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Development of a CRISPR/Cas gene editing technique for the coccolithophore Chrysotila carterae
(2024)
The plant cell wall plays several crucial roles during plant development with its integrity acting as key signalling component for growth regulation during biotic and abiotic stresses. Cellulose microfibrils, the principal load-bearing components is the major component of the primary cell wall, whose synthesis is mediated by microtubule-associated CELLULOSE SYNTHASE (CESA) COMPLEXES (CSC). Previous studies have shown that CSC interacting proteins COMPANION OF CELLULOSE SYNTHASE (CC) facilitate sustained cellulose synthesis during salt stress by promoting repolymerization of cortical microtubules. However, our understanding of cellulose synthesis during salt stress remains incomplete.
In this study, a pull-down of CC1 protein led to the identification of a novel interactor, termed LEA-like. Phylogenetic analysis revealed that LEA-like belongs to the LATE EMBRYOGENESIS ABUNDANT (LEA) protein family, specifically to the LEA_2 subgroup, showing a close relationship with the CC proteins. Roots of the double mutants lea-like and its closest homolog emb3135 exhibited hypersensitivity when grown on cellulose synthesis inhibitors. Further analysis of higher-order mutants of lea-like, emb3135, and cesa6 demonstrated a genetic interaction between them indicating a significant role in cellulose synthesis.
Live-cell imaging revealed that both LEA-like and EMB3135 migrated with the CSC at the plasma membrane along microtubule tracks in control and oryzalin-treated conditions which destabilize microtubules, suggesting a tight interaction. Investigation of fluorescently labeled lines of different domains of the LEA-like protein revealed that the N-terminal cytosolic domain of LEA-like colocalizes with microtubules, suggesting a physical association between the two.
Considering the established role of LEA proteins in abiotic stress tolerance, we performed phenotypic analysis of the mutant under various stresses. Growth of double mutants of lea-like and emb3135 on NaCl containing media resulted in swelling of root cell indicating a putative role in salt stress tolerance. Supportive of this the quadruple mutant, lacking LEA-like, EMB3135, CC1, and CC2 proteins, exhibited a severe root growth defect on NaCl media compared to control conditions. Live-cell imaging revealed that under salt stress, the LEA-like protein forms aggregates in the plasma membrane.
In conclusion, this study has unveiled two novel interactors of the CSC that act with the CC proteins that regulate plant growth in response to salt stress providing new insights into the intricate regulation of cellulose synthesis, particularly under such conditions.
The inclusion of exotic germplasm serves as a crucial means to enhance allelic and
consequently phenotypic diversity in inbred crop species. Such species have experienced a reduction in diversity due to artificial selection focused on a limited set of traits. The natural biodiversity within ecosystems presents an opportunity to explore various traits influencing plant survival, reproductive fitness and yield potential. In agricultural research, the study of wild species closely related to cultivated plants serves as a means to comprehend the genetic foundations of past domestication events and the polymorphisms essential for future breeding efforts to develop superior varieties. In order to examine the metabolic composition, pinpoint quantitative trait loci (QTL) and facilitate their resolution an extensive large-scale analysis of metabolic QTL (mQTL) was conducted on tomato backcross inbred lines (BILs) derived from a cross between the wild species S. pennellii (5240) incorporated into the background of S. lycopersicum cv. LEA determinate inbred which can be grown in open fields and cv. TOP indeterminate which can be grown in greenhouse conditions. A large number of mQTL associated with primary secondary and lipid metabolism in fruit were identified across the two BIL populations. Epistasis, the interactions between genes at different loci, has been an interest in molecular and quantitative genetics for many decades. The study of epistasis requires the analysis of very large populations with multiple independent genotypes that carry specific genomic regions. In order to understand the genetic basis of tomato fruit metabolism, I extended the work to investigate epistatic interactions of the genomic regions. In addition, two candidate genes were identified through quantitative trait loci underlying fruit-specific sucrose and jasmonic acid derivatives. Finally, in this study, I assessed the genetic framework of fruit metabolic traits with a high level of detail, utilizing the newly created Solanum pennellii (5240) backcrossed introgression lines (n=3000). This investigation resulted in the discovery of promising candidate loci associated with significant fruit quality traits, including those to the abundance of glutamic acid and aspartic acid crucial elements contributing to the development of acidity and flavors.
Carbohydrates play a vital role in all living organisms; serving as a cornerstone in primary metabolism through the release of energy from their hydrolysis and subsequent re-utilization (Apriyanto et al., 2022). Starch is the principal carbohydrate reserve in plants, providing essential energy for plant growth. Furthermore, starch serves as a significant carbohydrate source in the human diet. Beyond its nutritional value, starch has extensive industrial application associated with many aspects of human society, such as feed, pharmacy, textiles, and the production of biodegradable plastics. Understanding the mechanisms underlying starch metabolism in plants carries multifaceted benefits. Not only does it contribute to increasing crop yield and refining grain quality, but also can improve the efficiency of industrial applications.
Starch in plants is categorized into two classes based on their location and function: transitory starch and storage starch. Transitory starch is produced in chloroplasts of autotrophic tissues/organs, such as leaves. It is synthesized during the day and degraded during the night. Storage starch is synthesized in heterotrophic tissues/organs, such as endosperm, roots and tubers, which is utilized for plant reproduction and industrial application in human life. Most studies aiming to comprehend starch metabolism of Arabidopsis thaliana primarily focus on transitory starch.
Starch is stored as granular form in chloroplast and amyloplast. The parameters of starch granules, including size, morphology, and quantity per chloroplast serve as indicators of starch metabolism status. However, the understanding of their regulatory mechanism is still incomplete. In this research, I initially employed a simple and adapted method based on laser confocal scanning microscopy (LCSM) to observe size, morphology and quantity of starch granules within chloroplasts in Arabidopsis thaliana in vivo. This method facilitated a rapid and versatile analysis of starch granule parameters across numerous samples. Utilizing this approach, I compared starch granule number per chloroplast between mesophyll cells and guard cells in both wild type plants (Col-0) and several starch related mutants. The results revealed that the granule number is distinct between mesophyll cells and guard cells, even within the same genetic background, suggesting that guard cells operate a unique regulatory mechanism of starch granule number.
Subsequently, I redirected my attention toward examining starch morphology. Through microscopy analyses, I observed a gradual alteration in starch granule morphology in certain mutants during leaf aging. Specifically, in mutants such as sex1-8 and dpe2phs1ss4, there was a progressive alteration in starch granule morphology over time. Conversely, in Col-0 and ss4 mutant, these morphological alterations were not evident. This discovery suggests a new perspective to understand the development of starch morphology.
Further investigation revealed that mutants lacking either Disproportionating enzyme 2 (DPE2) or MALTOSE-EXCESS 1 (MEX1) exhibited gradual alterations in starch morphology with leaf aging. Notably, the most severe effects on starch morphology occurred in double mutants lacking either DPE2 or MEX1 in conjunction with a lack of starch synthase 4 (SS4). In these mutations, a transformation of the starch granule morphology from the typical discoid morphology to oval and eventually to a spherical shape.
To investigate the changes in the internal structure of starch during this alteration, I analyzed the chain length distribution (CLD) of the amylopectin of young, intermediate and old leaves of the mutants. Throughout starch granule development, I found an increased presence of short glucan chains within the granules, particularly evident in dpe2ss4 and mex1ss4 mutants, as well as their parental single mutants. Notably, the single mutant ss4 also showed an affected granule morphology, albeit not influenced by leaf aging..
The CLD pattern of the amylopectin reflects an integrative regulation involving several participants in starch synthesis, including starch synthases (SSs), starch branching/debranching enzymes (SBEs/DBEs). Therefore, I further detected the expression of related genes on transcription level and the enzymatic activity of their respective proteins. Results indicated altered gene expression of several regulators in these mutants, particularly demonstrating dramatic alterations in dpe2 and dpe2ss4 with leaf aging. These changes corresponded with the observed alterations in starch granule morphology.
Taken together, I have identified and characterized a progressive alteration in starch granule morphology primarily resulting from the deficiencies in DPE2 and MEX1. Furthermore, I have associated the CLD pattern with the granule morphogenesis, as well as the gene expression and enzymatic activity of proteins involved in starch synthesis. Unlike SS4, which is implicated in starch initiation, MEX1 and DPE2 are involved into starch degradation. MEX1 is located in chloroplast envelope and DPE2 is situated in the cytosol. Considering the locations and known functions of DPE2/MEX1 and SS4, I infer that there might be two pathways influencing starch morphology: an initiation-affected pathway via SS4 and a degradation-affected pathway via DPE2/MEX1.
Sulfur is an important element that is incorporated into many biomolecules in humans. The incorporation and transfer of sulfur into biomolecules is, however, facilitated by a series of different sulfurtransferases. Among these sulfurtransferases is the human mercaptopyruvate sulfurtransferase (MPST) also designated as tRNA thiouridine modification protein (TUM1). The role of the human TUM1 protein has been suggested in a wide range of physiological processes in the cell among which are but not limited to involvement in Molybdenum cofactor (Moco) biosynthesis, cytosolic tRNA thiolation and generation of H2S as signaling molecule both in mitochondria and the cytosol. Previous interaction studies showed that TUM1 interacts with the L-cysteine desulfurase NFS1 and the Molybdenum cofactor biosynthesis protein 3 (MOCS3). Here, we show the roles of TUM1 in human cells using CRISPR/Cas9 genetically modified Human Embryonic Kidney cells. Here, we show that TUM1 is involved in the sulfur transfer for Molybdenum cofactor synthesis and tRNA thiomodification by spectrophotometric measurement of the activity of sulfite oxidase and liquid chromatography quantification of the level of sulfur-modified tRNA. Further, we show that TUM1 has a role in hydrogen sulfide production and cellular bioenergetics.
In late summer, migratory bats of the temperate zone face the challenge of accomplishing two energy-demanding tasks almost at the same time: migration and mating. Both require information and involve search efforts, such as localizing prey or finding potential mates. In non-migrating bat species, playback studies showed that listening to vocalizations of other bats, both con-and heterospecifics, may help a recipient bat to find foraging patches and mating sites. However, we are still unaware of the degree to which migrating bats depend on con-or heterospecific vocalizations for identifying potential feeding or mating opportunities during nightly transit flights. Here, we investigated the vocal responses of Nathusius’ pipistrelle bats, Pipistrellus nathusii, to simulated feeding and courtship aggregations at a coastal migration corridor. We presented migrating bats either feeding buzzes or courtship calls of their own or a heterospecific migratory species, the common noctule, Nyctalus noctula. We expected that during migratory transit flights, simulated feeding opportunities would be particularly attractive to bats, as well as simulated mating opportunities which may indicate suitable roosts for a stopover. However, we found that when compared to the natural silence of both pre-and post-playback phases, bats called indifferently during the playback of conspecific feeding sounds, whereas P. nathusii echolocation call activity increased during simulated feeding of N. noctula. In contrast, the call activity of P. nathusii decreased during the playback of conspecific courtship calls, while no response could be detected when heterospecific call types were broadcasted. Our results suggest that while on migratory transits, P. nathusii circumnavigate conspecific mating aggregations, possibly to save time or to reduce the risks associated with social interactions where aggression due to territoriality might be expected. This avoidance behavior could be a result of optimization strategies by P. nathusii when performing long-distance migratory flights, and it could also explain the lack of a response to simulated conspecific feeding. However, the observed increase of activity in response to simulated feeding of N. noctula, suggests that P. nathusii individuals may be eavesdropping on other aerial hawking insectivorous species during migration, especially if these occupy a slightly different foraging niche.
The African weakly electric fish genus Campylomormyrus includes 15 described species mostly native to the Congo River and its tributaries. They are considered sympatric species, because their distribution area overlaps. These species generate species-specific electric organ discharges (EODs) varying in waveform characteristics, including duration, polarity, and phase number. They exhibit also pronounced divergence in their snout, i.e. the length, thickness, and curvature. The diversifications in these two phenotypical traits (EOD and snout) have been proposed as key factors promoting adaptive radiation in Campylomormyrus. The role of EODs as a pre-zygotic isolation mechanism driving sympatric speciation by promoting assortative mating has been examined using behavioral, genetical, and histological approaches. However, the evolutionary effects of the snout morphology and its link to species divergence have not been closely examined. Hence, the main objective of this study is to investigate the effect of snout morphology diversification and its correlated EOD to better understand their sympatric speciation and evolutionary drivers. Moreover, I aim to utilize the intragenus and intergenus hybrids of Campylomormyrus to better understand trait divergence as well as underlying molecular/genetic mechanisms involved in the radiation scenario. To this end, I utilized three different approaches: feeding behavior analysis, diet assessment, and geometric morphometrics analysis. I performed feeding behavior experiments to evaluate the concept of the phenotype-environment correlation by testing whether Campylomormyrus species show substrate preferences. The behavioral experiments showed that the short snout species exhibits preference to sandy substrate, the long snout species prefers a stone substrate, and the species with intermediate snout size does not exhibit any substrate preference. The experiments suggest that the diverse feeding apparatus in the genus Campylomormyrus may have evolved in adaptation to their microhabitats. I also performed diet assessments of sympatric Campylomormyrus species and a sister genus species (Gnathonemus petersii) with markedly different snout morphologies and EOD using NGS-based DNA metabarcoding of their stomach contents. The diet of each species was documented showing that aquatic insects such as dipterans, coleopterans and trichopterans represent the major diet component. The results showed also that all species are able to exploit diverse food niches in their habitats. However, comparing the diet overlap indices showed that different snout morphologies and the associated divergence in the EOD translated into different prey spectra. These results further support the idea that the EOD could be a ‘magic trait’ triggering both adaptation and reproductive isolation. Geometric morphometrics method was also used to compare the phenotypical shape traits of the F1 intragenus (Campylomormyrus) and intergenus (Campylomormyrus species and Gnathonemus petersii) hybrids relative to their parents. The hybrids of these species were well separated based on the morphological traits, however the hybrid phenotypic traits were closer to the short-snouted species. In addition, the likelihood that the short snout expressed in the hybrids increases with increasing the genetic distance of the parental species. The results confirmed that additive effects produce intermediate phenotypes in F1-hybrids. It seems, therefore, that morphological shape traits in hybrids, unlike the physiological traits, were not expressed straightforward.
Mitochondria and plastids are organelles with an endosymbiotic origin. During evolution, many genes are lost from the organellar genomes and get integrated in the nuclear genome, in what is known as intracellular/endosymbiotic gene transfer (IGT/EGT). IGT has been reproduced experimentally in Nicotiana tabacum at a gene transfer rate (GTR) of 1 event in 5 million cells, but, despite its centrality to eukaryotic evolution, there are no genetic factors known to influence the frequency of IGT in higher eukaryotes. The focus of this work was to determine the role of different DNA repair pathways of double strand break repair (DSBR) in the integration step of organellar DNA in the nuclear genome during IGT. Here, a CRISPR/Cas9 mutagenesis strategy was implemented in N. tabacum, with the aim of generating mutants in nuclear genes without expected visible phenotypes. This strategy led to the generation of a collection of independent mutants in the LIG4 (necessary for non-homologous end joining, NHEJ) and POLQ genes (necessary for microhomology mediated end joining, MMEJ). Targeting of other DSBR genes (KU70, KU80, RPA1C) generated mutants with unexpectedly strong developmental phenotypes.. These factors have telomeric roles, hinting towards a possible relationship between telomere length, and strength of developmental disruption upon loss of telomere structure in plants. The mutants were made in a genetic background encoding a plastid-encoded IGT reporter, that confers kanamycin resistance upon transfer to the nucleus. Through large scale independent experiments, increased IGT from the chloroplast to the nucleus was observed in lig4 mutants, as well as lines encoding a POLQ gene with a defective polymerase domain (polqΔPol). This shows that NHEJ or MMEJ have a double-sided relationship with IGT: while transferred genes may integrate using either pathway, the presence of both pathways suppresses IGT in wild-type somatic cells, thus demonstrating for the first time the extent on which nuclear genes control IGT frequency in plants. The IGT frequency increases in the mutants are likely mediated by increased availability of double strand breaks for integration. Additionally, kinetic analysis reveals that gene transfer (GT) events accumulate linearly as a function of time spent under antibiotic selection in the experiment, demonstrating that, contrary to what was previously thought, there is no such thing as a single GTR in somatic IGT experiments. Furthermore, IGT in tissue culture experiments appears to be the result of a "race against the clock" for integration in the nuclear genome, that starts when the organellar DNA arrives to the nucleus granting transient antibiotic resistance. GT events and escapes of kanamycin selection may be two possible outcomes from this race: those instances where the organellar DNA gets to integrate are recovered as GT events, and in those cases where timely integration fails, antibiotic resistance cannot be sustained, and end up considered as escapes. In the mutants, increased opportunities for integration in the nuclear genome change the overall ratio between IGT and escape events. The resources generated here are promising starting points for future research: (1) the mutant collection, for the further study of processes that depend on DNA repair in plants (2) the collection of GT lines obtained from these experiments, for the study of the effect of DSBR pathways over integration patterns and stability of transferred genes and (3) the developed CRISPR/Cas9 workflow for mutant generation, to make N. tabacum meet its potential as an attractive model for answering complex biological questions.
The G protein-coupled estrogen receptor (GPER1) is acknowledged as an important mediator of estrogen signaling. Given the ubiquitous expression of GPER1, it is likely that the receptor plays a role in a variety of malignancies, not only in the classic hormonally regulated tissues (e.g., breast, ovary, and prostate), but also in the colon. As colorectal cancer (CRC) is the third most common cancer in both men and women worldwide and environmental factors and dietary habits are important risk factors, it is increasingly recognized that natural and synthetic hormones and their associated receptors might play a role in CRC. Through oral consumption, environmental contaminants with endocrine activity are in contact with the gastrointestinal mucosa, where they might exert their toxic effects. Although GPER1 has been shown to be engaged in physiological and pathophysiological processes, its role in CRC remains poorly understood. Thus, pro- as well as anti-tumorigenic effects are described in the literature. This thesis has uncovered novel roles of GPER1 in mediating major CRC-associated phenotypes in transformed and non-transformed colon cell lines. Exposure to the estrogens 17β-estradiol (E2), bisphenol-A (BPA) and diethylstilbestrol (DES) but also the androgen dihydrotestosterone (DHT) resulted in GPER1-dependent induction of supernumerary centrosomes, whole chromosomal instability (w-CIN) and aneuploidy. Indeed, both knockdown and inhibition of GPER1 attenuated the generation of (xeno)hormone-driven supernumerary centrosomes and karyotype instability. Mechanistically, (xeno)hormone-induced centrosome amplification was associated with transient multipolar mitosis and the generation of so called anaphase “lagging” chromosomes. The results of this thesis propose a GPER1/PKA/AKAP9-pathway in regulating centrosome numbers in colorectal cancer cells and the involvement of the centriolar protein centrin. Remarkably, exposure to (xeno)hormones resulted in atypical enlargement and unexpected phosphorylation of the centriole marker centrin in interphase. These findings provide a novel role for GPER1 in key CRC-prone lesions and shed light on underlying mechanisms that involve GPER1 function in the colon. Elucidating to what extent centrosomal proteins are involved in the GPER1-mediated aneugenic effect will be an important task for future studies. The present study was intended to lay a first foundation to understand the molecular basis and potential risk factors of CRC which might help to reduce the use of laboratory animals. Since numerous animal experiments are conducted in biomedical research, the development of alternative methods is indispensable. The Federal Institute for Risk Assessment (BfR) as the German Center for the Protection of Laboratory Animals (Bf3R) addresses this issue by uncovering underlying mechanisms leading to colorectal cancer as necessary prerequisite in order to develop alternative methods.
Photosynthesis converts light into metabolic energy which fuels plant growth. In nature, many factors influence light availability for photosynthesis on different time scales, from shading by leaves within seconds up to seasonal changes over months. Variability of light energy supply for photosynthesis can limit a plant´s biomass accumulation. Plants have evolved multiple strategies to cope with strongly fluctuation light (FL). These range from long-term optimization of leaf morphology and physiology and levels of pigments and proteins in a process called light acclimation, to rapid changes in protein activity within seconds. Therefore, uncovering how plants deal with FL on different time scales may provide key ideas for improving crop yield. Photosynthesis is not an isolated process but tightly integrates with metabolism through mutual regulatory interactions. We thus require mechanistic understanding of how long-term light acclimation shapes both, dynamic photosynthesis and its interactions with downstream metabolism. To approach this, we analyzed the influence of growth light on i) the function of known rapid photosynthesis regulators KEA3 and VCCN1 in dynamic photosynthesis (Chapter 2-3) and ii) the interconnection of photosynthesis with photorespiration (PR; Chapter 4).
We approached topic (i) by quantifying the effect of different growth light regimes on photosynthesis and photoprotection by using kea3 and vccn1 mutants. Firstly, we found that, besides photosynthetic capacity, the activities of VCCN1 and KEA3 during a sudden high light phase also correlated with growth light intensity. This finding suggests regulation of both proteins by the capacity of downstream metabolism. Secondly, we showed that KEA3 accelerated photoprotective non-photochemical quenching (NPQ) kinetics in two ways: Directly via downregulating the lumen proton concentration and thereby de-activating pH-dependent NPQ, and indirectly via suppressing accumulation of the photoprotective pigment zeaxanthin.
For topic (ii), we analyzed the role of PR, a process which recycles a toxic byproduct of the carbon fixation reactions, in metabolic flexibility in a dynamically changing light environment. For this we employed the mutants hpr1 and ggt1 with a partial block in PR. We characterized the function of PR during light acclimation by tracking molecular and physiological changes of the two mutants. Our data, in contrast to previous reports, disprove a generally stronger physiological relevance of PR under dynamic light conditions. Additionally, the two different mutants showed pronounced and distinct metabolic changes during acclimation to a condition inducing higher photosynthetic activity. This underlines that PR cannot be regarded purely as a cyclic detoxification pathway for 2PG. Instead, PR is highly interconnected with plant metabolism, with GGT1 and HPR1 representing distinct metabolic modulators.
In summary, the presented work provides further insight into how energetic and metabolic flexibility is ensured by short-term regulators and PR during long-term light acclimation.
The light reactions of photosynthesis are carried out by a series of multiprotein complexes embedded in thylakoid membranes. Among them, photosystem I (PSI), acting as plastocyanin-ferderoxin oxidoreductase, catalyzes the final reaction. Together with light-harvesting antenna I, PSI forms a high-molecular-weight supercomplex of ~600 kDa, consisting of eighteen subunits and nearly two hundred co-factors. Assembly of the various components into a functional thylakoid membrane complex requires precise coordination, which is provided by the assembly machinery. Although this includes a small number of proteins (PSI assembly factors) that have been shown to play a role in the formation of PSI, the process as a whole, as well as the intricacy of its members, remains largely unexplored.
In the present work, two approaches were used to find candidate PSI assembly factors. First, EnsembleNet was used to select proteins thought to be functionally related to known PSI assembly factors in Arabidopsis thaliana (approach I), and second, co-immunoprecipitation (Co-IP) of tagged PSI assembly factors in Nicotiana tabacum was performed (approach II).
Here, the novel PSI assembly factors designated CO-EXPRESSED WITH PSI ASSEMBLY 1 (CEPA1) and Ycf4-INTERACTING PROTEIN 1 (Y4IP1) were identified. A. thaliana null mutants for CEPA1 and Y4IP1 showed a growth phenotype and pale leaves compared with the wild type. Biophysical experiments using pulse amplitude modulation (PAM) revealed insufficient electron transport on the PSII acceptor side. Biochemical analyses revealed that both CEPA1 and Y4IP1 are specifically involved in PSI accumulation in A. thaliana at the post-translational level but are not essential. Consistent with their roles as factors in the assembly of a thylakoid membrane protein complex, the two proteins localize to thylakoid membranes. Remarkably, cepa1 y4ip1 double mutants exhibited lethal phenotypes in early developmental stages under photoautotrophic growth. Finally, co-IP and native gel experiments supported a possible role for CEPA1 and Y4IP1 in mediating PSI assembly in conjunction with other PSI assembly factors (e.g., PPD1- and PSA3-CEPA1 and Ycf4-Y4IP1). The fact that CEPA1 and Y4IP1 are found exclusively in green algae and higher plants suggests eukaryote-specific functions. Although the specific mechanisms need further investigation, CEPA1 and Y4IP1 are two novel assembly factors that contribute to PSI formation.
The musculoskeletal system provides support and enables movement to the body, and its deterioration is a crucial aspect of age-related functional decline. Mesenchymal stromal cells (MSCs) play an important role in musculoskeletal homeostasis due to their broad differentiation potentials and their ability to support osteogenic and myogenic tissue maintenance and regeneration. In the bone, MSCs differentiate either into osteochondrogenic progenitors to form osteocytes and chondrocytes, or increasingly with age into adipogenic progenitors which give rise to bone-resident adipocytes. In skeletal muscle, during healthy regeneration MSCs provide regulatory signals that activate local, tissue-specific stem cells, known as satellite cells, which regenerate contractile myofibres. This process involves a significant cross-talk to immune cells stemming from both lymphoid and myeloid lineages. During ageing, muscle-resident MSCs undergo increased adipogenic lineage commitment, causing niche changes that contribute to fatty infiltration in muscles. These shifts in cell populations in bone lead to the loss of osteogenic cells and subsequently osteoporosis, or in muscle to impaired regeneration and to the development of sarcopenia. However, the signals that drive transition of MSCs into their respective cellular fates remain elusive.
This thesis aims to elucidate the transcriptional shifts modulating cell states and cell types in musculoskeletal MSC fate determination. Single-cell RNA-sequencing (scRNA-seq) was used to characterise cell type-specific transcript regulation. State-of-the-art bioinformatics tools were combined with different analytical platforms that include both droplet-based scRNA-seq for large heterogeneous populations, and microfluidics-based scRNA-seq to assess small, rare subpopulations. For each platform, distinct computational pipelines were established including filtering steps to exclude low-quality cells, and data visualisation was performed by dimensionality reduction. Downstream analysis included clustering, cell type annotation, and differential gene expression to investigate transcriptional states in defined cell types during ageing and injury in the muscle and bone. Finally, a novel tool to assess publication activities in defined areas of research for the identified marker genes was developed.
The results in the bone indicate that ageing MSCs increasingly commit towards an adipogenic fate at the expense of osteogenic specialisation. The data also suggests that significant cell population shifts of MSC-type fibro-adipogenic progenitors during muscle ageing underlie the pathologies observed in homeostatic and post-injury regenerative conditions. High-throughput visualisation of publication activity for candidate genes enabled more effective biological evaluation of scRNA-seq data. These results expose critical age-related changes in the stem cell niches of skeletal muscle and bone, highlight their respective sensitivity to nutrition and pathology, and elucidate novel factors that modulate stem cell-based regeneration. Targeting these processes might improve musculoskeletal health in the context of ageing and prevent the negative effects of pathological lineage determination.
Pichia pastoris (syn. Komagataella phaffi) is a distinguished expression system widely used in industrial production processes. Recent molecular research has focused on numerous approaches to increase recombinant protein yield in P. pastoris. For example, the design of expression vectors and synthetic genetic elements, gene copy number optimization, or co-expression of helper proteins
(transcription factors, chaperones, etc.). However, high clonal variability of transformants and low screening throughput have hampered significant success.
To enhance screening capacities, display-based methodologies inherit the potential for efficient isolation of producer clones via fluorescence-activated cell sorting (FACS). Therefore, this study focused on developing a novel clone selection method that is based on the non-covalent attachment of Fab fragments on the P. pastoris cell surface to be applicable for FACS.
Initially, a P. pastoris display system was developed, which is a prerequisite for the surface capture of secreted Fabs. A Design of Experiments approach was applied to analyze the influence of various genetic elements on antibody fragment display. The combined P. pastoris formaldehyde dehydrogenase promoter (PFLD1), Saccharomyces cerevisiae invertase 2 signal peptide (ScSUC2), - agglutinin (ScSAG1) anchor protein, and the ARS of Kluyveromyces lactis (panARS) conferred highest display levels.
Subsequently, eight single-chain variable fragments (scFv) specific for the constant part of the Fab heavy or light chain were individually displayed in P. pastoris. Among the tested scFvs, the anti-human CH1 IgG domain scFv allowed the most efficient Fab capture detected by flow cytometry.
Irrespective of the Fab sequence, exogenously added as well as simultaneously secreted Fabs were successfully captured on the cell surface. Furthermore, Fab secretion capacities were shown to correlate to the level of surface-bound Fabs as demonstrated for characterized producer clones.
Flow-sorted clones presenting high amounts of Fabs showed an increase in median Fab titers (factor of 21 to 49) compared to unsorted clones when screened in deep-well plates. For selected candidates, improved functional Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask production. Since the scFv capture matrix was encoded on an episomal plasmid with inherently unstable autonomously replicating sequences (ARS), efficient plasmid curing was observed after removing the selective pressure. Hence, sorted clones could be immediately used for production without the need to modify the expression host or vector. The resulting switchable display/secretion system provides a streamlined approach for the isolation of Fab producers and subsequent Fab production.
The genetic structure of Bryde's whale (Balaenoptera brydei) on the central and western North Pacific feeding grounds was investigated using a total of 1195 mitochondrial control region sequences and 1182 microsatellite genotypes at 17 loci in specimens collected from three longitudinal areas, 1W (135 degrees E-165 degrees E), 1E (165 degrees E-180 degrees), and 2 (180 degrees-155 degrees W). Genetic diversities were similar among areas and a haplotype network did not show any geographic structure, while an analysis of molecular variance found evidence of genetic structure in this species. Pairwise FST and G'ST estimates and heterogeneity tests attributed this structure to weak but significant differentiation between areas 1W/1E and 2. A Mantel test and a high-resolution analysis of genetic diversity statistics showed a weak spatial cline of genetic differentiation. These findings could be reconciled by two possible stock structure scenarios: (1) a single population with kin-association affecting feeding ground preference and (2) two populations with feeding ground preference for either area 1W or area 2. An estimated dispersal rate between areas 1W and 2 indicates that both scenarios should be considered as a precautionary principle in stock assessments.
Potato FLC-like and SVP-like proteins jointly control growth and distinct developmental processes
(2023)
Based on worldwide consumption, Solanum tuberosum L. (potato) is the most important non-grain food crop. Potato has two ways of stable propagation: sexually via flowering and vegetatively via tuberization. Remarkably, these two developmental processes are controlled by similar molecular regulators and mechanisms. Given that FLC and SVP genes act as key flowering regulators in the model species Arabidopsis and in various other crop species, this study aimed at identifying FLC and SVP homologs in potato and investigating their roles in the regulation of plant development, with a particular focus on flowering and tuberization. Our analysis demonstrated that there are five FLC-like and three SVP like proteins encoded in the potato genome. The expression profiles of StFLCs and StSVPs throughout potato development and the detected interactions between their proteins indicate tissue specificity of the individual genes and distinct roles of a variety of putative protein complexes. In particular, we discovered that StFLC-D, as well as StFLC-B, StSVP-A, and StSVP-B play a complex role in the regulation of flowering time, as not only increased but also decreased levels of their transcripts promote earlier flowering. Most importantly, StFLC-D has a marked impact on tuberization under non-inductive conditions and susceptibility to temperature-induced tuber malformation, also known as second growth. Plants with decreased levels of StFLC-D demonstrated a strong ability to produce tubers under long days and appeared to be insensitive to temperature-induced second growth. Lastly, our data also suggests that StFLCs and StSVPs may be involved in the nitrogen-dependent regulation of potato development. Taken together, this study highlights the functional importance of StFLC and StSVP genes in the regulation of distinct developmental processes in potato.
Functional characterization of ROS-responsive genes, ANAC085 and ATR7, in Arabidopsis thaliana
(2023)
Biostimulant SuperFifty based molecular priming to increase plant strength and stress tolerance
(2023)
In times of ongoing biodiversity loss, understanding how communities are structured and what mechanisms and local adaptations underlie the patterns we observe in nature is crucial for predicting how future ecological and anthropogenic changes might affect local and regional biodiversity. Aquatic zooplankton are a group of primary consumers that represent a critical link in the food chain, providing nutrients for the entire food web. Thus, understanding the adaptability and structure of zooplankton communities is essential. In this work, the genetic basis for the different temperature adaptations of two seasonally shifted (i.e., temperature-dependent) occurring freshwater rotifers of a formerly cryptic species complex (Brachionus calyciflorus) was investigated to understand the overall genetic diversity and evolutionary scenario for putative adaptations to different temperature regimes. Furthermore, this work aimed to clarify to what extent the different temperature adaptations may represent a niche partitioning process thus enabling co-existence. The findings were then embedded in a metacommunity context to understand how zooplankton communities assemble in a kettle hole metacommunity located in the northeastern German "Uckermark" and which underlying processes contribute to the biodiversity patterns we observe. Using a combined approach of newly generated mitochondrial resources (genomes/cds) and the analysis of a candidate gene (Heat Shock Protein 40kDa) for temperature adaptation, I showed that the global representatives of B. calyciflorus s.s.. are genetically more similar than B. fernandoi (average pairwise nucleotide diversity: 0.079 intraspecific vs. 0.257 interspecific) indicating that both species carry different standing genetic variation. In addition to differential expression in the thermotolerant B. calyciflorus s.s. and thermosensitive B. fernandoi, the HSP 40kDa also showed structural variation with eleven fixed and six positively selected sites, some of which are located in functional areas of the protein. The estimated divergence time of ~ 25-29 Myr combined with the fixed sites and a prevalence of ancestral amino acids in B. calyciflorus s.s. indicate that B. calyciflorus s.s. remained in the ancestral niche, while B. fernandoi partitioned into a new niche. The comparison of mitochondrial and nuclear markers (HPS 40kDa, ITS1, COI) revealed a hybridisation event between the two species. However, as hybridisation between the two species is rare, it can be concluded that the temporally isolated niches (i.e., seasonal-shifted occurrence) they inhabit based on their different temperature preferences most likely represent a pre-zygotic isolation mechanism that allows sympatric occurrence while maintaining species boundaries. To determine the processes underlying zooplankton community assembly, a zooplankton metacommunity comprising 24 kettle holes was sampled over a two-year period. Active (i.e., water samples) and dormant communities (i.e., dormant eggs hatched from sediment) were identified using a two-fragment DNA metabarcoding approach (COI and 18S). Species richness and diversity as well as community composition were analysed considering spatial, temporal and environmental parameters. The analysis revealed that environmental filtering based on parameters such as pH, size and location of the habitat patch (i.e., kettle hole) and surrounding field crops largely determined zooplankton community composition (explained variance: Bray-Curtis dissimilarities: 10.5%; Jaccard dissimilarities: 12.9%), indicating that adaptation to a particular habitat is a key feature of zooplankton species in this system. While the spatial configuration of the kettle holes played a minor role (explained variance: Bray-Curtis dissimilarities: 2.8% and Jaccard dissimilarities: 5.5%), the individual kettle hole sites had a significant influence on the community composition. This suggests monopolisation/priority effects (i.e., dormant communities) of certain species in individual kettle holes. As environmental filtering is the dominating process structuring zooplankton communities, this system could be significantly influenced by future land-use change, pollution and climate change.
Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.
Heat stress (HS) is one of the major abiotic stresses which adversely affects the survival and growth of plants due to their sessile nature. To combat the detrimental effects of HS and develop thermotolerance, plants have evolved several defense mechanisms. Thermomemory is one such molecular mechanism whereby plants that have been acclimated (or primed/P) by a moderate HS can respond more efficiently and continue their growth after exposure to a severe or lethal HS (called triggering/T), while unprimed plants cannot survive. Thermomemory is known to be regulated by several transcription factors (TFs), epigenetic changes, chromatin remodellers, post-transcriptional changes and it also involves protein stability control and primary metabolism adjustment. Recent research has suggested that the shoot apical meristem (SAM) in Arabidopsis thaliana has a distinct transcriptional thermomemory which is possibly regulated by eight TFs called HEAT SHOCK FACTORS (HSFs). The main objective of this PhD thesis is to investigate the role of HSFA7b (one of the eight HSFs), in regulating thermomemory at the SAM by identifying the molecular networks it regulates. HSFA7a, a close homolog of HSFA7b, is also one of the eight HSFs that are involved in regulating thermomemory at the SAM. Thermomemory was found to be defective in the hsfa7b and hsfa7a hsfa7b mutants; the percentage survival of these seedlings was significantly lower than in wild-type (WT) seedlings after the priming and triggering (PT) treatment. Transcriptome and ChIP analyses were performed to identify the molecular networks controlled by HSFA7b and its close homolog HSFA7a, in regulating thermomemory at the SAM. The chromatin regulator SPLAYED (SYD) was found to be regulated by both HSFA7a and HSFA7b at the SAM during thermomemory. SYD is directly involved in SAM maintenance by directly regulating WUSCHEL (WUS), a master regulator of stem cell maintenance. WUS expression was down-regulated at the SAM of PT treated hsfa7a/b mutants compared to WT-Col-0 seedlings. HSFA7a and HSFA7b also jointly regulate the expression of orphan gene QUA QUINE STARCH (QQS) during thermomemory. Starch accumulation negatively correlates with QQS expression and this trend was observed in WT plants in response to thermopriming. The remobilization of starch was affected in the hsfa7a/b mutants compared to WT plants during the recovery period after T treatment. These findings indicate that defects in SAM maintenance and starch remobilization could possibly contribute to the reduced thermomemory in the hsfa7a/b mutants. Moreover, transcriptome and ChIP analysis indicate that ethylene signaling genes are directly regulated by HSFA7b during thermomemory. Transcriptome analysis of the HSFA7b-IOE line indicates that HSFA7b positively regulates the expression of HEAT STRESS ASSOCIATED 32 (HSA32), an important thermomemory gene, and HSFA7b strongly suppresses the expression of the reactive oxygen species (ROS) responsive REDOX RESPONSIVE TRANSCRIPTION FACTOR 1 (RRTF1) gene, which is also a repressed target of SYD. In Arabidopsis, the HSFA7b transcript undergoes alternative splicing at high temperatures to form two splice variants: one correctly/constitutively spliced variant which is functional and codes for the HSFA7b protein and one intron retained splice variant. Higher accumulation of the functional HSFA7b splice variant was found at the SAM compared to other tissues. Moreover, accumulation of the functional splice variant was higher in P and PT plants compared to control plants, whereas higher levels of the intron retained splice variant is found in plants subjected directly to the T treatment. The intron retained HSFA7b splice variant is degraded by the non-sense mediated decay (NMD) pathway as a means of regulating transcript level essential for protein synthesis at high temperatures. Importantly, HSFA7b protein accumulation was observed in plants subjected to PT treatment that survive and continue growth, but not in plants subjected directly to T treatment that do not survive, indicating that constitutive/ correct splicing of the HSFA7b transcript is a component of thermomemory. Taken together, these findings suggest that HSFA7a and HSFA7b jointly regulate SAM maintenance via the chromatin remodeller SYD and starch remobilization via QQS. In addition to them, HSFA7b also regulates the expression of ethylene signaling genes, heat responsive genes and the ROS responsive RRTF1. Furthermore, constitutive/correct splicing in the HSFA7b transcript is also an essential component of thermomemory.
Biofilms are heterogeneous structures made of microorganisms embedded in a self-secreted extracellular matrix. Recently, biofilms have been studied as sustainable living materials with a focus on the tuning of their mechanical properties. One way of doing so is to use metal ions. In particular biofilms have been shown to stiffen in presence of some metal cations and to soften in presence of others. However, the specificity and the determinants of those interactions vary between species. While Escherichia coli is a widely studied model organism, little is known concerning the response of its biofilms to metal ions. In this work, we aimed at tuning the mechanics of E. coli biofilms by acting on the interplay between matrix composition and metal cations. To do so, we worked with E. coli strains producing a matrix composed of curli amyloid fibres or phosphoethanolamine-cellulose (pEtN-cellulose) fibres or both. The viscoelastic behaviour of the resulting biofilms was investigated with rheology after incubation with one of the following metal ion solutions: FeCl3, AlCl3, ZnCl2 and CaCl2 or ultrapure water. We observed that the strain producing both fibres stiffen by a factor of two when exposed to the trivalent metal cations Al(III) and Fe(III) while no such response is observed for the bivalent cations Zn(II) and Ca(II). Strains producing only one matrix component did not show any stiffening in response to either cation, but even a small softening. In order to investigate further the contribution of each matrix component to the mechanical properties, we introduced additional bacterial strains producing curli fibres in combination with non-modified cellulose, non-modified cellulose only or neither component. We measured biofilms produced by those different strains with rheology and without any solution. Since rheology does not preserve the architecture of the matrix, we compared those results to the mechanical properties of biofilms probed with the non-destructive microindentation. The microindentation results showed that biofilm stiffness is mainly determined by the presence of curli amyloid fibres in the matrix. However, this clear distinction between biofilm matrices containing or not containing curli is absent from the rheology results, i.e. following partial destruction of the matrix architecture. In addition, rheology also indicated a negative impact of curli on biofilm yield stress and flow stress. This suggests that curli fibres are more brittle and therefore more affected by the mechanical treatments. Finally, to examine the molecular interactions between the biofilms and the metal cations, we used Attenuated total reflectance - Fourier transform infrared spectroscopy (ATR-FTIR) to study the three E.coli strains producing a matrix composed of curli amyloid fibres, pEtN-cellulose fibres or both. We measured biofilms produced by those strains in presence of each of the aforementioned metal cation solutions or ultrapure water. We showed that the three strains cannot be distinguished based on their FTIR spectra and that metal cations seem to have a non-specific effect on bacterial membranes in absence of pEtN-cellulose. We subsequently conducted similar experiments on purified curli or pEtN-cellulose fibres. The spectra of the pEtN-cellulose fibres revealed a non-valence-specific interaction between metal cations and the phosphate of the pEtN-modification. Altogether, these results demonstrate that the mechanical properties of E. coli biofilms can be tuned via incubation with metal ions. While the mechanism involving curli fibres remains to be determined, metal cations seem to adsorb onto pEtN-cellulose and this is not valence-specific. This work also underlines the importance of matrix architecture to biofilm mechanics and emphasises the specificity of each matrix composition.
Twenty-four scientists met for the annual Auxological conference held at Krobielowice castle, Poland, to discuss the diverse influences of the environment and of social behavior on growth following last year’s focus on growth and public health concerns (Hermanussen et al., 2022b). Growth and final body size exhibit marked plastic responses to ecological conditions. Among the shortest are the pygmoid people of Rampasasa, Flores, Indonesia, who still live under most secluded insular conditions. Genetics and nutrition are usually considered responsible for the poor growth in many parts of this world, but evidence is accumulating on the prominent impact of social embedding on child growth. Secular trends not only in the growth of height, but also in body proportions, accompany the secular changes in the social, economic and political conditions, with major influences on the emotional and educational circumstances under which the children grow up (Bogin, 2021). Aspects of developmental tempo and aspects of sports were discussed, and the impact of migration by the example of women from Bangladesh who grew up in the UK. Child growth was considered in particular from the point of view of strategic adjustments of individual size within the network of its social group. Theoretical considerations on network characteristics were presented and related to the evolutionary conservation of growth regulating hypothalamic neuropeptides that have been shown to link behavior and physical growth in the vertebrate species. New statistical approaches were presented for the evaluation of short term growth measurements that permit monitoring child growth at intervals of a few days and weeks.
Background: Assessing short-term growth in humans is still fraught with difficulties. Especially when looking for small variations and increments, such as mini growth spurts, high precision instruments or frequent measurements are necessary. Daily measurements however require a lot of effort, both for anthropologists and for the subjects. Therefore, new sophisticated approaches are needed that reduce fluctuations and reveal underlying patterns.
Objectives: Changepoints are abrupt variations in the properties of time series data. In the context of growth, such variations could be variation in mean height. By adjusting the variance and using different growth models, we assessed the ability of changepoint analysis to analyse short-term growth and detect mini growth spurts.
Sample and Methods: We performed Bayesian changepoint analysis on simulated growth data using the bcp package in R. Simulated growth patterns included stasis, linear growth, catch-up growth, and mini growth spurts. Specificity and a normalised variant of the Matthews correlation coefficient (MCC) were used to assess the algorithm’s performance. Welch’s t-test was used to compare differences of the mean.
Results: First results show that changepoint analysis can detect mini growth spurts. However, the ability to detect mini growth spurts is highly dependent on measurement error. Data preparation, such as ranking and rotating time series data, showed negligible improvements. Missing data was an issue and may affect the prediction quality of the classification metrics.
Conclusion: Changepoint analysis is a promising tool to analyse short-term growth. However, further optimisation and analysis of real growth data is needed to make broader generalisations.
Human growth data analysis and statistics – the 5th Gülpe International Student Summer School
(2023)
The Summer School in Gülpe (Ecological Station of the University of Potsdam) offers an exceptional learning opportunity for students to apply their knowledge and skills to real-world problems. With the guidance of experienced human biologists, statisticians, and programmers, students have the unique chance to analyze their own data and gain valuable insights. This interdisciplinary setting not only bridges different research areas but also leads to highly valuable outputs. The progress of students within just a few days is truly remarkable, especially when they are motivated and receive immediate feedback on their questions, problems, and results. The Summer School covers a wide range of topics, with this year’s focus mainly on two areas: understanding the impact of socioeconomic and physiological factors on human development and mastering statistical techniques for analyzing data such as changepoint analysis and the St. Nicolas House Analysis (SNHA) to visualize interacting variables. The latter technique, born out of the Summer School’s emphasis on gaining comprehensive data insights and understanding major relationships, has proven to be a valuable tool for researchers in the field. The articles in this special issue demonstrate that the Summer School in Gülpe stands as a testament to the power of practical learning and collaboration. Students who attend not only gain hands-on experience but also benefit from the expertise of professionals and the opportunity to engage with peers from diverse disciplines.
No evidence of growth impairment after forced migration in Polish school children after World War II
(2023)
Background: Migration is omnipresent. It can come hand in hand with emotional stress which is known to influence the growth of children.
Objective: The aim of this study was to analyse whether type of migration (forced or voluntary) and the geographic direction had influenced the growth of Polish children after World War II.
Sample and Methods: A sub dataset of 2,208 individuals between the ages of 2-20, created from data of the 2nd Polish Anthropological Survey carried out in 1966–1969, including anthropometrical data and social and demographic information based on questionnaire, was used to analyse migration effects.
Results: No association could be found between the direction of migration and the height of the children. The confidence intervals of the means of all classified migration categories overlap significantly and the effect size of the influence of migration category on height is ds=.140, which is too low to see any effects, even if there were one.
Conclusion: Neither forced nor voluntary migration in Poland after World War II led to a change in height in children of migrating families.
Nutrition, size, and tempo
(2023)
Nutrition is a prerequisite, but not a regulator of growth. Growth is defined as increase in size over time. The understanding of growth includes an understanding of the binary concept of physical time and individual tempo. Excess food causes tempo acceleration. Food restriction delays tempo. Tempo reflects the pace of life. It is a dynamic physical response to a broad spectrum of social, economic, political, and emotional (SEPE) factors and can affect life expectancy. Variations in tempo create distortions of the z-score patterns of height and weight. Illness or intermediate food shortage lead to intermediate halts in development and create short dips in the z-score patterns. Children who develop throughout life at delayed pace usually run at lower z-scores for height and weight, and show a characteristic adolescent trough; children who develop throughout life at faster than average pace usually run at higher z-scores and show a characteristic adolescent peak in their z-score patterns. During adolescence, almost half of the height variance is due to tempo variation. There is not one tempo for the whole body. Different organ systems grow and mature at different pace.
What does stunting tell us?
(2023)
Stunting is commonly linked with undernutrition. Yet, already after World War I, German pediatricians questioned this link and stated that no association exists between nutrition and height. Recent analyses within different populations of Low- and middle-income countries with high rates of stunted children failed to support the assumption that stunted children have a low BMI and skinfold sickness as signs of severe caloric deficiency. So, stunting is not a synonym of malnutrition. Parental education level has a positive influence on body height in stunted populations, e.g., in India and in Indonesia. Socially disadvantaged children tend to be shorter and lighter than children from affluent families.
Humans are social mammals; they regulate growth similar to other social mammals. Also in humans, body height is strongly associated with the position within the social hierarchy, reflecting the personal and group-specific social, economic, political, and emotional environment. These non-nutritional impact factors on growth are summarized by the concept of SEPE (Social-Economic-Political-Emotional) factors. SEPE reflects on prestige, dominance-subordination, social identity, and ego motivation of individuals and social groups.
Light is the essential energy source for plants to drive photosynthesis. In nature, light availability is highly variable and often fluctuates on very short time scales. As a result, plants developed mechanisms to cope with these fluctuations. Understanding how to improve light use efficiency in natural fluctuating light (FL) conditions is a major target for agronomy.
In the first project, we identified an Arabidopsis thaliana plant that showed reduced levels of rapidly inducible non-photochemical quenching (NPQ). This plant was devoid of any T-DNA insertion. Using a mapping-by-sequencing approach, we successfully located the causal genomic region near the end of chromosome 4. Through variant investigations in that region, we identified a deletion of about 20 kb encompassing 9 genes. By complementation analysis, we confirmed that one of the deleted genes, VTC2, is the causal gene responsible for the low NPQ. Loss of VTC2 decreased NPQ particularly in old leaves, with young leaves being only slightly affected. Additionally, ascorbate levels were almost abolished in old leaves, likely causing the NPQ decrease by reducing the activity of the xanthophyll cycle. Although ascorbate levels in younger leaves were reduced compared to wild-type plants, they remained at a comparably higher level. This difference may be due to the VTC2 paralog VTC5, which is expressed at a higher level in young leaves than in old ones.
Plants require the PROTON GRADIENT REGULATION 5 (PGR5) protein for survival in FL. pgr5 mutants die because they fail to increase the luminal proton concentration in response to high light (HL) phases. A rapid elevation in ∆pH is needed to slow down electron transport through the Cytochrome b6 f complex (photosynthetic control). In FL, such lack of control in the pgr5 mutants results in photosystem I (PSI) overreduction, reactive oxygen species (ROS) production, and cell death. Decreases in photosystem II (PSII) activity introduced by crossing pgr5 with PSII deficient mutants
rescued the lethality of pgr5 in FL. PGR5 was suggested to act as part of the ferredoxin-plastoquinone reductase (FQR), involved in cyclic electron transfer around PSI. However, the proposed molecular role of PGR5 remains highly debated. To learn more about PGR5 function, we performed a forward genetic screen in Arabidopsis thaliana to identify EMS-induced suppressor mutants surviving longer when grown in FL compared to pgr5 mutants (referred to as ”suppressor of pgr5 lethality in fluctuating light”, splf ). 11 different candidate genes were identified in a total of 22 splf plants.
Mutants of seven of these genes in the pgr5 background showed low Fv/Fm values when grown in non-fluctuating low light (LL). Five of these 4genes were previously reported to have a role in PSII biogenesis or function. Two others, RPH1 and a DEAD/DEAH box helicase (AT3G02060), have not been linked to PSII function before. Three of splf candidate genes link to primary metabolism, fructose-2,6-bisphosphatase (F2KP ), udp-glucose pyrophosphorylase 1 (UGP1 ) and ferredoxin-dependent glutamate synthase (Fd-GOGAT ). They are characterized by the fact that they survive longer in FL than pgr5 mutants but do not procede beyond the early vegetative
phase and then die.
The St. Nicolas House Algorithm (SNHA) finds association chains of direct dependent variables in a data set. The dependency is based on the correlation coefficient, which is visualized as an undirected graph. The network prediction is improved by a bootstrap routine. It enables the computation of the empirical p-value, which is used to evaluate the significance of the predicted edges. Synthetic data generated with the Monte Carlo method were used to firstly compare the Python package with the original R package, and secondly to evaluate the predicted network using the sensitivity, specificity, balanced classification rate and the Matthew's correlation coefficient (MCC). The Python implementation yields the same results as the R package. Hence, the algorithm was correctly ported into Python. The SNHA scores high specificity values for all tested graphs. For graphs with high edge densities, the other evaluation metrics decrease due to lower sensitivity, which could be partially improved by using bootstrap,while for graphs with low edge densities the algorithm achieves high evaluation scores. The empirical p-values indicated that the predicted edges indeed are significant.
A biological trade-off situation denotes the dependence between traits whereby an increase in the value of one of the traits leads to a decrease in the value of at least one of the others. Understanding trade-offs in cellular systems is relevant to understanding the limits and constraints to tuning desired phenotypes. Therefore, it is mainly the case for rates (i.e. fluxes) of biochemical reactions that shape not only molecular traits, like metabolite concentrations but also determine physiological traits, like growth. Intracellular fluxes are the final phenotype from transcriptional and (post)translational regulation. Quantifying intracellular fluxes provides insights into cellular physiology under particular growth conditions and can be used to characterize the metabolic activity of different pathways. However, estimating fluxes from labelling experiments is labour-intensive; therefore, developing approaches to accurately and precisely predict intracellular fluxes is essential. This thesis addresses two main problems: (i) identifying flux trade-offs and (ii) predicting accurate and precise reaction flux at a genome-scale level. To this end, the concept of an absolute flux trade-off is defined, and a constraint-based approach, termed FluTO, was developed to identify absolute flux trade-offs. FluTO is cast as a mixed integer programming approach applied to genome-scale metabolic models of E. coli, S. cerevisiae, and A. thaliana, imposing realistic constraints on growth and nutrient uptake.. The findings showed that trade-offs are not only species-specific but also specific to carbon sources. In addition, we found that different models of a single species have a different number of reactions in trade-offs. We also showed that absolute flux trade-offs depend on the biomass reaction used to model the growth of A. thaliana under different carbon and nitrogen conditions. Findings reflect the strong relation between nitrogen, carbon, and sulphur metabolisms in the leaves of C3 plants. The concept of relative trade-offs was introduced to further study trade-offs in metabolic networks. A constraint-based approach, FluTOr, was proposed to identify reactions whose fluxes are in relative trade-off concerning an optimized fitness-related cellular task, like growth. FluTOr was employed to find the relative flux trade-offsin the genome-scale metabolic networks of E. coli, S. cerevisiae, and A. thaliana. The results showed that in contrast to the A. thaliana model, the relative trade-offs in the two microorganisms depend on the carbon source, reflecting the differences in the underlying metabolic network. Furthermore, applying FluTOr also showed that reactions that participated in relative trade-offs were implicated in cofactor biosynthesis in the two microorganisms. Prediction of reaction fluxes in the constraint-based metabolic framework is usually performed by parsimonious flux balance analysis (pFBA), employing the principle of efficient usage of protein resources. However, we argued that principles related to the coordination of flux values, neglected in previous studies, provide other means to predict intracellular fluxes. To this end, we designed a constraint-based approach, termed complex-balanced FBA (cbFBA), to predict steady-state flux distributions that maximize the number of balanced complexes in a flux distribution, whereby multi-reaction dependencies are maximized. The comparative analysis showed a better agreement of the flux distributions resulting from cbFBA compared to pFBA with experimentally measured fluxes from 17 E. coli strains and 26 S. cerevisiae knock-out mutants. The results also showed that the predictions from cbFBA are more precise than those from pFBA since cbFBA results in a smaller space of alternative solutions than pFBA.
The emerging threat of antibiotic-resistant bacteria has become a global challenge in the last decades, leading to a rising demand for alternative treatments for bacterial infections. One approach is to target the bacterial cell envelope, making understanding its biophysical properties crucial. Specifically, bacteriophages use the bacterial envelope as an entry point to initiate infection, and they are considered important building blocks of new antibiotic strategies against drug-resistant bacteria.. Depending on the structure of the cell wall, bacteria are classified as Gram-negative and Gram-positive. Gram-negative bacteria are equipped with a complex cell envelope composed of two lipid membranes enclosing a rigid peptidoglycan layer. The synthesis machinery of the Gram-negative cell envelope is the target of antimicrobial agents, including new physical sanitizing procedures addressing the outer membrane (OM). It is therefore very important to study the biophysical properties of the Gram-negative bacterial cell envelope. The high complexity of the Gram-negative OM sets the demand for a model system in which the contribution of individual components can be evaluated separately. In this respect, giant unilamellar vesicles (GUVs) are promising membrane systems to study membrane properties while controlling parameters such as membrane composition and surrounding medium conditions.
The aim of this work was to develop methods and approaches for the preparation and characterization of a GUV-based membrane model that mimics the OM of the Gram-negative cell envelope. A major component of the OM is the lipopolysaccharide (LPS) on the outside of the OM heterobilayer. The vesicle model was designed to contain LPS in the outer leaflet and lipids in the inner leaflet. Furthermore, the interaction of the prepared LPS-GUVs with bacteriophages was tested. LPS containing GUVs were prepared by adapting the inverted emulsion technique to meet the challenging properties of LPS, namely their high self-aggregation rate in aqueous solutions. Notably, an additional emulsification step together with the adaption of solution conditions was employed to asymmetrically incorporate LPS containing long polysaccharide chains into the artificial membranes. GUV membrane asymmetry was verified with a fluorescence quenching assay. Since the necessary precautions for handling the quenching agent sodium dithionite are often underestimated and poorly described, important parameters were tested and identified to obtain a stable and reproducible assay. In the context of varied LPS incorporation, a microscopy-based technique was introduced to determine the LPS content on individual GUVs and to directly compare vesicle properties and LPS coverage. Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.
Employing LPS-GUVs we could demonstrate that a Salmonella bacteriophage bound with high specificity to its LPS receptor when presented at the GUV surface, and that the number of bound bacteriophages scaled with the amount of presented LPS receptor. In addition to binding, the bacteriophages were able to eject their DNA into the vesicle lumen. LPS-GUVs thus provide a starting platform for bottom-up approaches for the generation of more complex membranes, in which the effects of individual components on the membrane properties and the interaction with antimicrobial agents such as bacteriophages could be explored.
Protein-protein-interactions play an important role in many cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced "blinking" and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N & B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for protein-protein-interactions quantification via fluorescence fluctuation-based methods.
Genomic and epigenomic determinants of heat stress-induced transcriptional memory in Arabidopsis
(2023)
Background
Transcriptional regulation is a key aspect of environmental stress responses. Heat stress induces transcriptional memory, i.e., sustained induction or enhanced re-induction of transcription, that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crop plants is an important breeding goal. However, not all heat stress-inducible genes show transcriptional memory, and it is unclear what distinguishes memory from non-memory genes. To address this issue and understand the genome and epigenome architecture of transcriptional memory after heat stress, we identify the global target genes of two key memory heat shock transcription factors, HSFA2 and HSFA3, using time course ChIP-seq.
Results
HSFA2 and HSFA3 show near identical binding patterns. In vitro and in vivo binding strength is highly correlated, indicating the importance of DNA sequence elements. In particular, genes with transcriptional memory are strongly enriched for a tripartite heat shock element, and are hallmarked by several features: low expression levels in the absence of heat stress, accessible chromatin environment, and heat stress-induced enrichment of H3K4 trimethylation. These results are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes.
Conclusions
Our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants, enabling the prediction and engineering of genes with transcriptional memory behavior.
Spatial and temporal variation in perceived predation risk is an important determinant of movement and foraging activity of animals. Foraging in this landscape of fear, individuals need to decide where and when to move, and what resources to choose. Foraging theory predicts the outcome of these decisions based on energetic trade-offs, but complex interactions between perceived predation risk and preferences of foragers for certain functional traits of their resources are rarely considered. Here, we studied the interactive effects of perceived predation risk on food trait preferences and foraging behavior in bank voles (Myodes glareolus) in experimental landscapes. Individuals (n = 19) were subjected for periods of 24 h to two extreme, risk-uniform landscapes (either risky or safe), containing 25 discrete food patches, filled with seeds of four plant species in even amounts. Seeds varied in functional traits: size, nutrients, and shape. We evaluated whether and how risk modifies forager preference for functional traits. We also investigated whether perceived risk and distance from shelter affected giving-up density (GUD), time in patches, and number of patch visits. In safe landscapes, individuals increased time spent in patches, lowered GUD and visited distant patches more often compared to risky landscapes. Individuals preferred bigger seeds independent of risk, but in the safe treatment they preferred fat-rich over carb-rich seeds. Thus, higher densities of resource levels remained in risky landscapes, while in safe landscapes resource density was lower and less diverse due to selective foraging. Our results suggest that the interaction of perceived risk and dietary preference adds an additional layer to the cascading effects of a landscape of fear which affects biodiversity at resource level.
The oil palm (Elaeis guineensis Jacq.) produces a large amount of oil from the fruit. However, increasing the oil production in this fruit is still challenging. A recent study has shown that starch metabolism is essential for oil synthesis in fruit-producing species. Therefore, the transcriptomic analysis by RNA-seq was performed to observe gene expression alteration related to starch metabolism genes throughout the maturity stages of oil palm fruit with different oil yields. Gene expression profiles were examined with three different oil yields group (low, medium, and high) at six fruit development phases (4, 8, 12, 16, 20, and 22 weeks after pollination). We successfully identified and analyzed differentially expressed genes in oil palm mesocarps during development. The results showed that the transcriptome profile for each developmental phase was unique. Sucrose flux to the mesocarp tissue, rapid starch turnover, and high glycolytic activity have been identified as critical factors for oil production in oil palms. For starch metabolism and the glycolytic pathway, we identified specific gene expressions of enzyme isoforms (isozymes) that correlated with oil production, which may determine the oil content. This study provides valuable information for creating new high-oil-yielding palm varieties via breeding programs or genome editing approaches.
Starch has been a convenient, economically important polymer with substantial applications in the food and processing industry. However, native starches present restricted applications, which hinder their industrial usage. Therefore, modification of starch is carried out to augment the positive characteristics and eliminate the limitations of the native starches. Modifications of starch can result in generating novel polymers with numerous functional and value-added properties that suit the needs of the industry. Here, we summarize the possible starch modifications in planta and outside the plant system (physical, chemical, and enzymatic) and their corresponding applications. In addition, this review will highlight the implications of each starch property adjustment.
Keeping cool on hot days
(2023)
Long-lived organisms are likely to respond to a rapidly changing climate with behavioral flexibility. Animals inhabiting the arid parts of southern Africa face a particularly rapid rise in temperature which in combination with food and water scarcity places substantial constraints on the ability of animals to tolerate heat. We investigated how three species of African antelope-springbok Antidorcas marsupialis, kudu Tragelaphus strepsiceros and eland T. oryx-differing in body size, habitat preference and movement ecology, change their activity in response to extreme heat in an arid savanna. Serving as a proxy for activity, dynamic body acceleration data recorded every five minutes were analyzed for seven to eight individuals per species for the three hottest months of the year. Activity responses to heat during the hottest time of day (the afternoons) were investigated and diel activity patterns were compared between hot and cool days. Springbok, which prefer open habitat, are highly mobile and the smallest of the species studied, showed the greatest decrease in activity with rising temperature. Furthermore, springbok showed reduced mean activity over the 24 h cycle on hot days compared to cool days. Large-bodied eland seemed less affected by afternoon heat than springbok. While eland also reduced diurnal activity on hot days compared to cool days, they compensated for this by increasing nocturnal activity, possibly because their predation risk is lower. Kudu, which are comparatively sedentary and typically occupy shady habitat, seemed least affected during the hottest time of day and showed no appreciable difference in diel activity patterns between hot and cool days. The interplay between habitat preference, body size, movement patterns, and other factors seems complex and even sub-lethal levels of heat stress have been shown to impact an animal's long-term survival and reproduction. Thus, differing heat tolerances among species could result in a shift in the composition of African herbivore communities as temperatures continue to rise, with significant implications for economically important wildlife-based land use and conservation.
The heat is on
(2023)
Climate conditions severely impact the activity and, consequently, the fitness of wildlife species across the globe. Wildlife can respond to new climatic conditions, but the pace of human-induced change limits opportunities for adaptation or migration. Thus, how these changes affect behavior, movement patterns, and activity levels remains unclear. In this study, we investigate how extreme weather conditions affect the activity of European hares (Lepus europaeus) during their peak reproduction period. When hares must additionally invest energy in mating, prevailing against competitors, or lactating, we investigated their sensitivities to rising temperatures, wind speed, and humidity. To quantify their activity, we used the overall dynamic body acceleration (ODBA) calculated from tri-axial acceleration measurements of 33 GPS-collared hares. Our analysis revealed that temperature, humidity, and wind speed are important in explaining changes in activity, with a strong response for high temperatures above 25 & DEG;C and the highest change in activity during temperature extremes of over 35 & DEG;C during their inactive period. Further, we found a non-linear relationship between temperature and activity and an interaction of activity changes between day and night. Activity increased at higher temperatures during the inactive period (day) and decreased during the active period (night). This decrease was strongest during hot tropical nights. At a stage of life when mammals such as hares must substantially invest in reproduction, the sensitivity of females to extreme temperatures was particularly pronounced. Similarly, both sexes increased their activity at high humidity levels during the day and low wind speeds, irrespective of the time of day, while the effect of humidity was stronger for males. Our findings highlight the importance of understanding the complex relationships between extreme weather conditions and mammal behavior, critical for conservation and management. With ongoing climate change, extreme weather events such as heat waves and heavy rainfall are predicted to occur more often and last longer. These events will directly impact the fitness of hares and other wildlife species and hence the population dynamics of already declining populations across Europe.
Progressive habitat fragmentation threatens plant species with narrow habitat requirements. While local environmental conditions define population growth rates and recruitment success at the patch level, dispersal is critical for population viability at the landscape scale. Identifying the dynamics of plant meta-populations is often confounded by the uncertainty about soil-stored population compartments. We combined a landscape-scale assessment of an amphibious plant's population structure with measurements of dispersal complexity in time to track dispersal and putative shifts in functional connectivity. Using 13 microsatellite markers, we analyzed the genetic structure of extant Oenanthe aquatica populations and their soil seed banks in a kettle hole system to uncover hidden connectivity among populations in time and space. Considerable spatial genetic structure and isolation-by-distance suggest limited gene flow between sites. Spatial isolation and patch size showed minor effects on genetic diversity. Genetic similarity found among extant populations and their seed banks suggests increased local recruitment, despite some evidence of migration and recent colonization. Results indicate stepping-stone dispersal across adjacent populations. Among permanent and ephemeral demes the resulting meta-population demography could be determined by source-sink dynamics. Overall, these spatiotemporal connectivity patterns support mainland-island dynamics in our system, highlighting the importance of persistent seed banks as enduring sources of genetic diversity.
Metabolic engineering of microalgae offers a promising solution for sustainable biofuel production, and rational design of engineering strategies can be improved by employing metabolic models that integrate enzyme turnover numbers. However, the coverage of turnover numbers for Chlamydomonas reinhardtii, a model eukaryotic microalga accessible to metabolic engineering, is 17-fold smaller compared to the heterotrophic cell factory Saccharomyces cerevisiae. Here we generate quantitative protein abundance data of Chlamydomonas covering 2337 to 3708 proteins in various growth conditions to estimate in vivo maximum apparent turnover numbers. Using constrained-based modeling we provide proxies for in vivo turnover numbers of 568 reactions, representing a 10-fold increase over the in vitro data for Chlamydomonas. Integration of the in vivo estimates instead of in vitro values in a metabolic model of Chlamydomonas improved the accuracy of enzyme usage predictions. Our results help in extending the knowledge on uncharacterized enzymes and improve biotechnological applications of Chlamydomonas.
The physiological dependence of animals on dietary intake of vitamins, amino acids, and fatty acids is ubiquitous. Sharp differences in the availability of these vital dietary biomolecules among different resources mean that consumers must adopt a range of strategies to meet their physiological needs. We review the emerging work on omega-3 long-chain polyunsaturated fatty acids, focusing predominantly on predator-prey interactions, to illustrate that trade-off between capacities to consume resources rich in vital biomolecules and internal synthesis capacity drives differences in phenotype and fitness of consumers. This can then feedback to impact ecosystem functioning. We outline how focus on vital dietary biomolecules in eco-eco-devo dynamics can improve our understanding of anthropogenic changes across multiple levels of biological organization.
The European Union is highly dependent on soybean imports from overseas to meet its protein demands. Individual Member States have been quick to declare self-sufficiency targets for plant-based proteins, but detailed strategies are still lacking. Rising global temperatures have painted an image of a bright future for soybean production in Europe, but emerging climatic risks such as drought have so far not been included in any of those outlooks.
Here, we present simulations of future soybean production and the most prominent risk factors across Europe using an ensemble of climate and soybean growth models. Projections suggest a substantial increase in potential soybean production area and productivity in Central Europe, while southern European production would become increasingly dependent on supplementary irrigation. Average productivity would rise by 8.3% (RCP 4.5) to 8.7% (RCP 8.5) as a result of improved growing conditions (plant physiology benefiting from rising temperature and CO2 levels) and farmers adapting to them by using cultivars with longer phenological cycles. Suitable production area would rise by 31.4% (RCP 4.5) to 37.7% (RCP 8.5) by the mid-century, contributing considerably more than productivity increase to the production potential for closing the protein gap in Europe.
While wet conditions at harvest and incidental cold spells are the current key challenges for extending soybean production, the models and climate data analysis anticipate that drought and heat will become the dominant limitations in the future. Breeding for heat-tolerant and water-efficient genotypes is needed to further improve soybean adaptation to changing climatic conditions.
This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.
Background
Teleost fishes comprise more than half of the vertebrate species. Within teleosts, most phylogenies consider the split between Osteoglossomorpha and Euteleosteomorpha/Otomorpha as basal, preceded only by the derivation of the most primitive group of teleosts, the Elopomorpha. While Osteoglossomorpha are generally species poor, the taxon contains the African weakly electric fish (Mormyroidei), which have radiated into numerous species. Within the mormyrids, the genus Campylomormyrus is mostly endemic to the Congo Basin. Campylomormyrus serves as a model to understand mechanisms of adaptive radiation and ecological speciation, especially with regard to its highly diverse species-specific electric organ discharges (EOD). Currently, there are few well-annotated genomes available for electric fish in general and mormyrids in particular. Our study aims at producing a high-quality genome assembly and to use this to examine genome evolution in relation to other teleosts. This will facilitate further understanding of the evolution of the osteoglossomorpha fish in general and of electric fish in particular.
Results
A high-quality weakly electric fish (C. compressirostris) genome was produced from a single individual with a genome size of 862 Mb, consisting of 1,497 contigs with an N50 of 1,399 kb and a GC-content of 43.69%. Gene predictions identified 34,492 protein-coding genes, which is a higher number than in the two other available Osteoglossomorpha genomes of Paramormyrops kingsleyae and Scleropages formosus. A Computational Analysis of gene Family Evolution (CAFE5) comparing 33 teleost fish genomes suggests an overall faster gene family turnover rate in Osteoglossomorpha than in Otomorpha and Euteleosteomorpha. Moreover, the ratios of expanded/contracted gene family numbers in Osteoglossomorpha are significantly higher than in the other two taxa, except for species that had undergone an additional genome duplication (Cyprinus carpio and Oncorhynchus mykiss). As potassium channel proteins are hypothesized to play a key role in EOD diversity among species, we put a special focus on them, and manually curated 16 Kv1 genes. We identified a tandem duplication in the KCNA7a gene in the genome of C. compressirostris.
Conclusions
We present the fourth genome of an electric fish and the third well-annotated genome for Osteoglossomorpha, enabling us to compare gene family evolution among major teleost lineages. Osteoglossomorpha appear to exhibit rapid gene family evolution, with more gene family expansions than contractions. The curated Kv1 gene family showed seven gene clusters, which is more than in other analyzed fish genomes outside Osteoglossomorpha. The KCNA7a, encoding for a potassium channel central for EOD production and modulation, is tandemly duplicated which may related to the diverse EOD observed among Campylomormyrus species.
The development of seeds in angiosperms starts with a complex process of double fertilization, involving the fusion of the maternal egg cell and central cell with two paternal sperm cells. This gives rise to the embryo and the nourishing endosperm, which are then enclosed by the seed coat, derived from the maternal integuments. The growth of the seed coat in Arabidopsis thaliana (Arabidopsis) is actively inhibited before fertilization by epigenetic regulators known as Polycomb Group (PcG) proteins. These proteins deposit a repressive histone mark called H3K27me3, which must be removed to enable seed coat formation. In this thesis, I explored the mechanism of removal of H3K27me3 marks from the integument cells following fertilization, which allows for seed coat formation. We hypothesized that this removal should be primarily facilitated by histone demethylases from the JMJ family and potentially influenced by the plant hormones Brassinosteroids (BRs). This hypothesis was supported by the expression patterns of the JMJ protein REF6 and of BR related genes, which are specifically expressed in the integuments and in the seed coat. Moreover, mutations in both these pathways lead to developmental defects, such as reduced ovule viability and delayed seed coat growth. Our research provides evidence suggesting that BR signalling is likely involved in recruiting JMJ-type histone demethylases to target loci responsible for seed coat growth. Moreover, we have discovered an additional pathway through which BRs regulate seed coat development, independent of their influence on H3K27me3 marks. This finding emphasizes the diverse roles of BRs in coordinating seed development, extending beyond their well-known involvement in plant growth and development. Furthermore, I explored the role of another epigenetic mark, DNA methylation, in fertilization-independent (or autonomous) seed formation in Arabidopsis. For this, we utilized epigenetic Recombinant Inbred Lines (epiRILs) and thus identified an epigenetic Quantitative Trait Locus (epiQTL) on chromosome II, potentially responsible for the larger autonomous seed size observed in DNA methylation mutants. Overall, this thesis significantly enhances our comprehension of the intricate relationship between epigenetic modifications, hormonal signaling, and plant reproductive processes. It offers valuable insights into the genetic mechanisms governing both sexual and asexual seed formation, while also presenting potential avenues for the engineer of advantageous traits in agricultural crops.
In nature, plants often encounter biotic and abiotic stresses, which can cause reduced crop yield and quality, and threaten the nutrition of a growing human population. As heat stress (HS) is one of the main abiotic stresses, and is projected to increase due to global warming, it is necessary to better understand how plants respond and survive under HS. In Arabidopsis thaliana, plants can survive under severe HS if primed by a non-lethal HS, a process called acquisition of thermotolerance. This primed stated can be maintained for several days, and the ability of plants to maintain the primed state is called maintenance of acquired thermotolerance (mATT) or HS memory. According to current research, two Heat shock factors (HSFs) HSFA2 and HSFA3 are known to account for the majority of mATT capability, and there are other HSFs e.g. HSFA1b and HSFA6b in HSF complexes containing HSFA2 and/or HSFA3, however, the roles of these HSFs in HS memory is not clearly understood. Moreover, the mechanism of these HSFs in regulating HS memory is unclear, whether transcriptional machinery e.g. the Mediator complex contributes to transcriptional memory. This work investigates the role of HSFs and Mediator subunits in HS memory in A. thaliana. For the role of HSFs, the interaction between HSFA1b and HSFA2 during HS memory phase was confirmed by in vivo co- immunoprecipitation (Co-IP). HSFA1b, HSFA2, HSFA3 and HSFA6b targeted HS memory-related genes according to DNA affinity purification sequencing (DAP-seq) data, and targets of HSFA1b were confirmed in vivo by chromatin immunoprecipitation qPCR (ChIP-qPCR). The mutant of hsfa6b showed an HS memory deficiency phenotype in mATT survival assay. These data confirmed the role for HSFA2 and HSFA3 in HS memory, and suggest that HSFA1b and HSFA6b also function in HS memory. The Mediator complex functions as an RNA Polymerase II (RNA Pol II) co-regulator, and includes Head, Middle, Tail and Kinase modules. Both MED23 and MED32 belong to the Tail module, and they have a positive role in HS memory. MED23 interacted with HSFA3, as determined by yeast two hybrid (Y2H) and in vivo Co-IP assays. The med23 mutant showed a decreased HS memory phenotype, reduced expression of Type I (sustained expression) memory genes following HS, and reduced accumulation of the memory-associated Tri-methylation of histone H3 lysine 4 (H3K4me3)histone modification at HS memory-related gene loci after HS. MED23 was recruited to HS-inducible memory and non-memory genes after HS, as determined by ChIP-qPCR. The med32
mutant showed a reduced HS memory phenotype, decreased expression of Type I and Type II (hyper-induction) memory genes, and lower accumulation of H3K4me3 at memory gene lociafter HS. However, MED32 did not show interaction with any tested HSF in Y2H or in vivo Co-IP. MED32 regulated the recruitment of RNA Pol II at HS-inducible genes after HS, but was not itself recruited to HS memory genes after HS. These results provided more evidence
that the Mediator subunits MED23 and MED32 regulate HS memory on transcriptional and epigenetic levels. In general, this work provides a better insight into the molecular mechanism of how HSFs and Mediator subunits regulate HS memory in plants and will provide new perspectives to breed crops with improved thermotolerance.
The capillary-venous pathology cerebral cavernous malformation (CCM) is caused by loss of CCM1/Krev interaction trapped protein 1 (KRIT1), CCM2/MGC4607, or CCM3/PDCD10 in some endothelial cells. Mutations of CCM genes within the brain vasculature can lead to recurrent cerebral hemorrhages. Pharmacological treatment options are urgently needed when lesions are located in deeply-seated and in-operable regions of the central nervous system. Previous pharmacological suppression screens in disease models of CCM led to the discovery that treatment with retinoic acid improved CCM phenotypes. This finding raised a need to investigate the involvement of retinoic acid in CCM and test whether it has a curative effect in preclinical mouse models. Here, we show that components of the retinoic acid synthesis and degradation pathway are transcriptionally misregulated across disease models of CCM. We complemented this analysis by pharmacologically modifying retinoic acid levels in zebrafish and human endothelial cell models of CCM, and in acute and chronic mouse models of CCM. Our pharmacological intervention studies in CCM2-depleted human umbilical vein endothelial cells (HUVECs) and krit1 mutant zebrafish showed positive effects when retinoic acid levels were increased. However, therapeutic approaches to prevent the development of vascular lesions in adult chronic murine models of CCM were drug regiment-sensitive, possibly due to adverse developmental effects of this hormone. A treatment with high doses of retinoic acid even worsened CCM lesions in an adult chronic murine model of CCM. This study provides evidence that retinoic acid signaling is impaired in the CCM pathophysiology and suggests that modification of retinoic acid levels can alleviate CCM phenotypes.
Cell-level systems biology model to study inflammatory bowel diseases and their treatment options
(2023)
To help understand the complex and therapeutically challenging inflammatory bowel diseases (IBDs), we developed a systems biology model of the intestinal immune system that is able to describe main aspects of IBD and different treatment modalities thereof. The model, including key cell types and processes of the mucosal immune response, compiles a large amount of isolated experimental findings from literature into a larger context and allows for simulations of different inflammation scenarios based on the underlying data and assumptions. In the context of a large and diverse virtual IBD population, we characterized the patients based on their phenotype (in contrast to healthy individuals, they developed persistent inflammation after a trigger event) rather than on a priori assumptions on parameter differences to a healthy individual. This allowed to reproduce the enormous diversity of predispositions known to lead to IBD. Analyzing different treatment effects, the model provides insight into characteristics of individual drug therapy. We illustrate for anti-TNF-alpha therapy, how the model can be used (i) to decide for alternative treatments with best prospects in the case of nonresponse, and (ii) to identify promising combination therapies with other available treatment options.
The deficiency of a (bio)chemical reaction network can be conceptually interpreted as a measure of its ability to support exotic dynamical behavior and/or multistationarity. The classical definition of deficiency relates to the capacity of a network to permit variations of the complex formation rate vector at steady state, irrespective of the network kinetics. However, the deficiency is by definition completely insensitive to the fine details of the directionality of reactions as well as bounds on reaction fluxes. While the classical definition of deficiency can be readily applied in the analysis of unconstrained, weakly reversible networks, it only provides an upper bound in the cases where relevant constraints on reaction fluxes are imposed. Here we propose the concept of effective deficiency, which provides a more accurate assessment of the network’s capacity to permit steady state variations at the complex level for constrained networks of any reversibility patterns. The effective deficiency relies on the concept of nonstoichiometric balanced complexes, which we have already shown to be present in real-world biochemical networks operating under flux constraints. Our results demonstrate that the effective deficiency of real-world biochemical networks is smaller than the classical deficiency, indicating the effects of reaction directionality and flux bounds on the variation of the complex formation rate vector at steady state.
Introduction Flux phenotypes from different organisms and growth conditions allow better understanding of differential metabolic networks functions. Fluxes of metabolic reactions represent the integrated outcome of transcription, translation, and post-translational modifications, and directly affect growth and fitness. However, fluxes of intracellular metabolic reactions cannot be directly measured, but are estimated via metabolic flux analysis (MFA) that integrates data on isotope labeling patterns of metabolites with metabolic models. While the application of metabolomics technologies in photosynthetic organisms have resulted in unprecedented data from 13CO2-labeling experiments, the bottleneck in flux estimation remains the application of isotopically nonstationary MFA (INST-MFA). INST-MFA entails fitting a (large) system of coupled ordinary differential equations, with metabolite pools and reaction fluxes as parameters. Here, we focus on the Calvin-Benson cycle (CBC) as a key pathway for carbon fixation in photosynthesizing organisms and ask if approaches other than classical INST-MFA can provide reliable estimation of fluxes for reactions comprising this pathway.
Methods First, we show that flux estimation with the labeling patterns of all CBC intermediates can be formulated as a single constrained regression problem, avoiding the need for repeated simulation of time-resolved labeling patterns.
Results We then compare the flux estimates of the simulation-free constrained regression approach with those obtained from the classical INST-MFA based on labeling patterns of metabolites from the microalgae Chlamydomonas reinhardtii, Chlorella sorokiniana and Chlorella ohadii under different growth conditions.
Discussion Our findings indicate that, in data-rich scenarios, simulation-free regression-based approaches provide a suitable alternative for flux estimation from classical INST-MFA since we observe a high qualitative agreement (rs=0.89) to predictions obtained from INCA, a state-of-the-art tool for INST-MFA.
Plant metabolism serves as the primary mechanism for converting assimilated carbon into essential compounds crucial for plant growth and ultimately, crop yield. This renders it a focal point of research with significant implications. Despite notable strides in comprehending the genetic principles underpinning metabolism and yield, there remains a dearth of knowledge regarding the genetic factors responsible for trait variation under varying environmental conditions. Given the burgeoning global population and the advancing challenges posed by climate change, unraveling the intricacies of metabolic and yield responses to water scarcity became increasingly important in safeguarding food security.
Our research group has recently started to work on the genetic resources of legume species. To this end, the study presented here investigates the metabolic diversity across five different legume species at a tissue level, identifying species-specific biosynthesis of alkaloids as well as iso-/flavonoids with diverse functional groups, namely prenylation, phenylacylation as well as methoxylation, to create a resource for follow up studies investigation the metabolic diversity in natural diverse populations of legume species.
Following this, the second study investigates the genetic architecture of drought-induced changes in a global common bean population. Here, a plethora of quantitative trait loci (QTL) associated with various traits are identified by performing genome-wide association studies (GWAS), including for lipid signaling. On this site, overexpression of candidates highlighted the induction of several oxylipins reported to be pivotal in coping with harsh environmental conditions such as water scarcity.
Diverging from the common bean and GWAS, the following study focuses on identifying drought-related QTL in tomato using a bi-parental breeding population. This descriptive study highlights novel multi-omic QTL, including metabolism, photosynthesis as well as fruit setting, some of which are uniquely assigned under drought. Compared to conventional approaches using the bi-parental IL population, the study presented improves the resolution by assessing further backcrossed ILs, named sub-ILs.
In the final study, a photosynthetic gene, namely a PetM subunit of the cytochrome b6f complex encoding gene, involved in electron flow is characterized in an horticultural important crop. While several advances have been made in model organisms, this study highlights the transition of this fundamental knowledge to horticultural important crops, such as tomato, and investigates its function under differing light conditions. Overall, the presented thesis combines different strategies in unveiling the genetic components in multi-omic traits under drought using conventional breeding populations as well as a diverse global population. To this end, it allows a comparison of either approach and highlights their strengths and weaknesses.
Leaves exhibit cells with varying degrees of shape complexity along the proximodistal axis. Heterogeneities in growth directions within individual cells bring about such complexity in cell shape. Highly complex and interconnected gene regulatory networks and signaling pathways have been identified to govern these processes. In addition, the organization of cytoskeletal networks and cell wall mechanical properties greatly influences the regulation of cell shape. Research has shown that microtubules are involved in regulating cellulose deposition and direc-tion of cell growth. However, comprehensive analysis of the regulation of the actin cytoskele-ton in cell shape regulation has not been well studied.
This thesis provides evidence that actin regulates aspects of cell growth, division, and direction-al expansion that impacts morphogenesis of developing leaves. The jigsaw puzzle piece mor-phology of epidermal pavement cells further serves as an ideal system to investigate the com-plex process of morphogenetic processes occurring at the cellular level. Here we have em-ployed live cell based imaging studies to track the development of pavement cells in actin com-promised conditions. Genetic perturbation of two predominantly expressed vegetative actin genes ACTIN2 and ACTIN7 results in delayed emergence of the cellular protrusions in pave-ment cells. Perturbation of actin also impacted the organization of microtubule in these cells that is known to promote emergence of cellular protrusions. Further, live-cell imaging of actin or-ganization revealed a correlation with cell shape, suggesting that actin plays a role in influencing pavement cell morphogenesis.
In addition, disruption of actin leads to an increase in cell size along the leaf midrib, with cells being highly anisotropic due to reduced cell division. The reduction of cell division further im-pacted the morphology of the entire leaf, with the mutant leaves being more curved. These re-sults suggests that actin plays a pivotal role in regulating morphogenesis at the cellular and tis-sue scales thereby providing valuable insights into the role of the actin cytoskeleton in plant morphogenesis.
Starch is an essential biopolymer produced by plants. Starch can be made inside source tissue (such as leaves) and sink tissue (such as fruits and tubers). Nevertheless, understanding how starch metabolism is regulated in source and sink tissues is fundamental for improving crop production.
Despite recent advances in the understanding of starch and its metabolism, there is still a knowledge gap in the source and sink metabolism. Therefore, this study aimed to summarize the state of the art regarding starch structure and metabolism inside plants. In addition, this study aimed to elucidate the regulation of starch metabolism in the source tissue using the leaves of a model organism, Arabidopsis thaliana, and the sink tissue of oil palm (Elaeis guineensis) fruit as a commercial crop.
The research regarding the source tissue will focus on the effect of the blockage of starch degradation on the starch parameter in leaves, especially in those of A. thaliana, which lack both disproportionating enzyme 2 (DPE2) and plastidial glucan phosphorylase 1 (PHS1) (dpe2/phs1). The additional elimination of phosphoglucan water dikinase (PWD), starch excess 4 (SEX4), isoamylase 3 (ISA3), and disproportionating enzyme 1 (DPE1) in the dpe2/phs1 mutant background demonstrates the alteration of starch granule number per chloroplast. This study provides insights into the control mechanism of granule number regulation in the chloroplast.
The research regarding the sink tissue will emphasize the relationship between starch metabolism and the lipid metabolism pathway in oil palm fruits. This study was conducted to observe the alteration of starch parameters, metabolite abundance, and gene expression during oil palm fruit development with different oil yields. This study shows that starch and sucrose can be used as biomarkers for oil yield in oil palms. In addition, it is revealed that the enzyme isoforms related to starch metabolism influence the oil production in oil palm fruit.
Overall, this thesis presents novel information regarding starch metabolism in the source tissue of A.thaliana and the sink tissue of E.guineensis. The results shown in this thesis can be applied to many applications, such as modifying the starch parameter in other plants for specific needs.
The use of automated tools to reconstruct lipid metabolic pathways is not warranted in plants. Here, the authors construct Plant Lipid Module for Arabidopsis rosette using constraint-based modeling, demonstrate its integration in other plant metabolic models, and use it to dissect the genetic architecture of lipid metabolism.
Lipids play fundamental roles in regulating agronomically important traits. Advances in plant lipid metabolism have until recently largely been based on reductionist approaches, although modulation of its components can have system-wide effects. However, existing models of plant lipid metabolism provide lumped representations, hindering detailed study of component modulation. Here, we present the Plant Lipid Module (PLM) which provides a mechanistic description of lipid metabolism in the Arabidopsis thaliana rosette. We demonstrate that the PLM can be readily integrated in models of A. thaliana Col-0 metabolism, yielding accurate predictions (83%) of single lethal knock-outs and 75% concordance between measured transcript and predicted flux changes under extended darkness. Genome-wide associations with fluxes obtained by integrating the PLM in diel condition- and accession-specific models identify up to 65 candidate genes modulating A. thaliana lipid metabolism. Using mutant lines, we validate up to 40% of the candidates, paving the way for identification of metabolic gene function based on models capturing natural variability in metabolism.
Wild bee species are important pollinators in agricultural landscapes. However, population decline was reported over the last decades and is still ongoing. While agricultural intensification is a major driver of the rapid loss of pollinating species, transition zones between arable fields and forest or grassland patches, i.e., agricultural buffer zones, are frequently mentioned as suitable mitigation measures to support wild bee populations and other pollinator species. Despite the reported general positive effect, it remains unclear which amount of buffer zones is needed to ensure a sustainable and permanent impact for enhancing bee diversity and abundance. To address this question at a pollinator community level, we implemented a process-based, spatially explicit simulation model of functional bee diversity dynamics in an agricultural landscape. More specifically, we introduced a variable amount of agricultural buffer zones (ABZs) at the transition of arable to grassland, or arable to forest patches to analyze the impact on bee functional diversity and functional richness. We focused our study on solitary bees in a typical agricultural area in the Northeast of Germany. Our results showed positive effects with at least 25% of virtually implemented agricultural buffer zones. However, higher amounts of ABZs of at least 75% should be considered to ensure a sufficient increase in Shannon diversity and decrease in quasi-extinction risks. These high amounts of ABZs represent effective conservation measures to safeguard the stability of pollination services provided by solitary bee species. As the model structure can be easily adapted to other mobile species in agricultural landscapes, our community approach offers the chance to compare the effectiveness of conservation measures also for other pollinator communities in future.
Artificial light at night (ALAN) is altering the behaviour of nocturnal animals in a manifold of ways. Nocturnal invertebrates are particularly affected, due to their fatal attraction to ALAN. This selective pressure has the potential to reduce the strength of the flight-to-light response in insects, as shown recently in a moth species. Here we investigated light attraction of ground beetles (Coleoptera: Carabidae).We compared among animals (three genera) from a highly light polluted (HLP) grassland in the centre of Berlin and animals collected at a low-polluted area in a Dark Sky Reserve (DSR), captured using odour bait. In an arena setting tested at night time, HLP beetles (n = 75 across all genera) showed a reduced attraction towards ALAN. Tested during daytime, HLP beetles were less active in an open field test (measured as latency to start moving), compared to DSR (n = 143). However, we did not observe a reduced attraction towards ALAN within the species most common at both sides, Calathus fuscipes (HLP = 37, DSR = 118 individuals) indicating that not all species may be equally affected by ALAN. Reduced attraction to ALAN in urban beetles may either be a result of phenotypic selection in each generation removing HLP individuals that are attracted to light, or an indication for ongoing evolutionary differentiation among city and rural populations in their light response. Reduced attraction to light sources may directly enhance survival and reproductive success of urban individuals. However, decrease in mobility may negatively influence dispersal, reproduction and foraging success, highlighting the selective pressure that light pollution may have on fitness, by shaping and modifying the behaviour of insects.
The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA.
Extreme habitats often harbor specific communities that differ substantially from non-extreme habitats. In many cases, these communities are characterized by archaea, bacteria and protists, whereas the number of species of metazoa and higher plants is relatively low. In extremely acidic habitats, mostly prokaryotes and protists thrive, and only very few metazoa thrive, for example, rotifers. Since many studies have investigated the physiology and ecology of individual species, there is still a gap in research on direct, trophic interactions among extremophiles. To fill this gap, we experimentally studied the trophic interactions between a predatory protist (Actinophrys sol, Heliozoa) and its prey, the rotifers Elosa woralli and Cephalodella sp., the ciliate Urosomoida sp. and the mixotrophic protist Chlamydomonas acidophila (a green phytoflagellate, Chlorophyta). We found substantial predation pressure on all animal prey. High densities of Chlamydomonas acidophila reduced the predation impact on the rotifers by interfering with the feeding behaviour of A. sol. These trophic relations represent a natural case of intraguild predation, with Chlamydomonas acidophila being the common prey and the rotifers/ciliate and A. sol being the intraguild prey and predator, respectively. We further studied this intraguild predation along a resource gradient using Cephalodella sp. as the intraguild prey. The interactions among the three species led to an increase in relative rotifer abundance with increasing resource (Chlamydomonas) densities. By applying a series of laboratory experiments, we revealed the complexity of trophic interactions within a natural extremophilic community.
Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis.
Malagasy shrew tenrecs (Microgale) have increasingly been used to study speciation genetics over the last years. A previous study recently uncovered gene flow between the Shrew-toothed shrew tenrec (M. soricoides) and sympatric southern population of the Pale shrew tenrec (M. fotsifotsy). This gene flow has been suggested to be accompanied by complete mitochondrial replacement in M. fotsifotsy. To explore the temporal framework of this replacement, we assembled mitogenomes from publicly available sequencing data of ultra-conserved elements. We were able to assemble complete and partial mitogenomes for 19 specimens from five species of shrew tenrecs, which represents a multifold increase in mitogenomic resources available for all tenrecs. Phylogenetic inferences and sequence simulations support the close relationship between the mitochondrial lineages of M. soricoides and the southern population of M. fotsifotsy. Based on the nuclear divergence of northern and southern populations of M. fotsifotsy and the mitochondrial divergence between the latter and M. soricoides, there was a mean time window for replacement of similar to 350,000 years. This timeframe implies that the effective size of the ancestral M. fotsifotsy southern population was less 70,000.
Uncovering the interplay between nutrient availability and cellulose biosynthesis inhibitor activity
(2022)
All plant cells are surrounded by a dynamic, carbohydrate-rich extracellular matrix known as the cell wall. Nutrient availability affects cell wall composition via uncharacterized regulatory mechanisms, and cellulose deficient mutants develop a hypersensitive root response to growth on high concentrations of nitrate. Since cell walls account for the bulk of plant biomass, it is important to understand how nutrients regulate cell walls. This could provide important knowledge for directing fertilizer treatments and engineering plants with higher nutrient use efficiency. The direct effect of nitrate on cell wall synthesis was investigated through growth assays on varying concentrations of nitrate, measuring cellulose content of roots and shoots, and assessing cellulose synthase activity (CESA) using live cell imaging with spinning disk confocal microscopy. A forward genetic screen was developed to isolate mutants impaired in nutrient-mediated cell wall regulation, revealing that cellulose biosynthesis inhibitor (CBI) activity is modulated by nutrient availability. Various non-CESA mutants were isolated that displayed CBI resistance, with the majority of mutations causing perturbation of mitochondria-localized proteins. To investigate mitochondrial involvement, the CBI mechanism of action was investigated using a reverse genetic screen, a targeted pharmacological screen, and -omics approaches. The results generated suggest that CBI-induced cellulose inhibition is due to off-target effects. This provides the groundwork to investigate uncharacterized processes of CESA regulation and adds valuable knowledge to the understanding of CBI activity, which could be harnessed to develop new and improved herbicides.
Phenotypic plasticity can increase individual fitness when environmental conditions change over time. Inducible defences are a striking example, allowing species to react to fluctuating predation pressure by only expressing their costly defended phenotype under high predation risk. Previous theoretical investigations have focused on how this affects predator–prey dynamics, but the impact on competitive outcomes and broader community dynamics has received less attention. Here we use a small food web model, consisting of two competing plastic autotrophic species exploited by a shared consumer, to study how the speed of inducible defences across three trade-off constellations affects autotroph coexistence, biomasses across trophic levels, and temporal variability. Contrary to the intuitive idea that faster adaptation increases autotroph fitness, we found that higher switching rates reduced individual fitness as it consistently provoked more maladaptive switching towards undefended phenotypes under high predation pressure. This had an unexpected positive impact on the consumer, increasing consumer biomass and lowering total autotroph biomass. Additionally, maladaptive switching strongly reduced autotroph coexistence through an emerging source-sink dynamic between defended and undefended phenotypes. The striking impact of maladaptive switching on species and food web dynamics indicates that this mechanism may be of more critical importance than previously recognized.
Patterns of space use are often subject to large temporal and individual-level variation, due to seasonality in behaviour and environmental conditions as well as age- or sex-specific needs. Especially in temperate regions, seasonality likely influences space use even in non-migratory birds. In waterfowl of the family Anatidae, however, few studies have analyzed space use of the same individuals across the full annual cycle. We used a resident population of Mandarin Ducks (Aix galericulata) in northeast Germany to study their year-round space use in relation to season, sex, and age. We marked 172 birds with colour rings and surveyed relevant water bodies for re-encounters for several years. As space-use patterns we derived home ranges from minimum convex polygons and the number of water bodies used by individual birds. Our analysis revealed that individuals shifted their space use between seasons, in particular extending their home ranges during the non-breeding season. Between years, in contrast, birds tended to show season-specific site fidelity. Sex differences were apparent during both breeding and non-breeding season, males consistently having larger home ranges and using slightly more water bodies. No difference was found between first-year and adult birds. Our study demonstrates that mark-resighting can provide valuable information about space use in species with suitable behaviour and readily accessible habitat. In such cases, it may be a valid alternative to more expensive GPS-tracking or short-term manual radio telemetry, particularly within citizen-science projects.
Throughout their lifetime plants need to adapt to temperature changes. Plants adapt to nonfreezing cold temperatures in a process called cold priming (cold acclimation) and lose the acquired freezing tolerance during warmer temperatures through deacclimation. The alternation of both processes is essential for plants to achieve optimal fitness in response to different temperature conditions. Cold acclimation has been extensively studied, however, little is known about the regulation of deacclimation. This thesis elucidates the process of deacclimation on a physiological and molecular level in Arabidopsis thaliana. Electrolyte leakage measurements during cold acclimation and up to four days of deacclimation enabled the identification of four knockout mutants (hra1, lbd41, mbf1c and jub1) with a slower rate of deacclimation compared to the wild type. A transcriptomic study using RNA-Sequencing in A. thaliana Col-0, jub1 and mbf1c identified the importance of the inhibition of stress responsive and Jasmonate-ZIM-domain genes as well as the regulation of cell wall modifications during deacclimation. Moreover, measurements of alcohol dehydrogenase activity and gene expression changes of hypoxia markers during the first four days of deacclimation evidently showed that a hypoxia response is activated during deacclimation. Epigenetic regulation was observed to be extensively involved during cold acclimation and 24 h of deacclimation in A. thaliana. Further, both deacclimation studies showed that the previous hypothesis that heat stress might play a role in early deacclimation, is not likely. A number of DNA- and histone demethylases as well as histone variants were upregulated during deacclimation suggesting a role in plant memory. Recently, multiple studies have shown that plants are able to retain memory of a previous cold stress even after a week of deacclimation. In this work, transcriptomic and metabolomic analyses of Arabidopsis during 24 h of priming (cold acclimation) and triggering (recurring cold stress after deacclimation) revealed a uniquely significant and transient induction of DREB1D, DREB1E and DREB1F transcription factors during triggering contributing to fine-tuning of the second cold stress response. Furthermore, genes encoding Late Embryogenesis Abundant (LEA) and antifreeze proteins and proteins detoxifying reactive oxygen species were higher induced during late triggering (24 h) compared to primed samples, while cell wall remodelers of the class xyloglucan endotransglucosylase/hydrolase were early responders of triggering. The high induction of cell wall remodelers during deacclimation as well as triggering proposes that these proteins play an essential role in the stabilization of the cells during growth as well as the response to recurring stresses. Collectively this work gives new insights on the regulation of deacclimation and cold stress memory in A. thaliana and opens the door to future targeted studies of essential genes in this process.
Heat stress (HS) is one of the most common abiotic stresses, frequently affecting plant growth and crop production. With its fluctuating nature, HS episodes are frequently interspersed by stress-free intervals. Plants can be primed by HS, allowing them to survive better a recurrent stress episode. A memory of this priming can be maintained during stress-free intervals and this memory is closely correlated with transcriptional memory at several HS-inducible loci. This transcriptional memory is evident from hyper-induction of a locus upon a recurrent HS. ASCORBATE PEROXIDASE 2 (APX2) shows such hyper-induction upon recurring HS, however, the molecular basis of this transcriptional memory is not understood. Previous research showed that the HSinduced transcriptional memory at APX2 can last for up to seven days, and that it is controlled by cis-regulatory elements within the APX2 promoter.
To identify regulators involved in HS transcriptional memory, an unbiased forward genetic screening using EMS mutated seeds of pAPX2::LUC was performed from this screen. Two EMS mutants with affected transcriptional memory of LUC were identified. I confirmed that both two EMS mutants resulted from the gene mutations of HISTONE ACETYLTRANSFERASE 1 (HAC1). Besides pAPX2::LUC, the HS-induced transcription of other HS memory genes were also affected in hac1 mutants. Moreover, HAC1 may promote HS transcriptional memory by acetylating promoters of HS memory genes.
On the other hand, to identify cis-regulatory elements that are required for transcriptional memory of APX2, I performed promoter analysis of the four conserved HSEs identified within a functional APX2 promoter. I found out that one of the HSEs (HSE1) is necessary for both HS-induced APX2 transcription and transcriptional memory, while another one of HSEs (HSE2) is important for HS-induced APX2 transcriptional memory. I also found out that the HSE1 itself (with 10 bp of flanking sequence) is sufficient to confer HS-induced APX2 transcriptional memory, and HSE1 is also necessary for HSFA2 to bind on APX2 promoter and activate APX2 transcription. The findings will provide important clues for the molecular mechanism of transcriptional memory and will enable engineering of enhanced stress tolerance in crops.
Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set
(2022)
A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material.
Transcriptomic dataset for early inflorescence stages of oil palm in response to defoliation stress
(2022)
Oil palm breeding and seed development have been hindered due to the male parent's incapacity to produce male inflorescence as a source of pollen under normal conditions. On the other hand, a young oil palm plantation has a low pollination rate due to a lack of male flowers. These are the common problem of sex ratio in the oil palm industry. Nevertheless, the regulation of sex ratio in oil palm plants is a complex mechanism and remains an open question until now. Researchers have previously used complete defoliation to induce male inflorescences, but the biological and molecular mechanisms underlying this morphological change have yet to be discovered. Here, we present an RNA-seq dataset from three early stages of an oil palm inflorescence under normal conditions and complete defoliation stress. This transcriptomic dataset is a valuable resource to improve our understanding of sex determination mechanisms in oil palm inflorescence.
Predation is a strong species interaction causing severe harm or death to prey. Thus, prey species have evolved various defence strategies to minimize predation risk, which may be immediate (e.g., a change in behaviour) or transgenerational (morphological defence structures). We studied the behaviour of two strains of a rotiferan prey (Brachionus calyciflorus) that differ in their ability to develop morphological defences in response to their predator Asplanchna brightwellii. Using video analysis, we tested: (a) if two strains differ in their response to predator presence and predator cues when both are undefended; (b) whether defended individuals respond to live predators or their cues; and (c) if the morphological defence (large spines) per se has an effect on the swimming behaviour. We found a clear increase in swimming speed for both undefended strains in predator presence. However, the defended specimens responded neither to the predator presence nor to their cues, showing that they behave indifferently to their predator when they are defended. We did not detect an effect of the spines on the swimming behaviour. Our study demonstrates a complex plastic behaviour of the prey, not only in the presence of their predator, but also with respect to their defence status.
Trade-off for survival
(2022)
The environmental micmbiota is increasingly exposed to chemical pollution. While the emergence of multi-resistant pathogens is recognized as a global challenge, our understanding of antimicrobial resistance (AMR) development from native microbiomes and the risks associated with chemical exposure is limited. By implementing a lichen as a bioindicator organism and model for a native microbiome, we systematically examined responses towards antimicrobials (colistin, tetracycline, glyphosate, and alkylpyrazine). Despite an unexpectedly high resilience, we identified potential evolutionary consequences of chemical exposure in terms of composition and functioning of native bacterial communities. Major shifts in bacterial composition were observed due to replacement of naturally abundant taxa; e.g. Chthoniobacterales by Pseudomonadales. A general response, which comprised activation of intrinsic resistance and parallel reduction of metabolic activity at RNA and protein levels was deciphered by a multi-omics approach. Targeted analyses of key taxa based on metagenome-assembled genomes reflected these responses but also revealed diversified strategies of their players. Chemical-specific responses were also observed, e.g., glyphosate enriched bacterial r-strategists and activated distinct ARGs. Our work demonstrates that the high resilience of the native micmbiota toward antimicrobial exposure is not only explained by the presence of antibiotic resistance genes but also adapted metabolic activity as a trade-off for survival. Moreover, our results highlight the importance of native microbiomes as important but so far neglected AMR reservoirs. We expect that this phenomenon is representative for a wide range of environmental microbiota exposed to chemicals that potentially contribute to the emergence of antibiotic-resistant bacteria from natural environments.
Environmental pollution by microplastics has become a severe problem in terrestrial and aquatic ecosystems and, according to actual prognoses, problems will further increase in the future. Therefore, assessing and quantifying the risk for the biota is crucial. Standardized short-term toxicological procedures as well as methods quantifying potential toxic effects over the whole life span of an animal are required. We studied the effect of the microplastic polystyrene on the survival and reproduction of a common freshwater invertebrate, the rotifer Brachionus calyciflorus, at different timescales. We used pristine polystyrene spheres of 1, 3, and 6 µm diameter and fed them to the animals together with food algae in different ratios ranging from 0 to 50% nonfood particles. As a particle control, we used silica to distinguish between a pure particle effect and a plastic effect. After 24 h, no toxic effect was found, neither with polystyrene nor with silica. After 96 h, a toxic effect was detectable for both particle types. The size of the particles played a negligible role. Studying the long-term effect by using life table experiments, we found a reduced reproduction when the animals were fed with 3 µm spheres together with similar-sized food algae. We conclude that the fitness reduction is mainly driven by the dilution of food by the nonfood particles rather than by a direct toxic effect.
Crop cultivation intensifies globally, which can jeopardize biodiversity and the resilience of cropping systems. We investigate changes in crop rotations as one intensification metric for half of the croplands in Germany with annual field-level land-use data from 2005 to 2018. We proxy crop rotations with crop sequences and compare how these sequences changed among three seven-year periods. The results reveal an overall high diversity of crop sequences in Germany. Half of the cropland has crop sequences with four or more crops within a seven-year period, while continuous cultivation of the same crop is present on only 2% of the cropland. Larger farms tend to have more diverse crop sequences and organic farms have lower shares of cereal crops. In three federal states, crop rotations became less structurally diverse over time, i.e. the number of crops and the number of changes between crops decreased. In one state, structural diversity increased and the proportion of monocropping decreased. The functional diversity of the crop sequences, which measures the share of winter and spring crops as well as the share of leaf and cereal crops per sequence, remained largely stable. Trends towards cereal-or leaf -crop dominated sequences varied between the states, and no clear overall dynamic could be observed. However, the share of winter crops per sequence decreased in all four federal states. Quantifying the dynamics of crop sequences at the field level is an important metric of land-use intensity and can reveal the patterns of land-use intensification.
Dictyostelium amoebae perform a semi-closed mitosis, in which the nuclear envelope is fenestrated at the insertion sites of the mitotic centrosomes and around the central spindle during karyokinesis. During late telophase the centrosome relocates to the cytoplasmic side of the nucleus, the central spindle disassembles and the nuclear fenestrae become closed. Our data indicate that Dictyostelium spastin (DdSpastin) is a microtubule-binding and severing type I membrane protein that plays a role in this process. Its mitotic localization is in agreement with a requirement for the removal of microtubules that would hinder closure of the fenestrae. Furthermore, DdSpastin interacts with the HeH/ LEM-family protein Src1 in BioID analyses as well as the inner nuclear membrane protein Sun1, and shows subcellular co-localizations with Src1, Sun1, the ESCRT component CHMP7 and the IST1-like protein filactin, suggesting that the principal pathway of mitotic nuclear envelope remodeling is conserved between animals and Dictyostelium amoebae.
The major histocompatibility complex (MHC) is a group of genes comprising one of the most important components of the vertebrate immune system. Consequently, there has been much interest in characterising MHC variation and its relationship with fitness in a variety of species. Due to the exceptional polymorphism of MHC genes, careful PCR primer design is crucial for capturing all of the allelic variation present in a given species. We therefore developed intronic primers to amplify the full-length 267 bp protein-coding sequence of the MHC class II DQB exon 2 in the Antarctic fur seal. We then characterised patterns of MHC variation among mother-offspring pairs from two breeding colonies and detected 19 alleles among 771 clone sequences from 56 individuals. The distribution of alleles within and among individuals was consistent with a single-copy, classical DQB locus showing Mendelian inheritance. Amino acid similarity at the MHC was significantly associated with genome-wide relatedness, but no relationship was found between MHC heterozygosity and genome-wide heterozygosity. Finally, allelic diversity was several times higher than reported by a previous study based on partial exon sequences. This difference appears to be related to allele-specific amplification bias, implying that primer design can strongly impact the inference of MHC diversity.
Understanding the environmental impact on the assembly of local communities in relation to their spatial and temporal connectivity is still a challenge in metacommunity ecology. This study aims to unravel underlying metacommunity processes and environmental factors that result in observed zooplankton communities. Unlike most metacommunity studies, we jointly examine active and dormant zooplankton communities using a DNA metabarcoding approach to overcome limitations of morphological species identification. We applied two-fragment (COI and 18S) metabarcoding to monitor communities of 24 kettle holes over a two-year period to unravel (i) spatial and temporal connectivity of the communities, (ii) environmental factors influencing local communities, and (iii) dominant underlying metacommunity processes in this system. We found a strong separation of zooplankton communities from kettle holes of different hydroperiods (degree of permanency) throughout the season, while the community composition within single kettle holes did not differ between years. Species richness was primarily dependent on pH and permanency, while species diversity (Shannon Index) was influenced by kettle hole location. Community composition was impacted by kettle hole size and surrounding field crops. Environmental processes dominated temporal and spatial processes. Sediment communities showed a different composition compared to water samples but did not differ between ephemeral and permanent kettle holes. Our results suggest that communities are mainly structured by environmental filtering based on pH, kettle hole size, surrounding field crops, and permanency. Environmental filtering based on specific conditions in individual kettle holes seems to be the dominant process in community assembly in the studied zooplankton metacommunity.
Many animals that have to cope with predation have evolved mechanisms to reduce their predation risk. One of these mechanisms is change in morphology, for example, the development of spines. These spines are induced, when mothers receive chemical signals of a predator (kairomones) and their daughters are then equipped with defensive spines. We studied the behaviour of a prey and its predator when the prey is either defended or undefended. We used common aquatic micro-invertebrates, the rotifers Brachionus calyciflorus (prey) and Asplanchna brightwellii (predator) as experimental animals. We found that undefended prey increased its swimming speed in the presence of the predator. The striking result was that the defended prey did not respond to the predator's presence. This suggests that defended prey has a different response behaviour to a predator than undefended conspecifics. Our study provides further insights into complex zooplankton predator-prey interactions. Predation is a strong species interaction causing severe harm or death to prey. Thus, prey species have evolved various defence strategies to minimize predation risk, which may be immediate (e.g., a change in behaviour) or transgenerational (morphological defence structures). We studied the behaviour of two strains of a rotiferan prey (Brachionus calyciflorus) that differ in their ability to develop morphological defences in response to their predator Asplanchna brightwellii. Using video analysis, we tested: (a) if two strains differ in their response to predator presence and predator cues when both are undefended; (b) whether defended individuals respond to live predators or their cues; and (c) if the morphological defence (large spines) per se has an effect on the swimming behaviour. We found a clear increase in swimming speed for both undefended strains in predator presence. However, the defended specimens responded neither to the predator presence nor to their cues, showing that they behave indifferently to their predator when they are defended. We did not detect an effect of the spines on the swimming behaviour. Our study demonstrates a complex plastic behaviour of the prey, not only in the presence of their predator, but also with respect to their defence status.
Although ecological networks are typically constructed based on a single type of interaction, e.g. trophic interactions in a food web, a more complete picture of ecosystem composition and functioning arises from merging networks of multiple interaction types. In this work, we consider tripartite networks constructed by merging two bipartite networks, one mutualistic and one antagonistic. Taking the interactions within each sub-network to be distributed randomly, we consider the stability of the dynamics of the network based on the spectrum of its community matrix. In the asymptotic limit of a large number of species, we show that the spectrum undergoes an eigenvalue phase transition, which leads to an abrupt destabilisation of the network as the ratio of mutualists to antagonists is increased. We also derive results that show how this transition is manifest in networks of finite size, as well as when disorder is introduced in the segregation of the two interaction types. Our random-matrix results will serve as a baseline for understanding the behaviour of merged networks with more realistic structures and/or more detailed dynamics.
Introduction: The ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications.
Objectives: In this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful tools for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity.
Methods: Five antibody candidates were selected out of a naïve camelid library by phage display and expressed as full length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron).
Results: All antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50).
Conclusion: Collectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy.
The generation of monoclonal antibodies using an in vitro immunization approach is a promising alternative to conventional hybridoma technology. As recently published, the in vitro approach enables an antigen-specific activation of B lymphocytes within 10-12 d followed by immortalization and subsequent selection of hybridomas. This in vitro process can be further improved by using a three-dimensional surrounding to stabilize the complex microenvironment required for a successful immune reaction. In this study, the suitability of Geltrex as a material for the generation of monoclonal antigen-specific antibodies by in vitro immunization was analyzed. We could show that dendritic cells, B cells, and T cells were able to travel through and interact inside of the matrix, leading to the antigen-specific activation of T and B cells. For cell recovery and subsequent hybridoma technique the suitability of dispase and Corning cell recovery solution (CRS) was compared. In our experiments, the use of dispase resulted in a severe alteration of cell surface receptor expression patterns and significantly higher cell death, while we could not detect an adverse effect of Corning CRS. Finally, an easy approach for high-density cell culture was established by printing an alginate ring inside a cell culture vessel. The ring was filled with Geltrex, cells, and medium to ensure a sufficient supply during cultivation. Using this approach, we were able to generate monoclonal hybridomas that produce antigen-specific antibodies against ovalbumin and the SARS-CoV-2 nucleocapsid protein.
The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.
Receptors predominantly expressed on tumor cells represent one of the key prerequisites of targeted radiotherapy. The gastric inhibitory polypeptide receptor (GIPR) has emerged as a promising target due to its substantial overexpression in neuroendocrine neoplasms (NENs) and virtual absence in healthy tissues (Waser 2012). So far, only radiolabeled peptides targeting the somatostatin receptor 2 (SSTR2) are approved for targeted radiotherapy of inoperable, metastatic NENs.
The aim of this thesis was to develop highly affine GIPR tracers for targeted radiotherapy by continuous in vitro and in vivo characterization of peptide sequence modifications. It was hypothesized that a GIPR antagonist might increase the sensitivity to detect GIPR-positive tumors relative to the agonist GIP(1-30), as shown for SSTR2 tracers (Reubi 2017). Further comparison to the SSTR2 agonist and antagonist (DOTATATE, JR11) should allow compound ranking regarding their ability to detect NENs.
The novel GIPR-targeting antagonists were conjugated to DOTA, enabling complexation of diagnostic (e. g. 111In) and therapeutic radionuclides (e. g. 177Lu). Among the high number of compounds screened, 3BP-3775 proved to be the most promising candidate for further preclinical and clinical development. High GIPR affinity and long receptor residence time in vitro were reflected in strong tumor uptake and retention in vivo. Low initial kidney accumulation and fast subsequent clearance represented a major advantage relative to previously described GIPR-targeting molecules (Gourni 2014). Furthermore, administration of 177Lu-3BP-3775 demonstrated for the first time strong therapeutic efficacy of a GIPR-targeting compound. In vitro receptor autoradiography with 111In-3BP-3626 (GIPR antagonist) demonstrated up to 6-fold higher signals in gastroenteropancreatic and bronchial NENs, relative to the clinically utilized SSTR2 agonist 111In-DOTATATE. Both receptor antagonists, however, revealed similar signal strength. In contrast to 111In-JR11, 111In-3BP-3626 showed no binding to non-target tissues, which led to higher tumor-to-background ratios for 111In-3BP-3626. Signal strength of the GIPR agonist 111In-GIP(1-30) was close to background in all investigated tumor samples. The ranking of compounds according to their ability to detect NENs based on autoradiographic signal intensities was determined to be: 111In-3BP-3626 ~ 111In-JR11> 111In-DOTATATE > 111In GIP(1-30).
In summary, this thesis proposes the application of the GIPR antagonist 3BP-3775 for a targeted radiotherapy in GEP- and bronchial NENs.
The morphogenesis of sessile plants is mainly driven by directional cell growth and cell division. The organization of their cytoskeleton and the mechanical properties of the cell wall greatly influence morphogenetic events in plants. It is well known that cortical microtubules (CMTs) contribute to directional growth by regulating the deposition of the cellulose microfibrils, as major cell wall fortifying elements. More recent findings demonstrate that mechanical stresses existing in cells and tissues influence microtubule organization. Also, in dividing cells, mechanical stress directions contribute to the orientation of the new cell wall. In comparison to the microtubule cytoskeleton, the role of the actin cytoskeleton in regulating shoot meristem morphogenesis has not been extensively studied.
This thesis focuses on the functional relevance of the actin cytoskeleton during cell and tissue scale morphogenesis in the shoot apical meristem (SAM) of Arabidopsis thaliana. Visualization of transcriptional reporters indicates that ACTIN2 and ACTIN7 are two highly expressed actin genes in the SAM. A link between the actin cytoskeleton and SAM development derives from the observation that the act2-1 act7-1 double mutant has abnormal cell shape and perturbed phyllotactic patterns. Live-cell imaging of the actin cytoskeleton further shows that its organization correlates with cell shape, which indicates a potential role of actin in influencing cellular morphogenesis.
In this thesis, a detailed characterization of the act2-1 act7-1 mutant reveals that perturbation of actin leads to more rectangular cellular geometries with more 90° cell internal angles, and higher incidences of four-way junctions (four cell boundaries intersecting together). This observation deviates from the conventional tricellular junctions found in epidermal cells. Quantitative cellular-level growth data indicates that such differences in the act2-1 act7-1 mutant arise due to the reduced accuracy in the placement of the new cell wall, as well as its mechanical maturation. Changes in cellular morphology observed in the act2-1 act7-1 mutant result in cell packing defects that subsequently compromise the flow of information among cells in the SAM.
We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (K-D) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.
Genetic divergence and the frequency of hybridization are central for defining species delimitations, especially among cryptic species where morphological differences are merely absent. Rotifers are known for their high cryptic diversity and therefore are ideal model organisms to investigate such patterns. Here, we used the recently resolved Brachionus calyciflorus species complex to investigate whether previously observed between species differences in thermotolerance and gene expression are also reflected in their genomic footprint. We identified a Heat Shock Protein gene (HSP 40 kDa) which exhibits cross species pronounced sequence variation. This gene exhibits species-specific fixed sites, alleles, and sites putatively under positive selection. These sites are located in protein binding regions involved in chaperoning and may therefore reflect adaptive diversification. By comparing three genetic markers (ITS, COI, HSP 40 kDa), we revealed hybridization events between the cryptic species. The low frequency of introgressive haplotypes/alleles suggest a tight, but not fully impermeable boundary between the cryptic species.