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The results of a 3-year tracking of a common crane equipped with a GPS-GSM transmitter have made it possible to better understand the geographical connectivity, migration routes, and key sites of summer stays and migration stopovers of individuals of this species that nest in the center of the European part of Russia. Flyways and wintering sites have been traced and key and short-term migration stopovers have been identified in the arid areas of Ukraine, Turkey, Israel, and Ethiopia. It has been established that the crane used different wintering grounds in different years. It has been revealed that immature cranes of the Dubna premigratory gathering (Moscow Region) can use areas where other premigratory gatherings are formed, in particular, in Smolensk Region. Successful nesting of a common crane at the age of 3 years noticed. published

Biophysical journal 122(9), 1646 - 1658 (2023). doi:10.1016/j.bpj.2023.03.030, Cells in living organisms are subjected to mechanical strains caused by external forces like overcrowding, resulting in strong deformations that affect cell function. We study the interplay between deformation and crowding of red blood cells (RBCs) in dispersions of nonabsorbing rod-like viruses. We identify a sequence of configurational transitions of RBC doublets, including configurations that can only be induced by long-ranged attraction: highly fluctuating T-shaped and face-to-face configurations at low, and doublets approaching a complete spherical configuration at high, rod concentrations. Complementary simulations are used to explore different energy contributions to deformation as well as the stability of RBC doublet configurations. Our advanced analysis of 3D reconstructed confocal images of RBC doublets quantifies the depletion interaction and the resulting deformation energy. Thus, we introduce a noninvasive, high-throughput platform that is generally applicable to investigate the mechanical response of biological cells to external forces and characterize their mechanical properties., Published by Soc., Bethesda, Md.

Hsp90 is a molecular chaperone responsible for regulating proteostasis under physiological and pathological conditions. Its central role in a range of diseases and potential as a drug target has focused efforts to understand its mechanisms and biological functions and to identify modulators that may form the basis for therapies. The 10th international conference on the Hsp90 chaperone machine was held in Switzerland in October 2022. The meeting was organized by Didier Picard (Geneva, Switzerland) and Johannes Buchner (Garching, Germany) with an advisory committee of Olivier Genest, Mehdi Mollapour, Ritwick Sawarkar, and Patricija van Oosten-Hawle. This was a much anticipated first in-person meeting of the Hsp90 community since 2018 after the COVID-19 pandemic led to the postponement of the 2020 meeting. The conference remained true to the tradition of sharing novel data ahead of publication, providing unparalleled depth of insight for both experts and newcomers to the field.

The fragile X protein (FXP) family comprises the multifunctional RNA-binding proteins FMR1, FXR1, and FXR2 that play an important role in RNA metabolism and regulation of translation, but also in DNA damage and cellular stress responses, mitochondrial organization, and more. FMR1 is well known for its implication in neurodevelopmental diseases. Recent evidence suggests substantial contribution of this protein family to amyotrophic lateral sclerosis (ALS) pathogenesis. ALS is a highly heterogeneous neurodegenerative disease with multiple genetic and unclear environmental causes and very limited treatment options. The loss of motoneurons in ALS is still poorly understood, especially because pathogenic mechanisms are often restricted to patients with mutations in specific causative genes. Identification of converging disease mechanisms evident in most patients and suitable for therapeutic intervention is therefore of high importance. Recently, deregulation of the FXPs has been linked to pathogenic processes in different types of ALS. Strikingly, in many cases, available data points towards loss of expression and/or function of the FXPs early in the disease, or even at the presymptomatic state. In this review, we briefly introduce the FXPs and summarize available data about these proteins in ALS. This includes their relation to TDP-43, FUS, and ALS-related miRNAs, as well as their possible contribution to pathogenic protein aggregation and defective RNA editing. Furthermore, open questions that need to be addressed before definitively judging suitability of these proteins as novel therapeutic targets are discussed.

We thank Goekce Ayan for sharing valuable protocols and experience with the experimental system. This work was supported by Junta de Andalucía projects P10-RNM-6630 and P11-FQM-7074 (Proyectos de Excelencia, Spain), MINECO CTM 2013-46951-R, MAT 2013-44429-R and PCIN 2015-051 projects (Spain) and by the European Regional Development Fund (ERDF)., Ecological risk assessment of chemicals focuses on the response of different taxa in isolation not taking ecological and evolutionary interplay in communities into account. Its consideration would, however, allow for an improved assessment by testing for implications within and across trophic levels and changes in the phenotypic and genotypic diversity within populations. We present a simple experimental system that can be used to evaluate the ecological and evolutionary responses to chemical exposure at microbial community levels. We exposed a microbial model system of the ciliate Tetrahymena thermophila (predator) and the bacterium Pseudomonas fluorescens (prey) to iron released from Magnetic Particles (MP-Fedis), which are Phosphorus (P) adsorbents used in lake restoration. Our results show that while the responses of predator single population size differed across concentrations of MP-Fedis and the responses of prey from communities differed also across concentration of MP-Fedis, the community responses (species ratio) were similar for the different MP-Fedis concentrations. Looking further at an evolutionary change in the bacterial preys' defence, we found that MP-Fedis drove different patterns and dynamics of defence evolution. Overall, our study shows how similar community dynamics mask changes at evolutionary levels that would be overlooked in the design of current risk assessment protocols where evolutionary approaches are not considered., Open Access funding enabled and organized by Projekt DEAL.

Global habitat degradation heightens the need to better understand patterns of genetic connectivity and diversity of marine biota across geographical ranges to guide conservation efforts. Corals across the Red Sea are subject to pronounced environmental differences, but studies so far suggest that animal populations are largely connected, excepting evidence for a genetic break between the northern-central and southern regions. Here, we investigated population structure and holobiont assemblage of two common pocilloporid corals, Pocillopora verrucosa and Stylophora pistillata, across the Red Sea. We found little evidence for population differentiation in P. verrucosa, except for the southernmost site. Conversely, S. pistillata exhibited a complex population structure with evidence for within-reef and regional genetic differentiation, in line with differences in their reproductive mode (P. verrucosa is a broadcast spawner and S. pistillata is a brooder). Analysis for genomic loci under positive selection identified 85 sites (18 of which were in coding sequences) that distinguished the southern P. verrucosa population from the remainder of the Red Sea population. By comparison, we found 128 loci (24 of which were residing in coding sequences) in S. pistillata with evidence for local adaptation at various sites. Functional annotation of the underlying proteins revealed putative roles in the response to stress, lipid metabolism, transport, cytoskeletal rearrangement, and ciliary function (among others). Microbial assemblages of both coral species showed pervasive association with microalgal symbionts from the genus Symbiodinium (former clade A) and bacteria from the genus Endozoicomonas that exhibited significant differences according to host genotype and environment. The disparity of population genetic and holobiont assemblage patterns even between closely related species (family Pocilloporidae) highlights the need for multispecies investigations to better understand the role of the environment in shaping evolutionary trajectories. It further emphasizes the importance of networks of reef reserves to achieve conservation of genetic variants critical to the future survival of coral ecosystems. published

Biotechnology & bioengineering 120(5), 1269-1287 (2023). doi:10.1002/bit.28334, Published by Wiley, New York, NY [u.a.]

In mammals, many germline genes are epigenetically repressed to prevent their illegitimate expression in somatic cells. To advance our understanding of the mechanisms restricting the expression of germline genes, we analyzed their chromatin signature and performed a CRISPR-Cas9 knock-out screen for genes involved in germline gene repression using a Dazl-GFP reporter system in mouse embryonic stem cells (mESCs). We show that the repression of germline genes mainly depends on the polycomb complex PRC1.6 and DNA methylation, which function additively in mESCs. Furthermore, we validated novel genes involved in the repression of germline genes and characterized three of them: Usp7, Shfm1 (also known as Sem1) and Erh. Inactivation of Usp7, Shfm1 or Erh led to the upregulation of germline genes, as well as retrotransposons for Shfm1, in mESCs. Mechanistically, USP7 interacts with PRC1.6 components, promotes PRC1.6 stability and presence at germline genes, and facilitates DNA methylation deposition at germline gene promoters for long term repression. Our study provides a global view of the mechanisms and novel factors required for silencing germline genes in embryonic stem cells.

Bi-allelic mutations in GBA1, the gene that encodes β-glucocerebrosidase (GCase), cause Gaucher disease (GD), whereas mono-allelic mutations do not cause overt pathology. Yet mono- or bi-allelic GBA1 mutations are the highest known risk factor for Parkinson’s disease (PD). GCase deficiency results in the accumulation of glucosylceramide (GluCer) and its deacylated metabolite glucosylsphingosine (GluSph). Brains from patients with neuronopathic GD have high levels of GluSph, and elevation of this lipid in GBA1-associated PD has been reported. To uncover the mechanisms involved in GBA1-associated PD, we used human induced pluripotent stem cell-derived dopaminergic (DA) neurons from patients harboring heterozygote mutations in GBA1 (GBA1/PD–DA neurons). We found that compared with gene-edited isogenic controls, GBA1/PD–DA neurons exhibit mammalian target of rapamycin complex 1 (mTORC1) hyperactivity, a block in autophagy, an increase in the levels of phosphorylated α-synuclein (129) and α-synuclein aggregation. These alterations were prevented by incubation with mTOR inhibitors. Inhibition of acid ceramidase, the lysosomal enzyme that deacylates GluCer to GluSph, prevented mTOR hyperactivity, restored autophagic flux and lowered α-synuclein levels, suggesting that GluSph was responsible for these alterations. Incubation of gene-edited wild type (WT) controls with exogenous GluSph recapitulated the mTOR/α-synuclein abnormalities of GBA1/PD neurons, and these phenotypic alterations were prevented when GluSph treatment was in the presence of mTOR inhibitors. We conclude that GluSph causes an aberrant activation of mTORC1, suppressing normal lysosomal functions, including the clearance of pathogenic α-synuclein species. Our results implicate acid ceramidase in the pathogenesis of GBA1-associated PD, suggesting that this enzyme is a potential therapeutic target for treating synucleinopathies caused by GCase deficiency.

Elongation factors modulate the efficiency of mRNA synthesis by RNA polymerase II (RNAPII) in the context of chromatin, thus contributing to implement proper gene expression programmes. The zinc-finger protein elongation factor 1 (ELF1) is a conserved transcript elongation factor (TEF), whose molecular function so far has not been studied in plants. Using biochemical approaches, we examined the interaction of Arabidopsis ELF1 with DNA and histones in vitro and with the RNAPII elongation complex in vivo. In addition, cytological assays demonstrated the nuclear localisation of the protein, and by means of double-mutant analyses, interplay with genes encoding other elongation factors was explored. The genome-wide distribution of ELF1 was addressed by chromatin immunoprecipitation. ELF1 isolated from Arabidopsis cells robustly copurified with RNAPII and various other elongation factors including SPT4-SPT5, SPT6, IWS1, FACT and PAF1C. Analysis of a CRISPR-Cas9-mediated gene editing mutant of ELF1 revealed distinct genetic interactions with mutants deficient in other elongation factors. Moreover, ELF1 associated with genomic regions actively transcribed by RNAPII. However, ELF1 occupied only c. 33% of the RNAPII transcribed loci with preference for inducible rather than constitutively expressed genes. Collectively, these results establish that Arabidopsis ELF1 shares several characteristic attributes with RNAPII TEFs.

Biological chemistry 404(2-3), 195 - 207 (2023). doi:10.1515/hsz-2022-0273, Oxalyl-CoA synthetase from Saccharomyces cerevisiae is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in a homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target., Published by de Gruyter, Berlin [u.a.]

The need for new options in lung cancer treatment inevitably leads back to basic research. The tumor itself and the tumor environment especially the interaction with the immune system need to be better understood to develop targeted therapies. In the context of lung cancer glucocorticoids (GC) are mainly known as a combination drug to attenuate side-effects of chemotherapies. However, endogenous extra-adrenal GC have been shown to substantially regulate local immune responses within various tissues, including the lung. In this study we investigated whether primary lung tumors have maintained the capacity to synthesize GC and may thereby regulate anti-tumor immune responses. We show that several non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) cell lines express key steroidogenic enzymes and synthesize bioactive GC under steady state conditions. We also show that tumor-derived GC can inhibit splenic T cell activation, thus demonstrating their immunoregulatory potential. Moreover, steroidogenic enzymes were detected by quantitative RT-PCR and immunohistochemistry in tissue sections of different human lung tumors, further strengthening the idea that human lung carcinomas regulate their microenvironment by releasing immunoregulatory GC, which potentially contributes to immune evasion and treatment resistance.

1. Colourful patterns on flowers are thought to benefit both pollinators and the plants they visit, by increasing the plants' pollination success via an increased foraging efficiency of the pollinators. This increased efficiency is assumed to result from a guidance effect of the flower patterns, correspondingly termed ‘nectar guides’, which indicate the position of the nectary to visiting pollinators, thus reducing their flower handling time.2. Although it is well established that flower patterns play an important role in flower choice, the mechanisms by which they improve the foraging efficiency of flower-visiting insects remain poorly understood.3. In this study, we quantified the contributions of patterns to all phases of flower interaction in the buff-tailed bumblebee Bombus terrestris. We analysed the bees' flight paths, as well as landing positions and walking tracks on artificial flowers with different pattern types.4. We reveal that flower patterns reduced the overall flower handling time of the bees by up to 30%, by guiding their approach flight, landing positions and departure decisions. Surprisingly, we observed no improvement in nectary discovery time after the bees landed on the flower.5. Since we tested experienced foragers, which represent the majority of insect pollinators active in nature, the newly described nectary-independent guidance effects of flower patterns are of high ecological relevance. published

Major regional gaps exist in the reporting and accessibility of naturalized plant species distribution data, especially within Southeast Asia. Here, we present the Malesian Naturalized Alien Flora database (MalNAF), the first standardized island-group level checklist of naturalized vascular plant species for the Malesian phytogeographical region. We used MalNAF to investigate the composition, origins, and habitat preferences of the naturalized flora. The naturalized vascular flora of Malesia consists of at least 1177 species. Richness is highest in the Philippines (539 spp.) and lowest in the Maluku Islands (87 spp.). But, the Lesser Sunda Islands had the highest naturalized species richness relative to native richness and Singapore has a higher naturalized plant species richness than would be expected given its size. When comparing the data for Malesia with a global dataset, we found that naturalized richness increased with area for islands but not for continental regions. Across the archipelago, 31 species are widespread, occurring in every island group, but the majority have a limited distribution of 2.4 ± 2.3 (mean ± SD) island groups per naturalized species. The naturalized plant species are representatives of 150 families, twenty of which are newly introduced to the region. Families richest in naturalized plant species in Malesia were Fabaceae (= Leguminosae) (160 spp.), Poaceae (= Gramineae) (138 spp.), and Asteraceae (= Compositae) (96 spp.). Most of these have a native range that includes tropical Asia, closely followed by those from Southern America (inclusive of the Caribbean, Central and South America), although at the island-group level, most have a higher proportion with a Southern American native range. Most naturalized species occur in anthropogenic habitats, but many are present in “natural” habitats with fewer species, such as Leucaena leucocephala, reported from specialized habitats like drylands. MalNAF provides a baseline for future studies of naturalized plant species distributions in the region.

Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR–protein interactions. Quantitative Kd values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53—and potentially also APLF—can form complex multivalent PAR–protein structures. In conclusion, our study gives detailed and quantitative insight into PAR–protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.

Chlorambucil (CLB) belongs to the class of nitrogen mustards (NMs), which are highly reactive bifunctional alkylating agents and were the first chemotherapeutic agents developed. They form DNA interstrand crosslinks (ICLs), which cause a blockage of DNA strand separation, inhibiting essential processes in DNA metabolism like replication and transcription. In fast replicating cells, e.g., tumor cells, this can induce cell death. The upregulation of ICL repair is thought to be a key factor for the resistance of tumor cells to ICL-inducing cytostatic agents including NMs. To monitor induction and repair of CLB-induced ICLs, we adjusted the automated reversed fluorometric analysis of alkaline DNA unwinding assay (rFADU) for the detection of ICLs in adherent cells. For the detection of monoalkylated DNA bases we established an LC-MS/MS method. We performed a comparative analysis of adduct formation and removal in five human cell lines and in peripheral blood mononuclear cells (PBMCs) after treatment with CLB. Dose-dependent increases in adduct formation were observed, and suitable treatment concentrations were identified for each cell line, which were then used for monitoring the kinetics of adduct formation. We observed significant differences in the repair kinetics of the cell lines tested. For example, in A2780 cells, hTERT immortalized VH10 cells, and in PBMCs a time-dependent repair of the two main monoalkylated DNA-adducts was confirmed. Regarding ICLs, repair was observed in all cell systems except for PBMCs. In conclusion, LC-MS/MS analyses combined with the rFADU technique are powerful tools to study the molecular mechanisms of NM-induced DNA damage and repair. By applying these methods to a spectrum of human cell systems of different origin and transformation status, we obtained insight into the cell-type specific repair of different CLB-induced DNA lesions, which may help identify novel resistance mechanisms of tumors and define molecular targets for therapeutic interventions. published

Understanding brain function remains challenging as work with human and animal models is complicated by compensatory mechanisms, while in vitro models have been too simple until now. With the advent of human stem cells and the bioengineering of brain microphysiological systems (MPS), understanding how both cognition and long-term memory arise is now coming into reach. We suggest combining cutting-edge AI with MPS research to spearhead organoid intelligence (OI) as synthetic biological intelligence. The vision is to realize cognitive functions in brain MPS and scale them to achieve relevant short- and long-term memory capabilities and basic information processing as the ultimate functional experimental models for neurodevelopment and neurological function and as cell-based assays for drug and chemical testing. By advancing the frontiers of biological computing, we aim to (a) create models of intelligence-in-a-dish to study the basis of human cognitive functions, (b) provide models to advance the search for toxicants contributing to neurological diseases and identify remedies for neurological maladies, and (c) achieve relevant biological computational capacities to complement traditional computing. Increased understanding of brain functionality, in some respects still superior to today’s supercomputers, may allow to imitate this in neuromorphic computer architectures or might even open up biological computing to complement silicon computers. At the same time, this raises ethical questions such as where sentience and consciousness start and what the relationship between a stem cell donor and the respective OI system is. Such ethical discussions will be critical for the socially acceptable advance of brain organoid models of cognition. published

Red fluorophores synthesized by oral bacteria are important for fluorescence-based diagnosis and treatment because they are used as markers for bacterially infected tissue, mature plaque, or calculus. A range of porphyrins have been identified as the source of this fluorescence in carious tissue. It is not clear which of these porphyrins are produced by individual oral bacteria or whether this ability depends on other factors. This study examined and compared the fluorescence spectra produced by selected cultured oral bacteria when grown on agars containing different nutrients with spectra for protoporphyrin IX, Zn-protoporphyrin IX, haematoporphyrin, and haematin. Actinomyces israelii (Deutsche Sammlung von Mikroorganismen [DSM], 43320), Actinomyces naeslundii (DSM 43013), Fusobacterium nucleatum (DSM, 20482), Lactobacillus casei (DSM, 20011), Prevotella intermedia (DSM, 20706), Streptococcus mutans (DSM, 20523), Streptococcus oralis (DSM, 20627), Streptococcus salivarius (DSM, 20560) and Streptococcus sobrinus (DSM, 20742) were rehydrated and grown anaerobically on caso, caso blood (containing 5% sheep blood), and caso chlorophyll (containing 5% spinach extract) agar for 3 days at 37°C in the dark. Colonies were harvested, transferred to ethanol, and centrifuged. Fluorescence emission spectra were recorded from the supernatant at 405 nm excitation (Fluorolog 3–22, Jobin Yvon-Spex ISA, Edison, NJ, USA). All Streptococci, L. casei, and F. nucleatum produced red fluorescence when grown on caso and caso chlorophyll agar but not on caso blood agar. A. naeslundii and P. intermedia emitted intense red fluorescence when grown on caso or caso blood agar but not on caso chlorophyll agar. Fluorescence emission spectra of A. naeslundii and P. intermedia grown on caso blood agar correlated exactly with both fluorescence peaks for protoporphyrin-IX at 632 and 701 nm. Most peaks observed could be correlated with at least one of the emission peaks of protoporphyrin IX, Zn-protoporphyrin IX, or haematoporphyrin. Oral bacteria emitted red fluorescence matching known porphyrins, but this depended on nutrients available in the agar.

The natriuretic peptides (NPs) ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide) mediate their widespread effects by activating the natriuretic peptide receptor-A (NPR-A), while C-type natriuretic peptide (CNP) acts via natriuretic peptide receptor-B (NPR-B). NPs are removed from the circulation by internalization via the natriuretic peptide clearance receptor natriuretic peptide receptor-C (NPR-C). In addition to their well-known functions, for instance on blood pressure, all three NPs confer significant cardioprotection and renoprotection. Since neither the NP-mediated renal functions nor the renal target cells of renoprotection are completely understood, we performed systematic localization studies of NP receptors using in situ hybridization (RNAscope) in mouse kidneys. NPR-A mRNA is highly expressed in glomeruli (mainly podocytes), renal arterioles, endothelial cells of peritubular capillaries, and PDGFR-receptor β positive (PDGFR-β) interstitial cells. No NPR-A mRNA was detected by RNAscope in the tubular system. In contrast, NPR-B expression is highest in proximal tubules. NPR-C is located in glomeruli (mainly podocytes), in endothelial cells and PDGFR-β positive cells. To test for a possible regulation of NPRs in kidney diseases, their distribution was studied in adenine nephropathy. Signal intensity of NPR-A and NPR-B mRNA was reduced while their spatial distribution was unaltered compared with healthy kidneys. In contrast, NPR-C mRNA signal was markedly enhanced in cell clusters of myofibroblasts in fibrotic areas of adenine kidneys. In conclusion, the primary renal targets of ANP and BNP are glomerular, vascular, and interstitial cells but not the tubular compartment, while the CNP receptor NPR-B is highly expressed in proximal tubules. Further studies are needed to clarify the function and interplay of this specific receptor expression pattern.

The African savannah ecosystem is populated by nomadic pastoralists who herd livestock in the day and corral them at night in temporary enclosures, called bomas, to protect them. The number and distribution of bomas on the savannah is important from an ecological perspective and may have a significant impact on wildlife movement. However, no study has yet examined this relationship. Here, using very high-resolution satellite imagery from two time periods, we quanitified changes in boma distribution and density across an area of 3377 km2 in the Laikipia-Samburu ecosystem of northern Kenya between 2011 and 2019. To assess wildlife movement in relation to bomas, we used a GPS data set on African bush elephant Loxodonta africana movement from 27 collared matriarchs representing herds of 9–15, covering 112 467 hourly GPS fixes over 31 months between 2018 and 2020. Our results showed a more than 46% increase in the total number of human-built structures between 2011 and 2019, the majority of which were bomas, representing a 21.9% increase in human-modified land area. Elephants readily adjusted their foraging habits and itineraries in this habitat shared with humans, who were also nomadic in space and time. Assessing the night–day activity ratio, we found elephants move more nocturnally when in closer proximity to bomas, particularly during the dry season. This temporal separation means elephants avoid the times humans are active in and around bomas while still accessing required resources—water and forage. The temporal shift was stronger during the dry season when shared resources are scarce. Using daily travel distance as a metric, we show elephants moved further in closer proximity to bomas which was likely linked to the need to travel between forage patches. Given the rise in human settlements, understanding the consequences of animals' behavioral adjustments is critical to understand the long-term population viability of elephant populations. published

published

Pathogenic bacteria possess a great potential of causing infectious diseases and represent a serious threat to human and animal health. Understanding the molecular basis of infection development can provide new valuable strategies for disease prevention and better control. In host‐pathogen interactions, actin‐cytoskeletal dynamics play a crucial role in the successful adherence, invasion, and intracellular motility of many intruding microbial pathogens. Cortactin, a major cellular factor that promotes actin polymerization and other functions, appears as a central regulator of host‐pathogen interactions and different human diseases including cancer development. Various important microbes have been reported to hijack cortactin signaling during infection. The primary regulation of cortactin appears to proceed via serine and/or tyrosine phosphorylation events by upstream kinases, acetylation, and interaction with various other host proteins, including the Arp2/3 complex, filamentous actin, the actin nucleation promoting factor N‐WASP, focal adhesion kinase FAK, the large GTPase dynamin‐2, the guanine nucleotide exchange factor Vav2, and the actin‐stabilizing protein CD2AP. Given that many signaling factors can affect cortactin activities, several microbes target certain unique pathways, while also sharing some common features. Here we review our current knowledge of the hallmarks of cortactin as a major target for eminent Gram‐negative and Gram‐positive bacterial pathogens in humans.

Species-rich calcareous grasslands in Europe strongly declined during the twentieth century due to drastic land use changes. Many grasslands were converted into more productive pastures or are covered by shrubs or forests today, since they were overgrown after abandonment or afforested. Restoration of calcareous grasslands by shrub or forest clearing and subsequent recolonization of grassland species from adjacent grasslands is, therefore, an important conservation approach. Restored populations of calcareous grassland species may, however, differ from their source populations in genetic diversity and differentiation due to potential founder and bottleneck effects. In our study we analyzed, therefore, the impact of restoration by forest clearing and natural recolonization on the genetic variation of three common calcareous grassland species (Agrimonia eupatoria, Campanula rotundifolia, and Knautia arvensis) without a contribution of persistent seed bank, in South Western Germany. We used molecular markers AFLPs (Amplified fragment length polymorphisms) to compare genetic diversity within and differentiation between spontaneously recovered subpopulations with adjacent historically old, natural subpopulations at eight study sites. Restored parts of the grasslands have been re-established during the 1990s. Molecular markers revealed broadly similar levels of genetic diversity in source and restored subpopulations of the study species. Only A. eupatoria exhibited slightly higher diversity in restored subpopulations, which may be explained by higher dispersal potential due to the hooky fruits of the species. Genetic differentiation between source and restored subpopulations was not significant, indicating strong gene flow between the subpopulations. Our study underlines, therefore, that restoration of calcareous grasslands by natural recolonization after forest clearing is an efficient method to re-establish genetically variable subpopulations comparable to their sources.

Aim Global warming is assumed to restructure mountain insect communities in space and time. Theory and observations along climate gradients predict that insect abundance and richness, especially of small‐bodied species, will increase with increasing temperature. However, the specific responses of single species to rising temperatures, such as spatial range shifts, also alter communities, calling for intensive monitoring of real‐world communities over time. Location German Alps and pre‐alpine forests in south‐east Germany. Methods We empirically examined the temporal and spatial change in wild bee communities and its drivers along two largely well‐protected elevational gradients (alpine grassland vs. pre‐alpine forest), each sampled twice within the last decade. Results We detected clear abundance‐based upward shifts in bee communities, particularly in cold‐adapted bumble bee species, demonstrating the speed with which mobile organisms can respond to climatic changes. Mean annual temperature was identified as the main driver of species richness in both regions. Accordingly, and in large overlap with expectations under climate warming, we detected an increase in bee richness and abundance, and an increase in small‐bodied species in low‐ and mid‐elevations along the grassland gradient. Community responses in the pre‐alpine forest gradient were only partly consistent with community responses in alpine grasslands. Main Conclusion In well‐protected temperate mountain regions, small‐bodied bees may initially profit from warming temperatures, by getting more abundant and diverse. Less severe warming, and differences in habitat openness along the forested gradient, however, might moderate species responses. Our study further highlights the utility of standardized abundance data for revealing rapid changes in bee communities over only one decade.

Background and Objectives Due to insufficient resolution, $^{1}$H nuclear magnetic resonance (NMR) spectroscopy-based methods are limited to quantify carbohydrates. In the past, heteronuclear single quantum coherence (HSQC)-based methods were demonstrated to be superior as the second dimension greatly improves resolution. However, whether these experiments are also suitable to determine structurally similar oligosaccharides such as raffinose family oligosaccharides (RFO) still needs to be demonstrated. Findings By optimizing NMR parameters, well resolved signals for the analysis of glucose, fructose, galactose, sucrose and the RFO raffinose, stachyose, and verbascose were identified. Application of fast HSQC methods in combination with nonuniform sampling enables analyses of sucrose and RFO in pulses (blue lupin seeds, red lentils, kidney beans) within 24 min. If the analytes are present at levels greater than 0.5 g/100 g, HSQC-based methods provide data equivalent to an anion-exchange chromatography-based reference method. Conclusions High resolution fast HSQC-based approaches are suitable tools to analyze complex carbohydrate mixtures as demonstrated for RFO in different pulses. Significance and Novelty Fast HSQC experiments were applied for the first time to analyze structurally similar oligosaccharides. In the future, this approach will be a most valuable tool to analyze complex mixtures of carbohydrates in food products.

Alzheimer’s disease (AD), the leading cause of dementia, has an estimated heritability of approximately 70%1. The genetic component of AD has been mainly assessed using genome-wide association studies, which do not capture the risk contributed by rare variants2. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals—16,036 AD cases and 16,522 controls. Next to variants in TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Additionally, the rare-variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential drivers of respective AD-genome-wide association study loci. Variants associated with the strongest effect on AD risk, in particular loss-of-function variants, are enriched in early-onset AD cases. Our results provide additional evidence for a major role for amyloid-β precursor protein processing, amyloid-β aggregation, lipid metabolism and microglial function in AD.

Plant-parasitic nematodes cause devastating agricultural damage worldwide. Only a few synthetic nematicides can be used and their application is limited in fields. Therefore, there is a need for sustainable and environment-friendly alternatives. Nematode-trapping fungi (NTF) are natural predators of nematodes. They capture and digest them with their hyphae and are starting to being used as bio-control agents. In this study, we applied the NTF Arthrobotrys flagrans (Duddingtonia flagrans) against the wine pathogenic nematode Xiphinema index. A. flagrans reduced the number of X. index juveniles in pot cultures of Ficus carica, an alternative host plant for X. index, significantly. Sodium-alginate pellets with A. flagrans spores were produced for vineyard soil inoculation under laboratory conditions. The NTF A. conoides, A. musiformis and A. superba were enriched from several soil samples, showing their natural presence. Trap formation is an energy-consuming process and depends upon various biotic and abiotic stimuli. Here, we show that bacteria of the genus Delftia, Bacillus, Pseudomonas, Enterobacter and Serratia induced trap formation in NTF like A. conoides and A. oligospora but not in A. flagrans in the absence of nematodes. The application of NTF along with such bacteria could be a combinatorial way of efficient biocontrol in nematode-infested soil.

Context Soaring birds depend on atmospheric uplifts and are sensitive to wind energy development. Predictive modelling is instrumental to forecast conflicts between human infrastructures and single species of concern. However, as multiple species often coexist in the same area, we need to overcome the limitations of single species approaches. Objectives We investigate whether predictive models of flight behaviour can be transferred across species boundaries. Methods We analysed movement data from 57 white storks, Ciconia ciconia, and 27 griffon vultures, Gyps fulvus. We quantified the accuracy of topographic features, correlates of collision risk in soaring birds, in predicting their soaring behaviour, and tested the transferability of the resulting suitability models across species. Results 59.9% of the total area was predicted to be suitable to vultures only, and 1.2% exclusively to storks. Only 20.5% of the study area was suitable to both species to soar, suggesting the existence of species-specific requirements in the use of the landscape for soaring. Topography alone could accurately predict 75% of the soaring opportunities available to storks across Europe, but was less efficient for vultures (63%). While storks relied on uplift occurrence, vultures relied on uplift quality, needing stronger uplifts to support their higher body mass and wing loading. Conclusions Energy landscapes are species-specific and more knowledge is required to accurately predict the behaviour of highly specialised soaring species, such as vultures. Our models provide a base to explore the effects of landscape changes on the flight behaviour of different soaring species. Our results suggest that there is no reliable and responsible way to shortcut risk assessment in areas where multiple species might be at risk by anthropogenic structures.

Extracellular production of target proteins simplifies downstream processing due to obsolete cell disruption. However, optimal combinations of a heterologous protein, suitable signal peptide and secretion host can currently not be predicted, resulting in large strain libraries that need to be tested. On the experimental side, this challenge can be tackled by miniaturization, parallelization and automation, which provide high-throughput screening data. These data need to be condensed into a candidate ranking for decision making to focus bioprocess development on the most promising candidates. We screened for Bacillus subtilis signal peptides mediating Sec secretion of two polyethylene terephthalate degrading enzymes (PETases), leaf-branch compost cutinase (LCC) and polyester hydrolase (PE-H) mutants, by Corynebacterium glutamicum. We developed a fully automated screening process and constructed an accompanying Bayesian statistical modeling framework, which we applied in screenings for highest activity in 4-nitrophenyl palmitate degradation. In contrast to classical evaluation methods, batch effects and biological errors are taken into account and their uncertainty is quantified. Within only two rounds of screening, the most suitable signal peptide was identified for each PETase. Results from LCC secretion in microliter-scale cultivation were shown to be scalable to laboratory-scale bioreactors. This work demonstrates an experiment-modeling loop that can accelerate early-stage screening in a way that experimental capacities are focused to the most promising strain candidates. Combined with high-throughput cloning, this paves the way for using large strain libraries of several hundreds of strains in a Design-Build-Test-Learn approach.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA’s 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU ‘on the ground’ in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

In low-income countries, the livelihoods of smallholders are affected to different extent by multiple issues, such as food insecurity or low soil fertility, depending on their multiple assets and farming characteristics. In this study, we aim at assessing the current sustainability of households in Tanzania to suggest potential changes in farming system to increase the household́ sustainability. Household survey data from 891 households in two regions of Tanzania (dry Dodoma and humid Morogoro) were used to build composite sustainability indices representing the three pillars of sustainability (economic, environmental and social), based on 46 basic indicators. Then, a household typology was developed through principal component analysis and hierarchical clustering analysis of descriptive variables, and regression analysis linked the sustainability of farms with household characteristics. The analysis revealed significant differences in household types that relied on different livelihood strategies including particularly a “Gathering-based” type in Dodoma and two “Crop-based” types in both regions. These livelihoods significantly influenced the level of farming system sustainability. Particularly, the households in the “Gathering-based” type performed worst on all three pillars of sustainability. By examining the level of capitals from the different household types, we identified that the “Gathering-based” type and “Crop-based” types could improve their livelihood and be better off if they adopt various upgrading solutions. Such solutions include intercropping and optimised weeding to increase productivity and resource-use efficiency, for which these households have sufficient labour resources. Additionally, livestock rearing and use of fertilisers coupled with rainwater harvesting can increase soil fertility and water use efficiency, and hence food security, without compromising the environmental component of sustainability. To facilitate the adoption of these sustainable intensification options, agricultural policies and appropriate training need to be implemented to fit the local context and diversity of household types.

Under environmental conditions, biofilms can oftentimes be found on different surfaces, accompanied by the structural degradation of the substrate. Since high-copper-content paints were banned in the EU, a solution for the protection of these surfaces has to be found. In addition to hydrophobation, making the surfaces inherently biofilm-repellent is a valid strategy. We want to accomplish this via the metal exchange in calcium silicate hydrate (CSH) substrates with transition metals. As has been shown with Europium, even small amounts of metal can have a great influence on the material properties. To effectively model CSH surfaces, ultrathin CSH films were grown on silicon wafers using Ca(OH)

Salt stress is a major abiotic stress, responsible for declining agricultural productivity. Roots are regarded as hubs for salt detoxification, however, leaf salt concentrations may exceed those of roots. How mature leaves manage acute sodium chloride (NaCl) stress is mostly unknown. To analyze the mechanisms for NaCl redistribution in leaves, salt was infiltrated into intact tobacco leaves. It initiated pronounced osmotically‐driven leaf movements. Leaf downward movement caused by hydro‐passive turgor loss reached a maximum within 2 h. Salt‐driven cellular water release was accompanied by a transient change in membrane depolarization but not an increase in cytosolic calcium ion (Ca\(^{2+}\)) level. Nonetheless, only half an hour later, the leaves had completely regained turgor. This recovery phase was characterized by an increase in mesophyll cell plasma membrane hydrogen ion (H\(^{+}\)) pumping, a salt uptake‐dependent cytosolic alkalization, and a return of the apoplast osmolality to pre‐stress levels. Although, transcript numbers of abscisic acid‐ and Salt Overly Sensitive pathway elements remained unchanged, salt adaptation depended on the vacuolar H\(^{+}\)/Na\(^{+}\)‐exchanger NHX1. Altogether, tobacco leaves can detoxify sodium ions (Na\(^{+}\)) rapidly even under massive salt loads, based on pre‐established posttranslational settings and NHX1 cation/H+ antiport activity. Unlike roots, signaling and processing of salt stress in tobacco leaves does not depend on Ca\(^{2+}\) signaling.

Plant cells can undergo regulated cell death in response to exogenous factors (often in a stress context), but also as regular element of development (often regulated by phytohormones). The cellular aspects of these death responses differ, which implies that the early signalling must be different. We use cytokinin-induced programmed cell death as paradigm to get insight into the role of the cytoskeleton for the regulation of developmentally induced cell death, using tobacco BY-2 cells as experimental model. We show that this PCD in response to kinetin correlates with an arrest of the cell cycle, a deregulation of DNA replication, a loss of plasma membrane integrity, a subsequent permeabilisation of the nuclear envelope, an increase of cytosolic calcium correlated with calcium depletion in the culture medium, an increase of callose deposition and the loss of microtubule and actin integrity. We discuss these findings in the context of a working model, where kinetin, mediated by calcium, causes the breakdown of the cytoskeleton, which, either by release of executing proteins or by mitotic catastrophe, will result in PCD.

Holins are generally believed to generate large membrane lesions that permit the passage of endolysins across the cytoplasmic membrane of prokaryotes, ultimately resulting in cell wall degradation and cell lysis. However, there are more and more examples known for non-lytic holin-dependent secretion of proteins by bacteria, indicating that holins somehow can transport proteins without causing large membrane lesions. Phage-derived holins can be used for a non-lytic endolysin translocation to permeabilize the cell wall for the passage of secreted proteins. In addition, clostridia, which do not possess the Tat pathway for transport of folded proteins, most likely employ non-lytic holin-mediated transport also for secretion of toxins and bacterioc-ins that are incompatible with the general Sec pathway. The mechanism for non-lytic holin-mediated transport is unknown, but the recent finding that the small holin TpeE mediates a non-lytic toxin secretion in Clostridium perfringens opened new perspec-tives. TpeE contains only one short transmembrane helix that is followed by an amphipathic helix, which is reminiscent of TatA, the membrane-permeabilizing component of the Tat translocon for folded proteins. Here we review the known cases of non-lytic holin-mediated transport and then focus on the structural and functional comparison of TatA and TpeE, resulting in a mechanistic model for holin-mediated transport. This model is strongly supported by a so far not recognized naturally occurring holin-endolysin fusion protein.

The exponential increase in the human population in tandem with increased food demand has caused agriculture to be the global‐dominant form of land use. Afrotropical drylands are currently facing the loss of natural savannah habitats and agricultural intensification with largely unknown consequences for bees. Here we investigate the effects of agricultural intensification on bee assemblages in the Afrotropical drylands of northern Tanzania. We disentangled the direct effects of agricultural intensification and temperature on bee richness from indirect effects mediated by changes in floral resources. We collected data from 24 study sites representing three levels of management intensity (natural savannah, moderate intensive and highly intensive agriculture) spanning an extensive gradient of mean annual temperature (MAT) in northern Tanzania. We used ordinary linear models and path analysis to test the effects of agricultural intensity and MAT on bee species richness, bee species composition and body‐size variation of bee communities. We found that bee species richness increased with agricultural intensity and with increasing temperature. The effects of agricultural intensity and temperature on bee species richness were mediated by the positive effects of agriculture and temperature on the richness of floral resources used by bees. During the off‐growing season, agricultural land was characterized by an extensive period of fallow land holding a very high density of flowering plants with unique bee species composition. The increase in bee diversity in agricultural habitats paralleled an increasing variation of bee body sizes with agricultural intensification that, however, diminished in environments with higher temperatures. Synthesis and applications. Our study reveals that bee assemblages in Afrotropical drylands benefit from agricultural intensification in the way it is currently practiced. However, further land‐use intensification, including year‐round irrigated crop monocultures and excessive use of agrochemicals, is likely to exert a negative impact on bee diversity and pollination services, as reported in temperate regions. Moreover, several bee species were restricted to natural savannah habitats. To conserve bee communities and guarantee pollination services in the region, a mixture of savannah and agriculture, with long periods of fallow land should be maintained.

Green plants are equipped with photoreceptors that are capable of sensing radiation in the ultraviolet‐to‐blue and the red‐to‐far‐red parts of the light spectrum. However, plant cells are not particularly sensitive to green light (GL), and light which lies within this part of the spectrum does not efficiently trigger the opening of stomatal pores. Here, we discuss the current knowledge of stomatal responses to light, which are either provoked via photosynthetically active radiation or by specific blue light (BL) signaling pathways. The limited impact of GL on stomatal movements provides a unique option to use this light quality to control optogenetic tools. Recently, several of these tools have been optimized for use in plant biological research, either to control gene expression, or to provoke ion fluxes. Initial studies with the BL‐activated potassium channel BLINK1 showed that this tool can speed up stomatal movements. Moreover, the GL‐sensitive anion channel GtACR1 can induce stomatal closure, even at conditions that provoke stomatal opening in wild‐type plants. Given that crop plants in controlled‐environment agriculture and horticulture are often cultivated with artificial light sources (i.e. a combination of blue and red light from light‐emitting diodes), GL signals can be used as a remote‐control signal that controls stomatal transpiration and water consumption.

Many studies show that photodynamic inactivation (PDI) is a powerful tool for the fight against pathogenic, multiresistant bacteria and the closing of hygiene gaps. However, PDI studies have been frequently performed under standardized in vitro conditions comprising artificial laboratory settings. Under real-life conditions, however, PDI encounters substances like ions, proteins, amino acids and fatty acids, potentially hampering the efficacy of PDI to an unpredictable extent. Thus, we investigated PDI with the phenalene-1-one-based photosensitizer SAPYR against Escherichia coli and Staphylococcus aureus in the presence of calcium or magnesium ions, which are ubiquitous in potential fields of PDI applications like in tap water or on tissue surfaces. The addition of citrate should elucidate the potential as a chelator. The results indicate that PDI is clearly affected by such ubiquitous ions depending on its concentration and the type of bacteria. The application of citrate enhanced PDI, especially for Gram-negative bacteria at certain ionic concentrations (e.g. CaCl2 or MgCl2: 7.5 to 75 mmol L−1). Citrate also improved PDI efficacy in tap water (especially for Gram-negative bacteria) and synthetic sweat solution (especially for Gram-positive bacteria). In conclusion, the use of chelating agents like citrate may facilitate the application of PDI under real-life conditions.

Chronotypes describe consistent differences between individuals in biological time-keeping. They have been linked both with underlying variation in the circadian system and fitness. Quantification of chronotypes is usually by time of onset, midpoint, or offset of a rhythmic behaviour or physiological process. However, diel activity patterns respond flexibly to many short-term environmental influences, which can make chronotypes hard to identify. In contrast, rhythmic patterns in physiological processes, such as body temperature, may provide more robust insights into the circadian basis of chronotypes. These can be telemetrically recorded from skin-mounted, temperature-sensitive transmitters, offering minimally invasive opportunities for working on free-ranging animals in the wild. Currently, computational methods for deriving chronotype from skin temperature require further development, as time series are often noisy and incomplete. Here, we investigate such methods using simultaneous radio telemetry recordings of activity and skin temperature in a wild songbird model (Great Tit Parus major) temporarily kept in outdoor aviaries. Our aims were to first develop standardised selection criteria to filter noisy time series of skin temperature and activity, to second assign chronotype based on the filtered recordings, and to third compare chronotype as assigned based on each of the two rhythms. After the selection of rhythmic data using periodicity and autocorrelation parameters, chronotype estimates (onset and offset) were extracted using four different changepoint approaches for skin temperature and one approach for activity records. The estimates based on skin temperature varied between different approaches but were correlated to each other (onset: correlation coefficient r = 0.099–0.841, offset: r = 0.131–0.906). In contrast, chronotype estimates from skin temperature were more weakly correlated to those from activity (onset: r = −0.131–0.612, offset: r = −0.040– −0.681). Overall, chronotype estimates were less variable and timed later in the day for activity than for skin temperature. The distinctions between physiological and behavioural chronotypes in this study might reflect differences in underlying mechanisms and in responsiveness to external and internal cues. Thus, studying each of these rhythms has specific strengths, while parallel studies of both could inform broadly on natural variation in biological time-keeping, and may allow assessment of how biological rhythms relate to changes in the environment.

Regulation of cellular catabolic metabolism in immune cells has recently become a major concept for resolution of inflammation. Nuclear receptors (NRs), including peroxisome proliferator activator receptors, 1,25-dihydroxyvitamin D (3) receptor, liver X receptors, glucocorticoid receptors, oestrogen-related receptor α and nuclear receptor 4A1, have been identified as major modulators of inflammation, affecting innate immune cells, such as macrophages. Evidence emerges on how NRs regulate cellular metabolism in macrophages during inflammatory processes and contribute to the resolution of inflammation. This could have new implications for our understanding of how NRs shape immune responses and inform anti-inflammatory drug design. This review will highlight the recent developments about NRs and their role in cellular metabolism in macrophages., publishedVersion

Major climatic changes in conjunction with animal movement may be associated with the spread of parasites and their vectors into new populations, with potentially important consequences for population persistence. Parasites can evolve to adapt to unsuitable ecological conditions and take up refuge within new host species, with consequences for the population growth of the new host species. One parasite species that has likely been increasing its geographic range, and potentially infecting new hosts, is the recently described air sac nematode Serratospiculoides amaculata, in great tits (Parus major) in Slovakia. In this study, we screened wild birds for potential air sac nematode infection in a woodland area of southern Germany. We identified four additional host species: Eurasian nuthatch, great spotted woodpecker, greenfinch and robin. As infection by this group of nematodes can be highly pathogenic, we recommend further investigation into its potential risk to these populations., International Journal for Parasitology. Parasites and Wildlife, 21, ISSN:2213-2244

HTT ist ein Protein, das durch seine Verbindung mit mehreren Interaktionspartnern an einer Vielzahl von zellulären Prozessen beteiligt ist. Darüber hinaus verursacht eine Mutation im HTT-Gen eine Krankheit, die als Huntington-Krankheit (HD) bezeichnet wird. Aufgrund der gerüstbildenden Eigenschaften von HTT wurde eine Vielzahl von Studien durchgeführt, um potenzielle therapeutische Ziele zu identifizieren. Die auf dem Resonanzenergietransfer (RET) basierenden Ansätze sind jedoch im Bereich der Huntington-Krankheit noch nicht vollständig genutzt worden. Daher habe ich versucht, solche Ansätze in Interaktionsstudien mit dem HTT-Exon 1 (HTTexon1) und dem Protein in voller Länge zu bewerten. Ich habe eine Benchmarking-Studie mit einem zuvor beschriebenen Huntingtin-interagierenden Protein (HIP) und HTTex1 (Wildtyp und mutiert) unter Verwendung eines BRET-Ansatzes durchgeführt. Meine Studien bestätigten die binäre Interaktion zwischen HTTex1 und sieben Proteinen. Ich habe auch drei Interaktionen mit der mutierten Version von HTTex1 bestätigt. Zusätzlich bewertete ich die Interaktionen durch FRET-Messungen mit Hilfe der Durchflusszytometrie. Der zweite Teil dieser Arbeit zielte darauf ab, ein Hochdurchsatz-Screening für den Nachweis von Protein-Protein-Interaktionen (PPIs) mit HTT in voller Länge (FL) unter Verwendung von Biolumineszenz-Resonanz-Energie-Transfer zu etablieren. Auf diese Weise konnte ich die Wechselwirkung zwischen FL HTT und einer Bibliothek von 580 Proteinkinasen bewerten. Schließlich analysierte ich die Spezifität der entdeckten Wechselwirkungen, indem ich die unspezifische Bindung durch Donor-Sättigungstests bewertete. Zusammenfassend belegen meine Ergebnisse die potenzielle Verwendung von Resonanzenergietransferansätzen zur Validierung von HTT-Wechselwirkungen. Außerdem wird ein neues Screening-Tool vorgestellt, das dazu beitragen soll, HTT-Interaktoren zu identifizieren und zu verifizieren. HTT is a protein involved in a plethora of cellular processes through its association with several interaction partners. Furthermore, a mutation in the HTT gene, causes a disease denominated Huntington’s disease (HD). Due to the scaffolding properties of HTT, a large variety of studies have been performed to identify potential therapeutical targets. However, resonance energy transfer-based (RET) approaches have not been fully exploited in the HD field. Therefore, I aimed to evaluate such approaches in interaction studies using the HTT exon 1 (HTTexon1) as well as the full-length protein. I performed a benchmarking study with a previously described huntingtin interacting protein (HIP) and HTTex1 (wild type and mutated) using a BRET approach. My studies confirmed the binary interaction between HTTex1 and seven proteins. I also confirmed three interactions with the mutated version of HTTex1. Additionally, I also evaluated the interactions by measuring FRET using flow cytometry. The second part of this work aimed to stablish a high-throughput screening for the detection of protein-protein interactions (PPIs) with full-length (FL) HTT using bioluminescence resonance energy transfer. With this, I was able to evaluate the interaction between FL HTT and a library composed by 580 protein kinases. Finally, I analysed the specificity of the detected interactions by assessing unspecific binding through donor saturation assays. In summary my results provide evidence of the potential use of resonance energy transfer approaches to validate HTT interactions. Additionally, a new screening tool is presented to contribute to identify and verify HTT interactors.

This study examined the effect of rare PDE5A variants on coronary artery disease risk from several genetic and pharmacological aspects. Our study provides evidence of a putative causal effect of PDE5A overexpression on coronary artery disease and myocardial infarction. Furthermore, these findings vigorously promote PDE5 inhibition as a promising strategy to encounter coronary artery disease. However, this remains to be studied. Im Rahmen dieser Studie wurde genetisch und pharmakologisch untersucht, wie sich das Tragen seltener PDE5A Varianten auf das Risiko für koronare Herzerkrankungen auswirkt. In diesem Zusammenhang konnten wir einen mutmaßlich kausalen Zusammenhang zwischen einer PDE5A Überexpression und einem erhöhtem Risiko für koronare Herzerkrankungen feststellen. Diese Erkenntnisse erwecken den Eindruck, dass die Anwendung eines PDE5-Hemmers bei koronaren Herzerkrankungen von großem Nutzen sein könnte, was jedoch weiterer Untersuchungen bedarf.

NTCP serves as species-specific key factor for HBV entry. Identification of suitable animal NTCP may allow discovering potential HBV animal models. In this study, we identified NTCP from rabbits, cats and horses as suitable to allow HBV binding and uptake. Of note, HBV infection was increased by expressing horse instead of human NTCP on HepG2 and AML12 cells. Thus, horse NTCP may allow to create new in vitro and in vivo models of HBV infection and help to clarify NTCPs mode of action during HBV entry. NTCP dient als spezies-spezifischer Schlüsselfaktor für die Bindung des HBV an Leberzellen. Die Identifikation geeigneter NTCP aus dem Tierreich sollte helfen, neue Infektionsmodelle zu generieren. In dieser Arbeit wurden Kaninchen-, Katzen- und Pferde-NTCP als Kandidaten für eine HBV-Infektion identifiziert. Nach Expression des NTCP vom Pferd in permissiven Zellen war eine HBV Infektion effizienter als mit dem humanen Analog, was es zu einem vielversprechenden Protein macht, um in vitro als auch in vivo Modelle zu schaffen.

Assessing the distribution of geographically restricted and evolutionarily unique species and their underlying drivers is key to understanding biogeographical processes and critical for global conservation prioritization. Here, we quantified the geographic distribution and drivers of phylogenetic endemism for ~320,000 seed plants worldwide and identified centers and drivers of evolutionarily young (neoendemism) and evolutionarily old endemism (paleoendemism). Tropical and subtropical islands as well as tropical mountain regions displayed the world’s highest phylogenetic endemism. Most tropical rainforest regions emerged as centers of paleoendemism, while most Mediterranean-climate regions showed high neoendemism. Centers where high neo- and paleoendemism coincide emerged on some oceanic and continental fragment islands, in Mediterranean-climate regions and parts of the Irano-Turanian floristic region. Global variation in phylogenetic endemism was well explained by a combination of past and present environmental factors (79.8 to 87.7% of variance explained) and most strongly related to environmental heterogeneity. Also, warm and wet climates, geographic isolation, and long-term climatic stability emerged as key drivers of phylogenetic endemism. Neo- and paleoendemism were jointly explained by climatic and geological history. Long-term climatic stability promoted the persistence of paleoendemics, while the isolation of oceanic islands and their unique geological histories promoted neoendemism. Mountainous regions promoted both neo- and paleoendemism, reflecting both diversification and persistence over time. Our study provides insights into the evolutionary underpinnings of biogeographical patterns in seed plants and identifies the areas on Earth with the highest evolutionary and biogeographical uniqueness—key information for setting global conservation priorities. published

Gene regulation is a tightly controlled process in Eukaryotes. Coding and non-coding regions work together to ensure proper spatiotemporal gene expression through 3D chromatin organization, nucleosome positioning, histone tail post-translational modifications, epigenetic DNA modifications, and non-canonical nucleic acid structures such as RNA:DNA triplexes. In this thesis, I investigate two of these mechanisms, nucleosomes and RNA:DNA triplexes. In the first chapter, I focus on characterizing nucleosomal properties, such as stability and accessibility, and explore how these properties change to regulate gene expression. I show that Eukaryotic organisms can modulate nucleosome stability and change the RNA polymerase pausing rate, which in turn regulates gene expression. Intriguingly, I find a distinct group of un-stable nucleosomes enriched at the TSS of promoters marked with motif one, an M1BP TF-specific motif, in D. melanogaster. Modulation of stability may ensure a fast response to environmental cues and proper spatiotemporal expression of developmental genes. Furthermore, I developed a bioinformatic pipeline called nucMACC, with which scientists can study nucleosomal properties and positioning in their projects. I show the nucMACC pipeline is consistent and robust and provide recommendations for minimum sequencing depth, MNase titrations, and spike-in use. In summary, the nucMACC pipeline provides high-resolution nucleosome positions and an automated way of calling non-canonical nucleosomes, un-stable nucleosomes, hyper-accessible nucleosomes, hypo-accessible nucleosomes, and stable canonical nucleosomes. In the second chapter, I study the triplex binding code and explore how the code changes based on the triplex motif (Purine, pyrimidine, and mixed), sequence Guanine content, length, and the nucleic acid (RNA or DNA). Triplexes are non-canonical DNA/RNA structures consisting of three nucleotide strands, most commonly a double-stranded DNA molecule and a single-stranded RNA molecule in its major groove. I show that major differences exist between the triplex motifs and between RNA:DNA and DNA:DNA triplexes. Interestingly, I discovered that the mixed RNA:DNA triplex motif permits triplex formation only at very narrow Guanine contents, while DNA:DNA mixed motif triplexes are able to form at almost any Guanine content. Moreover, I confirm the newly defined triplex code by testing published triplex pairs, of which half are unable to form a triplex under physiological conditions. Furthermore, I developed a high throughput method to investigate the triplex binding code in the context of molecular crowding and competition. I find that certain mismatches in the motif stabilize triplex formation and are preferentially selected for triplex formation over the complementary Hoogsten base-pairing motif. Moreover, I investigate how the location of a mismatch modifies triplex stability and show that the middle section of the triplex is more sensitive to mismatches than flanking regions. In summary, I add to the existing triplex code by delineating the differences in the binding code between triplex motifs and RNA:DNA and DNA:DNA triplexes. I show how the binding code changes in the context of molecular crowding and competition and moreover, show the differential effect of different mismatch locations., Die Genregulation ist ein genau kontrollierter Prozess in Eukaryonten. Kodierende und nicht-kodierende Regionen arbeiten zusammen, um eine korrekte räumlich-zeitliche Genexpression durch 3D-Chromatinorganisation, Nukleosomenpositionierung, posttranslationale Histonschwanzmodifikationen, epigenetische DNA-Modifikationen und nicht-kanonische Nukleinsäurestrukturen wie RNA:DNA-Triplexe sicherzustellen. In dieser Arbeit untersuche ich zwei dieser Mechanismen, Nukleosomen und RNA:DNA-Triplexe. Im ersten Kapitel konzentriere ich mich auf die Charakterisierung der Eigenschaften von Nukleosomen, wie Stabilität und Zugänglichkeit, und untersuche, wie sich diese Eigenschaften ändern, um die Genexpression zu regulieren. Ich zeige, dass eukaryotische Organismen die Nukleosomenstabilität modulieren und die Pausenrate der RNA-Polymerase verändern können, was wiederum die Genexpression reguliert. Interessanterweise finde ich in D. melanogaster eine bestimmte Gruppe instabiler Nukleosomen, die an der TSS von Promotoren mit dem „Motif one“ , einem M1BP TF-spezifischen Motiv, angereichert sind. Eine Modulation der Stabilität könnte eine schnelle Reaktion auf Umweltreize und eine angemessene räumlich-zeitliche Expression von Entwicklungsgenen gewährleisten. Darüber hinaus habe ich eine bioinformatische Pipeline namens nucMACC entwickelt, mit der Wissenschaftler die Eigenschaften und Positionierung von Nukleosomen in ihren Projekten untersuchen können. Ich zeige, dass die nucMACC-Pipeline konsistent und robust ist, und gebe Empfehlungen für die Mindestsequenzierungstiefe, MNase-Titrationen und die Verwendung von Spike-in DNA. Zusammenfassend erlaubt die nucMACC-Pipeline sowohl die Bestimmung von hochauflösenden Nukleosomenpositionen als auch eine automatische Kategorisierung von nicht-kanonischen, instabilen, hyper- und hypo-zugänglichen und stabilen kanonischen Nukleosomen. Im zweiten Kapitel erforsche ich den Triplex-Bindungscode und untersuche, wie sich der Code je nach Triplex-Motiv (Purin, Pyrimidin und gemischt), Sequenz, Guanin-Gehalt, Länge und Nukleinsäure (RNA oder DNA) verhält. Triplexe sind nicht-kanonische DNA/RNA-Strukturen, die aus drei Nukleotidsträngen bestehen, meist einem doppelsträngigen DNA-Molekül und einem einzelsträngigen RNA-Molekül in seiner großen Furche . Ich zeige, dass es große Unterschiede zwischen den Triplex-Motiven und zwischen RNA:DNA- und DNA:DNA-Triplexen gibt. Interessanterweise entdeckte ich, dass das gemischte RNA:DNA-Triplex-Motiv die Triplex-Bildung nur bei einem sehr geringen Guanin-Gehalt ermöglicht, während Triplexe mit gemischtem DNA:DNA-Motiv bei fast jedem Guanin-Gehalt gebildet werden können. Darüber hinaus bestätige ich den neu definierten Triplex-Code, indem ich schon veröffentlichte Triplex-Paare teste und zeige außerdem, dass von diesen Paaren die Hälfte unter physiologischen Bedingungen keine Triplex bildet. Zusätzlich habe ich eine Hochdurchsatzmethode entwickelt, um den Triplex-Bindungscode im Kontext von molekularem Gedränge und Kompetetion zu untersuchen. Ich stelle fest, dass bestimmte Fehlpaarungen im Motiv die Triplexbildung stabilisieren und gegenüber dem komplementären Hoogsten-Basenpaarungsmotiv bevorzugt für die Triplexbildung ausgewählt werden. Außerdem untersuche ich, wie die Lage einer Fehlpaarung die Triplex-Stabilität verändert, und zeige, dass der mittlere Abschnitt des Triplex empfindlicher auf Fehlpaarungen reagiert als flankierende Regionen. Zusammenfassend lässt sich sagen, dass ich den bestehenden Triplex-Code ergänze, indem ich die Unterschiede im Bindungscode zwischen Triplex-Motiven und RNA:DNA- und DNA:DNA-Triplexen beschreibe. Ich zeige, wie sich der Bindungscode im Kontext von molekularem Gedränge und Kompetetion verändert, und zeige darüber hinaus die unterschiedliche Wirkung verschiedener Mismatch-Positionen.

Cholera is an infectious disease caused by the pathogenic Vibrio cholerae bacteria. The transmission dynamics for the disease span multiple interconnected scales, making it challenging for traditional single-scale immunological or epidemiological models to capture the disease dynamics. In this thesis, we develop multi-scale models to study the transmission dynamics of cholera. Cholera ist eine Infektionskrankheit welche durch das pathogene Bakterium Vibrio cholerae verursacht wird. Die Übertragungsdynamik der Krankheit erstreckt sich über mehrere miteinander verknüpfte Skalen. Daher gestaltet sich die Erfassung der Krankheitsdynamik durch herkömmliche immunologische oder epidemiologische Modelle basierend auf einer einzigen Skala als schwierig. In dieser Arbeit entwickeln wir Multiskalenmodelle zur Untersuchung der Übertragungsdynamik von Cholera.

For many consumers, washing clothes in a washing machine is an everyday activity. In addition to removing visible dirt and noticeable odors, removing microorganisms is one of the most important functions of a washing machine. As in recent years, and due to the energy crisis of 2022, the quest for higher energy efficiency and energy savings by using lower washing machine programmes (especially 20 °C and 30 °C) has increased, many microorganisms now survive a wash cycle. In addition to inadequate removal of microorganisms, the consequences are above all textile malodors. The aim of this study was therefore to conduct a comprehensive analysis of the microbial community in washing machines, taking into account the microbial contamination of used textiles, in order to understand the development of textile malodors. So far, neither the composition of biofilms in washing machines that lead to the formation of or protect against malodor has been intensively investigated, nor have microbial communities on used towels after normal use been analysed. Furthermore, no standardised methods exist to transfer a household biofilm into a laboratory model. This would ensure testing under near-consumer conditions. The results established a correlation between the qualitative and quantitative analysis of microbial communities in washing machines and on used towels and the occurrence of malodor. The evaluation gives an indication of bacterial colonisers of washing machines that can prevent the formation of malodor. It could be shown that especially soil bacteria such as Rhizobium, Agrobacterium, Bosea and Microbaterium are present in non-odorous washing machines and that Rhizobium species can prevent odor formation in an in vitro model. Furthermore, a method for transferring a household washing machine biofilm to a laboratory model was established. The results showed that although not identical biofilms can be recultivated, they have a high diversity and a micorbial composition similar to a household biofilm. Furthermore, a statistical correlation between the Euclidean distance of the melting curve analysis and the Bray-Curtis dissimilarity of the metagenome analysis could be established. This enables a faster and more cost-effective analysis of the similarity between two DNA samples., Das Waschen von Wäsche in einer Waschmaschine ist für viele Verbraucher und Verbraucherinnen eine alltägliche Tätigkeit. Neben der Entfernung von sichtbarem Schmutz und wahrnehmbaren Gerüchen, ist die Entfernung von Mikroorganismen eine der wichtigsten Funktionen einer Waschmaschine. Durch die Energiekrise 2022 hat das Streben nach höherer Energieeffizeinz und Energieeinsparungen sowie das Verwenden niedriger Waschmaschinenprogramme zugenommen. Aus diesem Grund überleben viele Mikroroganismen einen Waschgang mittlerweile. Konsequenzen sind neben unzureichender Abtötung von Mikroorganismen vor allem textile Schlechtgerüche. Ziel dieser Studie war es daher eine umfassende Analyse der mikrobiellen Gemeinschaft in Waschmaschinen unter Berücksichtigung der mikrobiellen Kontamination gebrauchter Textilien durchzuführen, um die Entstehung von textilen Fehlgerüchen zu verstehen. Bisher wurden weder die Zusammensetzung der Biofilme in Waschmaschinen, welche für die Bildung von oder zum Schutz vor textilem Schlechtgeruch führen, intensiv untersucht, noch wurden mikrobielle Gemeinschaften auf gebrauchten Handtüchern nach normalem Gebrauch analysiert. Des Weiteren sind keine standardisierten Methoden bekannt, um einen Haushaltsbiofilm in ein Labormodell zu übertragen. Dieses würde eine Prüfung unter verbrauchernahen Bedingungen gewährleisten. Die Ergebnisse stellten einen Zusammenhang zwischen der qualitativen und quantitativen Analyse mikrobieller Gemeinschaften in Waschmaschinen und auf gebrauchten Handtüchern sowie dem Auftreten von textilem Schlechtgeruch her. Die Auswertung gibt einen Hinweis auf bakterielle Besiedler von Waschmaschinen, welche die Geruchsbildung verhindern können. Es konnte gezeigt warden, dass insbesondere Bodenbakterien wie Rhizobium, Agrobacterium, Bosea und Microbaterium in nicht riechenden Waschmaschinen vorkommen und dass Rhizobium-Arten in einem in-vitro-Modell die Geruchsbildung verhindern können. Es konnte eine Methode zur Übertragung eines Haushaltswaschmaschinenbiofilms in ein Labormodell etabliert werden. Die Ergebnisse zeigten, dass zwar nicht identische Biofilme rekultiviert werden können, diese aber eine hohe Diversität und eine mikorbielle Zusammensetzung aufweisen, welche einem Haushaltsbiofilm ähnelt. Des Weiteren konnte ein statistischer Zusammenhang zwischen der euklidischen Distanz der Schmelzkurvenanalyse und der Bray-Curtis-Dissimilarität der Metagenomanalyse identifiziert werden. Dieses ermöglicht eine schnellere und kostengünstigere Analyse der Ähnlichkeit zwischen zwei DNA-Proben.