induced proteotoxic stress.
Intriguingly, upon induction of lysosomal membrane permeabilization (LMP) with the lysosomotropic drug L-Leucyl-L-Leucine methyl ester (LLOMe), M1 poly-Ub strongly accumulated at damaged lysosomes and colocalized with TAX1BP1- and galectin-3-positive puncta. M1 poly-Ub-modified lysosomes formed a platform for NF-κB essential modulator (NEMO) and inhibitor of κB (IκB) kinase (IKK) complex recruitment and local NF-κB activation in a K63 poly-Ub- and OTULIN-dependent manner. Furthermore, inhibition of lysosomal degradation enhanced LLOMe-induced cell death, suggesting pro-survival functions of lysophagy following LMP. Enrichment of M1 poly-Ub at damaged lysosomes was also observed in human dopaminergic neurons and in primary mouse embryonic cortical neurons, confirming the importance of M1 poly-Ub in the response to lysosomal damage.
Together, these results identify OTULIN as a negative regulator of autophagy induction and the autophagic flux and reveal OTULIN-dependent autophagy cargo proteins.
Furthermore, this study uncovers novel and important roles of M1 poly-Ub in the response to lysosomal damage and local NF-κB activation at damaged lysosomes.
Muller's ratchet, in its prototype version, models a haploid, asexual population whose size~N is constant over the generations. Slightly deleterious mutations are acquired along the lineages at a constant rate, and individuals carrying less mutations have a selective advantage. The classical variant considers {\it fitness proportional} selection, but other fitness schemes are conceivable as well. Inspired by the work of Etheridge et al. ([EPW09]) we propose a parameter scaling which fits well to the ``near-critical'' regime that was in the focus of [EPW09] (and in which the mutation-selection ratio diverges logarithmically as N→∞). Using a Moran model, we investigate the``rule of thumb'' given in [EPW09] for the click rate of the ``classical ratchet'' by putting it into the context of new results on the long-time evolution of the size of the best class of the ratchet with (binary) tournament selection, which (other than that of the classical ratchet) follows an autonomous dynamics up to the time of its extinction. In [GSW23] it was discovered that the tournament ratchet has a hierarchy of dual processes which can be constructed on top of an Ancestral Selection graph with a Poisson decoration. For a regime in which the mutation/selection-ratio remains bounded away from 1, this was used in [GSW23] to reveal the asymptotics of the click rates as well as that of the type frequency profile between clicks. We will describe how these ideas can be extended to the near-critical regime in which the mutation-selection ratio of the tournament ratchet converges to 1 as N→∞.
Inorganic phosphate is one of the most abundant and essential nutrients in living organisms. It plays an indispensable role in energy metabolism and serves as a building block for major cellular components such as the backbones of DNA and RNA, headgroups of phospholipids and in posttranslational modifcations of many proteins. Disturbances in cellular phosphate homeostasis have a detrimental effect on the viability of cells. There- fore, both the import and export of phosphate is strictly regulated in eukaryotic cells. In the eukaryotic model organism Saccharomyces cerevisiae, the uptake of phosphate is carried out either by transporters with high affinity or by transporters with low affinity, depending on the cytosolic phosphate concentration. While structures are available for homologues of the high-affinity transporters, no structures of low-affinity transporters have been solved so far. Interestingly, only the low-affinity transporters have a regulatory SPX domain, which is found in various proteins involved in phosphate homeostasis.
In this work, structures of Pho90 from Saccharomyces cerevisiae, a low-affinity phosphate transporter, were solved by cryo-EM, providing insights into its transport mechanism. The dimeric structure resembles the structures of proteins of the divalent anion symporter superfamily (DASS) and of mammalian transporters of the solute carrier 13 (SLC13) family. The transmembrane domain of each protomer consists of 13 helical elements and can be subdivided into scaffold and transport domains. The structure of ScPho90 in the presence of phosphate shows the phosphate binding site within the transporter domain in an outward-open conformation with a bound phosphate ion and two sodium ions. In the absence of phosphate, an asymmetric dimer structure was determined, with one protomer adopting an inward-open conformation. While the dimer contact and the scaffold domain are identical in both conformations, the transport domain is rotated by about 30° and shifted by 11 Å towards the cytoplasmic side, leading to the accessibility of the binding pocket from the cytoplasm. Based on these findings and by comparison with known structures, a phosphate transport mechanism is proposed in the present work that involves substrate binding on the extracellular side, conformational change by a rigid-body motion of the transport domain, in an "elevator-like" motion, and substrate release into the cytoplasm. The regulatory SPX domain is not well resolved in the ScPho90 structures, so that no direct conclusions were drawn about its regulatory mechanism. The findings provide new insights into the function and mechanism of eukaryotic low-affinity phosphate transporters.
While eukaryotic cells express various phosphate import proteins, most eukaryotes have only a single highly conserved and essential phosphate exporter. These exporters show no sequence homology to other transporters of known structure, but also possess a regulatory SPX domain. In this work, the structural basis for eukaryotic phosphate export is investigated by elucidating the structures of the homologous phosphate exporters Syg1 from Saccharomyces cerevisiae and Xpr1 from Homo sapiens, using cryo-EM. The structures of ScSyg1 and HsXpr1 show a conserved homodimeric structure and the transmembrane part of each protomer consists of 10 TM helices. Helix TM1 establishes the dimer contact by means of a glycine zipper motif, which is a known oligomerization motif. Helices TM2-5 form a hydrophobic pocket that has density for a lipid molecule. Whether the lipid binding into the hydrophobic pocket has an allosteric effect on the phosphate export activity or only serves protein stabilization is not known. Helices TM5-10 form a six-helix bundle, which constitutes a putative phosphate translocation pathway in its center. This bundle is formed by the protein sequence annotated as EXS domain.
The respective phosphate translocation pathways of ScSyg1 and HsXpr1 show structural differences. While the translocation pathway in HsXpr1 is accessible from the cytoplasm, in ScSyg1 it is closed by a large loop of the SPX domain. Interestingly, this loop is not conserved in higher eukaryotes and is therefore not present in HsXpr1. Another difference are distinct conformations of helix TM9. In ScSyg1, TM9 adopts a kinked conformation, which results in the translocation pathway being open to the extracellular side. In contrast, TM9 adopts a straight conformation in HsXpr1, resulting in the placement of a highly conserved tryptophane residue in the middle of the translocation pathway. As a result, the translocation pathway in HsXpr1 is closed to the extracellular side.
Graph4Med: a web application and a graph database for visualizing and analyzing medical databases
(2022)
Background: Medical databases normally contain large amounts of data in a variety of forms. Although they grant significant insights into diagnosis and treatment, implementing data exploration into current medical databases is challenging since these are often based on a relational schema and cannot be used to easily extract information for cohort analysis and visualization. As a consequence, valuable information regarding cohort distribution or patient similarity may be missed. With the rapid advancement of biomedical technologies, new forms of data from methods such as Next Generation Sequencing (NGS) or chromosome microarray (array CGH) are constantly being generated; hence it can be expected that the amount and complexity of medical data will rise and bring relational database systems to a limit.
Description: We present Graph4Med, a web application that relies on a graph database obtained by transforming a relational database. Graph4Med provides a straightforward visualization and analysis of a selected patient cohort. Our use case is a database of pediatric Acute Lymphoblastic Leukemia (ALL). Along routine patients’ health records it also contains results of latest technologies such as NGS data. We developed a suitable graph data schema to convert the relational data into a graph data structure and store it in Neo4j. We used NeoDash to build a dashboard for querying and displaying patients’ cohort analysis. This way our tool (1) quickly displays the overview of patients’ cohort information such as distributions of gender, age, mutations (fusions), diagnosis; (2) provides mutation (fusion) based similarity search and display in a maneuverable graph; (3) generates an interactive graph of any selected patient and facilitates the identification of interesting patterns among patients.
Conclusion: We demonstrate the feasibility and advantages of a graph database for storing and querying medical databases. Our dashboard allows a fast and interactive analysis and visualization of complex medical data. It is especially useful for patients similarity search based on mutations (fusions), of which vast amounts of data have been generated by NGS in recent years. It can discover relationships and patterns in patients cohorts that are normally hard to grasp. Expanding Graph4Med to more medical databases will bring novel insights into diagnostic and research.
Climate forecasts show that in many regions the temporal distribution of precipitation events will become less predictable. Root traits may play key roles in dealing with changes in precipitation predictability, but their functional plastic responses, including transgenerational processes, are scarcely known. We investigated root trait plasticity of Papaver rhoeas with respect to higher versus lower intra-seasonal and inter-seasonal precipitation predictability (i.e., the degree of temporal autocorrelation among precipitation events) during a four-year outdoor multi-generation experiment. We first tested how the simulated predictability regimes affected intra-generational plasticity of root traits and allocation strategies of the ancestors, and investigated the selective forces acting on them. Second, we exposed three descendant generations to the same predictability regime experienced by their mothers or to a different one. We then investigated whether high inter-generational predictability causes root trait differentiation, whether transgenerational root plasticity existed and whether it was affected by the different predictability treatments. We found that the number of secondary roots, root biomass and root allocation strategies of ancestors were affected by changes in precipitation predictability, in line with intra-generational plasticity. Lower predictability induced a root response, possibly reflecting a fast-acquisitive strategy that increases water absorbance from shallow soil layers. Ancestors’ root traits were generally under selection, and the predictability treatments did neither affect the strength nor the direction of selection. Transgenerational effects were detected in root biomass and root weight ratio (RWR). In presence of lower predictability, descendants significantly reduced RWR compared to ancestors, leading to an increase in performance. This points to a change in root allocation in order to maintain or increase the descendants’ fitness. Moreover, transgenerational plasticity existed in maximum rooting depth and root biomass, and the less predictable treatment promoted the lowest coefficient of variation among descendants’ treatments in five out of six root traits. This shows that the level of maternal predictability determines the variation in the descendants’ responses, and suggests that lower phenotypic plasticity evolves in less predictable environments. Overall, our findings show that roots are functional plastic traits that rapidly respond to differences in precipitation predictability, and that the plasticity and adaptation of root traits may crucially determine how climate change will affect plants.
Johnson, Alex G. ; Mayer, Megan L. ; Schaefer, Stefan L. ; McNamara-Bordewick, Nora K. ; Hummer, Gerhard ; Kranzusch, Philip J.
In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1–3. Studies of human and mouse GSDM pores reveal the functions and architectures of 24–33 protomers assemblies4–9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing >50 protomers. We determine a 3.3 Å cryo-EM structure of a Vitiosangium bGSDM in an active slinky-like oligomeric conformation and analyze bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, we define a stepwise model of GSDM pore assembly and demonstrate that pore formation is driven by local unfolding of membrane-spanning β-strand regions and pre-insertion of a covalently bound palmitoyl into the target membrane. These results yield insights into the diversity of GSDM pores found in nature and the function of an ancient post-translational modification in enabling a programmed host cell death process.
Johnson, Alex G. ; Mayer, Megan L. ; Schaefer, Stefan L. ; McNamara-Bordewick, Nora K. ; Hummer, Gerhard ; Kranzusch, Philip J.
In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1–3. Studies of human and mouse GSDM pores reveal the functions and architectures of 24–33 protomers assemblies4–9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing >50 protomers. We determine a 3.3 Å cryo-EM structure of a Vitiosangium bGSDM in an active slinky-like oligomeric conformation and analyze bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning β-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.
Linker, Stephanie M. ; Urban, Lara ; Clark, Stephen ; Chhatriwala, Mariya ; Amatya, Shradha ; McCarthy, Davis J. ; Ebersberger, Ingo ; Vallier, Ludovic ; Reik, Wolf ; Stegle, Oliver ; Bonder, Marc Jan
Background: Alternative splicing is a key mechanism in eukaryotic cells to increase the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied both across human tissues and in genetically diverse individuals. This has identified disease-relevant splicing events, as well as associations between splicing and genomic variations, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue and its determinants remain poorly understood.
Results: We applied parallel DNA methylation and transcriptome sequencing to differentiating human induced pluripotent stem cells to characterize splicing variation (exon skipping) and its determinants. Our results shows that splicing rates in single cells can be accurately predicted based on sequence composition and other genomic features. We also identified a moderate but significant contribution from DNA methylation to splicing variation across cells. By combining sequence information and DNA methylation, we derived an accurate model (AUC=0.85) for predicting different splicing modes of individual cassette exons. These explain conventional inclusion and exclusion patterns, but also more subtle modes of cell-to-cell variation in splicing. Finally, we identified and characterized associations between DNA methylation and splicing changes during cell differentiation.
Conclusions: Our study yields new insights into alternative splicing at the single-cell level and reveals a previously underappreciated component of DNA methylation variation on splicing.
Linker, Stephanie M. ; Urban, Lara ; Clark, Stephen ; Chhatriwala, Mariya ; Amatya, Shradha ; McCarthy, Davis J. ; Ebersberger, Ingo ; Vallier, Ludovic ; Reik, Wolf ; Stegle, Oliver ; Bonder, Marc Jan
Background: Alternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic variations, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remain poorly understood.
Results: We applied parallel DNA methylation and transcriptome sequencing to differentiating human induced pluripotent stem cells to characterize splicing variation (exon skipping) and its determinants. Our results shows that variation in single-cell splicing can be accurately predicted based on local sequence composition and genomic features. We observe moderate but consistent contributions from local DNA methylation profiles to splicing variation across cells. A combined model that is built based on sequence as well as DNA methylation information accurately predicts different splicing modes of individual cassette exons (AUC=0.85). These categories include the conventional inclusion and exclusion patterns, but also more subtle modes of cell-to-cell variation in splicing. Finally, we identified and characterized associations between DNA methylation and splicing changes during cell differentiation.
Conclusions: Our study yields new insights into alternative splicing at the single-cell level and reveals a previously underappreciated link between DNA methylation variation and splicing.
Linker, Stephanie M. ; Urban, Lara ; Clark, Stephen ; Chhatriwala, Mariya ; Amatya, Shradha ; McCarthy, Davis J. ; Ebersberger, Ingo ; Vallier, Ludovic ; Reik, Wolf ; Stegle, Oliver ; Bonder, Marc Jan
Background: Alternative splicing is a key regulatory mechanism in eukaryotic cells and increases the effective number of functionally distinct gene products. Using bulk RNA sequencing, splicing variation has been studied across human tissues and in genetically diverse populations. This has identified disease-relevant splicing events, as well as associations between splicing and genomic features, including sequence composition and conservation. However, variability in splicing between single cells from the same tissue or cell type and its determinants remains poorly understood.
Results: We applied parallel DNA methylation and transcriptome sequencing to differentiating human induced pluripotent stem cells to characterize splicing variation (exon skipping) and its determinants. Our results show that variation in single-cell splicing can be accurately predicted based on local sequence composition and genomic features. We observe moderate but consistent contributions from local DNA methylation profiles to splicing variation across cells. A combined model that is built based on genomic features as well as DNA methylation information accurately predicts different splicing modes of individual cassette exons. These categories include the conventional inclusion and exclusion patterns, but also more subtle modes of cell-to-cell variation in splicing. Finally, we identified and characterized associations between DNA methylation and splicing changes during cell differentiation.
Conclusions: Our study yields new insights into alternative splicing at the single-cell level and reveals a previously underappreciated link between DNA methylation variation and splicing.
ABC transporters are found in all organisms and almost every cellular compartment. They mediate the transport of various solutes across membranes, energized by ATP binding and hydrolysis. Dysfunctions can result in severe diseases, such as cystic fibrosis or antibiotic resistance. In type IV ABC transporters, each of the two nucleotide-binding domains is connected to a transmembrane domain by two coupling helices, which are part of cytosolic loops. Although there are many structural snapshots of different conformations, the interdomain communication is still enigmatic. Therefore, we analyzed the function of three conserved, charged residues in the intra-cytosolic loop 1 of the human homodimeric, lysosomal peptide transporter TAPL. Substitution of D278 in coupling helix 1 by alanine interrupted peptide transport by impeding ATP hydrolysis. Alanine substitution of R288 and D292, both localized next to the coupling helix 1 extending to transmembrane helix 3, reduced peptide transport but increased basal ATPase activity. Surprisingly, the ATPase activity of the R288A variant dropped in a peptide-dependent manner while ATPase activity of wildtype and D292A was unaffected. Interestingly, R288A and D292A mutants did not differentiate between ATP and GTP in respect of hydrolysis. However, in contrast to wildtype TAPL, only ATP energized peptide transport. In sum, D278 seems to be involved in bidirectional interdomain communication mediated by network of polar interactions while the two residues in the cytosolic extension of TMH3 are involved in regulation of ATP hydrolysis, most likely by stabilization of the outward facing conformation.
Exploring strategies to improve the reverse beta-oxidation pathway in Saccharomyces cerevisiae
(2024)
Microbes are the most diverse living organisms on Earth, with various metabolic adaptations that allow them to live in different conditions and produce compounds with different chemical complexity. Microbial biotechnology exploits the metabolic diversity of microorganisms to manufacture products for different industries. Today, the chemical industry is a significant energy consumer and carbon dioxide emitter, with processes that harm natural ecosystems, like the extraction of medium-chain fatty acids (MCFAs). MCFAs are used as precursors for biofuels, volatile esters, surfactants, or polymers in materials with enhanced properties.
However, their current extraction process uses large, non-sustainable monocultures of coconut and palm trees. Therefore, the microbial production of MCFAs can help reduce the current environmental impact of obtaining these products and their derivatives.
In nature, fatty acids are mostly produced via fatty acid biosynthesis (FAB). However, the reverse β-oxidation (rBOX) is a more energy-efficient pathway compared to FAB. The rBOX pathway consists of four reactions, which result in the elongation of an acyl-CoA molecule by two carbon units from acetyl-CoA in each cycle. In this work we used Saccharomyces cerevisiae, an organism with a high tolerance towards toxic compounds, as the expression host of the rBOX pathway to produce MCFAs and medium-chain fatty alcohols (MCFOHs).
In the first part of this work, we expanded the length of the products from expressing the rBOX in the cytosol and increased the MCFAs titres. First, we deleted the major glycerol-3-phosphate dehydrogenase (GPD2). This resulted in a platform strain with significantly reduced glycerol fermentation and increased rBOX pathway activity, probably due to an increased availability of NADH. Then, we tested different combinations of rBOX enzymes to increase the length and titres of MCFA. Expressing the thiolase CnbktB and β-hydroxyacyl-CoA dehydrogenase CnpaaH1 from Cupriavidus necator, Cacrt from Clostridium acetobutylicum and the trans-enoyl-CoA reductase Tdter (Treponema denticola) resulted in hexanoic acid as the main product.
Expressing Cncrt2 (C. necator) or YlECH (Y. lipolytica) as enoyl-CoA hydratases resulted in octanoic acid as the main product. Then, we integrated the octanoic (Cncrt2 or YlECH) and the hexanoic acid (Cacrt)-producing variants in the genome of the platform strain and we achieved titers of ≈75 mg/L (hexanoic acid) and ≈ 60 mg/L (octanoic acid) when growing these strains in a complex, highly buffered medium. These are the highest titers of octanoic and hexanoic acid obtained in S. cerevisiae with the rBOX. Additionally, we deleted TES1 and FAA2 to prevent competition for butyryl-CoA and degradation of the produced fatty acids, respectively.
However, these deletions did not improve MCFA titers. In addition, we tested two dual acyl-CoA reductase/alcohol dehydrogenases (ACR/ADH), CaadhE2 from C. acetobutylicum and the putative ACR/ADH EceutE from Escherichia coli, in an octanoyl-CoA-producing strain to produce MCFOH. As a result, we produced 1-hexanol and 1-octanol for the first time in S. cerevisiae with these two enzymes. Nonetheless, the titres were low (<10 mg/L and <2 mg/L, respectively), and four-carbon 1-butanol was the main product in both cases (>80 mg/L). This showed the preference of these two enzymes for butyryl-CoA.
In the second part of this work, we expressed the rBOX in the mitochondria of S. cerevisiae to benefit from the high levels of acetyl-CoA and the reducing environment in that organelle. First, in an adh-deficient strain, we mutated MTH1, a transcription factor regulating the expression of hexose transporters, and deleted GPD2. This resulted in a strain with a reduced Crabtree effect and, therefore, an increased carbon flux to the mitochondria. We partially validated the increased flux to the mitochondria by expressing the ethanol-acetyltransferase EAT1 from Kluyveromyces marxianus in this organelle. This resulted in a higher isoamyl acetate production in the MTH1-mutant strain. Isoamyl acetate is synthesised by Eat1 from acetyl-CoA and isoamyl alcohol, a product of the metabolism of amino acids in the mitochondria. Then, we targeted different butyryl-CoA-producing rBOX variants to the mitochondria, and we used the production of 1-butanol and butyric acid as a proof-of-concept. The strong expression of all the enzymes was toxic for the cell, and the highest butyric acid titres (≈ 50 mg/L) in the mitochondria from the rBOX were obtained from the weak expression of the pathway. The highest 1-butanol titers (≈ 5 mg/L) were obtained with the downregulation of the mitochondrial NADH-oxidase NDI1. However, this downregulation led to a non-desirable petite phenotype.
In summary, we produced hexanoic and octanoic acid for the first time in S. cerevisiae using the rBOX and achieved the highest reported titers of hexanoic and octanoic acid so far using this pathway in S. cerevisiae. In addition, we successfully compartmentalised the rBOX in the mitochondria. However, competing reactions, some of them essential for the viability of the cell, limit the use of this organelle for the rBOX.
Deniz, Erhan ; Löffler, Jan. G. ; Kondratiev, Alexander ; Thun, Asok Rafael ; Shen, Yunxia ; Wille, Georg ; Bredenbeck, Jens
Alternating acquisition of background and sample spectra is often employed in conventional Fourier-transform infrared spectroscopy or ultraviolet–visible spectroscopy for accurate background subtraction. For example, for solvent background correction, typically a spectrum of a cuvette with solvent is measured and subtracted from a spectrum of a cuvette with solvent and solute. Ultrafast spectroscopies, though, come with many peculiarities that make the collection of well-matched, subtractable background and sample spectra challenging. Here, we present a demountable split-sample cell in combination with a modified Lissajous scanner to overcome these challenges. It allows for quasi-simultaneous measurements of background and sample spectra, mitigating the effects of drifts of the setup and maintaining the beam and sample geometry when swapping between background and sample measurements. The cell is moving between subsequent laser shots to refresh the excited sample volume. With less than 45 μl of solution for 150 μm optical thickness, sample usage is economical. Cell assembly is a key step and covered in an illustrated protocol.
Neuman, Manuela G. ; Seitz, Helmut K. ; Teschke, Rolf ; Malnick, Stephen ; Johnson-Davis, Kamisha L. ; Cohen, Lawrence B. ; German, Anit ; Hohmann, Nicolas ; Moreira, Bernhardo ; Moussa, George ; Opris, Mihai
Hepatic cells are sensitive to internal and external signals. Ethanol is one of the oldest and most widely used drugs in the world. The focus on the mechanistic engine of the alcohol-induced injury has been in the liver, which is responsible for the pathways of alcohol metabolism. Ethanol undergoes a phase I type of reaction, mainly catalyzed by the cytoplasmic enzyme, alcohol dehydrogenase (ADH), and by the microsomal ethanol-oxidizing system (MEOS). Reactive oxygen species (ROS) generated by cytochrome (CYP) 2E1 activity and MEOS contribute to ethanol-induced toxicity. We aimed to: (1) Describe the cellular, pathophysiological and clinical effects of alcohol misuse on the liver; (2) Select the biomarkers and analytical methods utilized by the clinical laboratory to assess alcohol exposure; (3) Provide therapeutic ideas to prevent/reduce alcohol-induced liver injury; (4) Provide up-to-date knowledge regarding the Corona virus and its affect on the liver; (5) Link rare diseases with alcohol consumption. The current review contributes to risk identification of patients with alcoholic, as well as non-alcoholic, liver disease and metabolic syndrome. Additional prevalence of ethnic, genetic, and viral vulnerabilities are presented.
Mitochondrial RNA granules (MRGs) are membraneless, highly specialized compartments that play an essential role in the post-transcriptional regulation of mitochondrial gene expression. This regulation is crucial for maintaining energy production, controlling metabolic functions and ensuring homeostasis in cells. Dysregulation of mitochondrial genes has been linked to various human diseases, including neurodegenerative and metabolic disorders as well as certain types of cancer.
MRGs are composed of different RNA species, including mitochondrial precursor RNA (pre-RNA), mature tRNAs, rRNAs and mRNAs complexed with multiple proteins involved in RNA processing and mitoribosome assembly. However, despite the significance of MRGs, their protein composition, structural organization, stability and dynamics during stress conditions remain elusive. In the study reported here, I adopted a three-step approach to address the aforementioned fundamental issues.
First and foremost, I identified the protein composition of MRGs and unveiled their architectural complexity. To characterize the MRG proteome, I applied the cutting-edge TurboID-based proximity labeling approach combined with quantitative mass spectrometry. Proximity labeling was conducted on 20 distinct MRG-associated human proteins, resulting in the identification of more than 1,700 protein-protein interactions. This expansive dataset enabled me to create a comprehensive network, providing valuable insights into both the (sub)architecture as well as the core structure of MRGs in-depth.
Secondly, I investigated the spatio-temporal dynamics of MRGs under various mitochondrial stress conditions. To monitor the morphological alterations and compositional changes of MRGs, I utilized time-resolved confocal fluorescence microscopy and proteomics, respectively. In this analysis, I applied IMT1, the first specific inhibitor that selectively targets mitochondrial transcription. Using this methodology, I pinpointed precise conditions that triggered MRGs’ disassembly during stress, followed by their reassembly when nascent RNA production was restored. The results of this examination elucidate that MRGs are highly dynamic and stress adaptive structures, capable of rapid dissolution and reassembly, a process closely connected to mitochondrial transcription.
Thirdly, I aimed to explore the impact of RNA turnover on MRGs’ integrity during stress, employing confocal fluorescence microscopy and quantitative real-time PCR. I observed that depletion of MRG proteins associated with RNA degradation counteracts MRGs’ disassembly under stress conditions, a phenomenon attributed to the accumulation of double-stranded RNA (dsRNA). These results emphasize the critical role of pre-RNA turnover in maintaining MRG integrity and reveal that MRGs can be stabilized by dsRNA.
Taken together, the comprehensive investigation reported in this thesis has substantially broadened and deepened our understanding of MRGs’ complexity. By identifying their molecular structure and dynamics, I have gained significant insights into the fundamental characteristics and biological functions of MRGs in cellular processes. This knowledge contributes to the identification of disease-related pathways linked to mitochondrial gene expression and may inspire future studies to develop novel therapeutic approaches.
Biedenbänder, Thomas ; Jesus, Vanessa de ; Schmidt-Dengler, Martina ; Helm, Mark ; Corzilius, Björn ; Fürtig, Boris
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.
Moore, Hannah ; Lutz, Lena ; Bernhardt, Victoria Christiane ; Drijfhout, Falko P. ; Cody, Robert ; Amendt, Jens
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.
Rico-Sánchez, Axel Eduardo ; Rodríguez-Romero, Alexis Joseph ; Sedeño-Díaz, Jacinto Elías ; López-López, Eugenia ; Sundermann, Andrea
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.
Liu, Ziwen ; Hirata-Miyasaki, Eduardo ; Pradeep, Soorya ; Rahm, Johanna Viola ; Foley, Christian ; Chandler, Talon ; Ivanov, Ivan ; Woosley, Hunter ; Lao, Tiger ; Balasubramanian, Akilandeswari ; Liu, Chad ; Leonetti, Manu ; Arias, Carolina ; Jacobo, Adrian ; Mehta, Shalin B.
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)
Maekiniemi, Anna ; Savakis, Philipp ; Snoep, Jacky L. ; Seiler, Markus ; Rossum, Kelly van ; Niekerk, David D. van ; Zarnack, Katharina ; Singer, Robert H. ; Tutucci, Evelina
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)
Brinkmann, Vanessa ; Romeo, Margherita ; Larigot, Lucie ; Hemmers, Anne ; Tschage, Lisa ; Kleinjohann, Jennifer ; Schiavi, Alfonso ; Steinwachs, Swantje ; Esser, Charlotte ; Menzel, Ralph ; Giani Tagliabue, Sara ; Bonati, Laura ; Cox, Fiona ; Ale-Agha, Niloofar ; Jakobs, Philipp ; Altschmied, Joachim ; Haendeler, Judith ; Coumoul, Xavier ; Ventura, Natascia
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.
Dalimot, Jill Jasmine ; Smith, Ilka Mc Cormick ; Gerkrath, Jasmin ; Hartmann, Sylvia ; Cornely, Oliver Andreas ; Lee, Soo Chan ; Heitman, Joseph ; Rickerts, Volker
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.
Gurarie, Eliezer ; Bracis, Chloe ; Brilliantova, Angelina ; Kojola, Ilpo ; Suutarinen, Johanna ; Ovaskainen, Otso ; Potluri, Sriya ; Fagan, William F.
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.
Liu, Ziwen ; Hirata-Miyasaki, Eduardo ; Pradeep, Soorya ; Rahm, Johanna Viola ; Foley, Christian ; Chandler, Talon ; Ivanov, Ivan ; Woosley, Hunter ; Lao, Tiger ; Balasubramanian, Akilandeswari ; Liu, Chad ; Leonetti, Manu ; Arias, Carolina ; Jacobo, Adrian ; Mehta, Shalin B.
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.