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Ecophysiological studies on Antarctic cryptophytes to assess whether climatic changes such as ocean acidification and enhanced stratification affect their growth in Antarctic coastal waters in the future are lacking so far. This is the first study that investigated the combined effects of increasing availability of pCO2 (400 and 1000 µatm) and irradiance (20, 200 and 500 μmol photons m−2 s −1) on growth, elemental composition and photophysiology of the Antarctic cryptophyte Geminigera cryophila. Under ambient pCO2, this species was characterized by a pronounced sensitivity to increasing irradiance with complete growth inhibition at the highest light intensity. Interestingly, when grown under high pCO2 this negative light effect vanished and it reached highest rates of growth and particulate organic carbon production at the highest irradiance compared to the other tested experimental conditions. Our results for G. cryophila reveal beneficial effects of ocean acidification in conjunction with enhanced irradiance on growth and photosynthesis. Hence, cryptophytes such as G. cryophila may be potential winners of climate change, potentially thriving better in more stratified and acidic coastal waters and contributing in higher abundance to future phytoplankton assemblages of coastal Antarctic waters.
Identification of unique cardiolipin and monolysocardiolipin species in Acinetobacter baumannii
(2017)
Acidic glycerophospholipids play an important role in determining the resistance of Gram-negative bacteria to stress conditions and antibiotics. Acinetobacter baumannii, an opportunistic human pathogen which is responsible for an increasing number of nosocomial infections, exhibits broad antibiotic resistances. Here lipids of A. baumannii have been analyzed by combined MALDI-TOF/MS and TLC analyses; in addition GC-MS analyses of fatty acid methyl esters released by methanolysis of membrane phospholipids have been performed. The main glycerophospholipids are phosphatidylethanolamine, phosphatidylglycerol, acyl-phosphatidylglycerol and cardiolipin together with monolysocardiolipin, a lysophospholipid only rarely detected in bacterial membranes. The major acyl chains in the phospholipids are C16:0 and C18:1, plus minor amounts of short chain fatty acids. The structures of the cardiolipin and monolysocardiolipin have been elucidated by post source decay mass spectrometry analysis. A large variety of cardiolipin and monolysocardiolipin species were found in A. baumannii. Similar lysocardiolipin levels were found in the two clinical strains A. baumannii ATCC19606T and AYE whereas in the nonpathogenic strain Acinetobacter baylyi ADP1 lysocardiolipin levels were highly reduced.
In the past two decades, an increasing body of studies has been published on the intersex phenomenon in separate-sexed crustaceans from marine and freshwater ecosystems. Various causes are being considered that could have an influence on the occurrence of intersex. Besides genetic factors, environmental conditions such as photoperiodicity, temperature, salinity and parasitism, but also environmental pollution with endocrine disrupting chemicals (EDCs) are discussed. As part of a long-term monitoring (2012 – 2020) in north-west Brittany, we recorded the occurrence of intersex in the marine amphipod Echinogammarus marinus. We quantified the intersex incidence at marine and estuarine sites and analyzed the incidence in relation to the endocrine potential of the sediments. Intersex occurred with mean frequencies between 0.87% and 12%. It was striking that the incidence of intersex increased with increasing distance from the sea. Since the highest incidence was observed at the range boundary of this stenohaline species, we assume that intersex is triggered by endocrine potential and increasing stress due to increasing freshwater content − and thus an interplay of different environmental factors.
Nanoplastics affect the inflammatory cytokine release by primary human monocytes and dendritic cells
(2022)
So far, the human health impacts of nano- and microplastics are poorly understood. Thus, we investigated whether nanoplastics exposure induces inflammatory processes in primary human monocytes and monocyte-derived dendritic cells. We exposed these cells in vitro to nanoplastics of different shapes (irregular vs. spherical), sizes (50–310 nm and polydisperse mixtures) and polymer types (polystyrene; polymethyl methacrylate; polyvinyl chloride, PVC) using concentrations of 30–300 particles cell−1. Our results show that irregular PVC particles induce the strongest cytokine release of these nanoplastics. Irregular polystyrene triggered a significantly higher pro-inflammatory response compared to spherical nanoplastics. The contribution of chemicals leaching from the particles was minor. The effects were concentration-dependent but varied markedly between cell donors. We conclude that nanoplastics exposure can provoke human immune cells to secrete cytokines as key initiators of inflammation. This response is specific to certain polymers (PVC) and particle shapes (fragments). Accordingly, nanoplastics cannot be considered one homogenous entity when assessing their health implications and the use of spherical polystyrene nanoplastics may underestimate their inflammatory effects.
Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in humans. Molecular mechanisms underlying mitochondrial dysfunction during acute injury in I/R are tissue-specific, but their details are not completely understood. A metabolic shift and accumulation of substrates of reverse electron transfer (RET) such as succinate are observed in tissue ischemia, making mitochondrial complex I of the respiratory chain (NADH:ubiquinone oxidoreductase) the most vulnerable enzyme to the following reperfusion. It has been shown that brain complex I is predisposed to losing its flavin mononucleotide (FMN) cofactor when maintained in the reduced state in conditions of RET both in vitro and in vivo. Here we investigated the process of redox-dependent dissociation of FMN from mitochondrial complex I in brain and heart mitochondria. In contrast to the brain enzyme, cardiac complex I does not lose FMN when reduced in RET conditions. We proposed that the different kinetics of FMN loss during RET is due to the presence of brain-specific long 50 kDa isoform of the NDUFV3 subunit of complex I, which is absent in the heart where only the canonical 10 kDa short isoform is found. Our simulation studies suggest that the long NDUFV3 isoform can reach toward the FMN binding pocket and affect the nucleotide affinity to the apoenzyme. For the first time, we demonstrated a potential functional role of tissue-specific isoforms of complex I, providing the distinct molecular mechanism of I/R-induced mitochondrial impairment in cardiac and cerebral tissues. By combining functional studies of intact complex I and molecular structure simulations, we defined the critical difference between the brain and heart enzyme and suggested insights into the redox-dependent inactivation mechanisms of complex I during I/R injury in both tissues.
Environmental gradients have emerged as important barriers to structuring populations and species distributions. We set out to test whether the strong salinity gradient from the marine North Sea to the brackish Baltic Sea in northern Europe represents an ecological and genetic break, and to identify life history traits that correlate with the strength of this break. We accumulated mitochondrial cytochrome oxidase subunit 1 sequence data, and data on the distribution, salinity tolerance, and life history for 28 species belonging to the Cnidaria, Crustacea, Echinodermata, Mollusca, Polychaeta, and Gastrotricha. We included seven non-native species covering a broad range of times since introduction, in order to gain insight into the pace of adaptation and differentiation. We calculated measures of genetic diversity and differentiation across the environmental gradient, coalescent times, and migration rates between North and Baltic Sea populations, and analyzed correlations between genetic and life history data. The majority of investigated species is either genetically differentiated and/or adapted to the lower salinity conditions of the Baltic Sea. Species exhibiting population structure have a range of patterns of genetic diversity in comparison with the North Sea, from lower in the Baltic Sea to higher in the Baltic Sea, or equally diverse in North and Baltic Sea. Two of the non-native species showed signs of genetic differentiation, their times since introduction to the Baltic Sea being about 80 and >700 years, respectively. Our results indicate that the transition from North Sea to Baltic Sea represents a genetic and ecological break: The diversity of genetic patterns points toward independent trajectories in the Baltic compared with the North Sea, and ecological differences with regard to salinity tolerance are common. The North Sea–Baltic Sea region provides a unique setting to study evolutionary adaptation during colonization processes at different stages by jointly considering native and non-native species.
Anthropogenic activities have a major impact on our planet and rapidly drive biodiversity loss in ecosystems at a global scale. Particularly over the last century, rising CO2 emissions significantly raised global temperatures and increased the intensity and frequency of droughts and heatwaves. Additionally, agricultural land use and fossil fuel combustion contribute to the continuous release of nitrogen (N) and phosphorus (P) into ecosystems worldwide through extensive fertilization and deposition from the atmosphere. It is important to understand how these rapid changes affect the evolution of plant populations and their adaptive potential. Adaptation by natural selection (i.e., adaptive evolution) within a few generations is an essential process as a response to rapid environmental changes. Rapid evolution of plant populations can be detected by using the so-called resurrection approach. Here, diaspores (i.e., seeds) from a population are collected before (ancestors) and after (descendants) a potential selection pressure (e.g., consecutive years of drought or changes in nutrient supply). Comparing phenotypes of ancestors and descendants in a common environment such as an outside garden, greenhouse, or climate chamber, may then reveal evolutionary changes. Ideally, plants are first grown in a common environment for an intermediate refresher generation to reduce parental and storage effects.
The aim of this thesis was to investigate the occurrence of adaptive evolution in natural plant populations in response to rapidly changing environments over the past three decades. I conducted three experiments using the resurrection approach to generate comprehensive data on the adaptive processes that acted on three plant populations from three different species over the last three decades. Furthermore, I filled knowledge gaps in plant evolutionary ecology and conceptually developed the resurrection approach further.
In Chapter I, I performed a novel approach by testing for adaptive evolution in natural plant populations using the resurrection approach in combination with in-situ transplantations. I cultivated seedlings from ancestors (23 – 26 years old) and contemporary descendants of three perennial species (Melica ciliata, Leontodon hispidus and Clinopodium vulgare) from calcareous grasslands in the greenhouse and In Chapter III, I assessed the reproducibility of phenotypic differences between genotypes among three different growth facilities (climate chamber, greenhouse, and outdoor garden). I also evaluated differences in phenotypic expression between plants grown after one vs. two intermediate generations (i.e., refresher generations). I performed this experiment within the framework of the resurrection approach and compared ancestors and descendants of the same population of Leontodon hispidus.
I observed very strong differences among plants growing in the different growth facilities. I found a significant interaction between the growth facility and the temporal origin (ancestors vs. descendants): descendants had significantly larger rosettes than ancestors only in the greenhouse and they flowered significantly later than ancestors exclusively in the climate chamber. I did not find significant differences between intermediate generations within the growth facilities. Overall, Chapter III shows that the use of a particular experimental system can dictate the presence and magnitude of phenotypic differences. This implies that absence of evidence is not evidence of absence when it comes to investigating genetically based trait differentiation among plant origins (in space or time). Experimental systems should be carefully designed to provide meaningful conditions, ideally mimicking the environmental conditions of the population’s origins. Finally, growing a second intermediate generation did not impact the genetic differences of ancestors and descendants within the environments, supporting the idea that only one intermediate generation may be sufficient to reduce detectable parental and storage effects.
The resurrection approach allows a better understanding of rapid plant adaptation, but some limitations deserve to be highlighted. I only studied one population per species, and Chapters II and III only focus on one population of L. hispidus, which is also hampering generalizations, as adaptive potential can vary greatly among populations of the same species. I only compared the ancestral genotypes to one descendant sample with a long time span in between (26 – 28 years), which makes it hard to pinpoint the selection agents that caused the genetic differentiation among the sampling years. Hence, closely monitoring biotic and abiotic factors of the studied populations between the ancestral and descendant sampling in future studies, would make identifying the responsible selection pressures more precise. I also recommend sampling multiple populations over consecutive years to improve the robustness of results and make generalizations more approachable.Furthermore, combining the resurrection approach with other methods such as in-situ transplantations will be valuable to offset the limitation that adaptations cannot be proven under artificial conditions (e.g., in the greenhouse).
In Arabidopsis thaliana, the stem cell niche (SCN) within the root apical meristem (RAM) is maintained by an intricate regulatory network that ensures optimal growth and high developmental plasticity. Yet, many aspects of this regulatory network of stem cell quiescence and replenishment are still not fully understood. Here, we investigate the interplay of the key transcription factors (TFs) BRASSINOSTEROID AT VASCULAR AND ORGANIZING CENTRE (BRAVO), PLETHORA 3 (PLT3) and WUSCHEL-RELATED HOMEOBOX 5 (WOX5) involved in SCN maintenance. Phenotypical analysis of mutants involving these TFs uncover their combinatorial regulation of cell fates and divisions in the SCN. Moreover, interaction studies employing fluorescence resonance energy transfer fluorescence lifetime imaging microscopy (FRET-FLIM) in combination with novel analysis methods, allowed us to quantify protein-protein interaction (PPI) affinities as well as higher-order complex formation of these TFs. We integrated our experimental results into a computational model, suggesting that cell type specific profiles of protein complexes and characteristic complex formation, that is also dependent on prion-like domains in PLT3, contribute to the intricate regulation of the SCN. We propose that these unique protein complex ‘signatures’ could serve as a read-out for cell specificity thereby adding another layer to the sophisticated regulatory network that balances stem cell maintenance and replenishment in the Arabidopsis root.
The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5′-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.
Alzheimer’s disease (AD) is characterized by the deposition of aggregated species of amyloid beta (Aβ) in the brain, which leads to progressive cognitive deficits and dementia. Aβ is generated by the successive cleavage of the amyloid precursor protein (APP), first by β-site APP cleaving enzyme 1 (BACE1) and subsequently by the γ-secretase complex. Those conditions which enhace or reduce its clearance predispose to Aβ aggregation and the development of AD. In vitro studies have demonstrated that Aβ assemblies spark a feed-forward loop heightening Aβ production. However, the underlying mechanism remains unknown. Here, we show that oligomers and fibrils of Aβ enhance colocalization and physical interaction of APP and BACE1 in recycling endosomes of human neurons derived from induced pluripotent stem cells and other cell types, which leads to exacerbated amyloidogenic processing of APP and intracellular accumulation of Aβ42. In cells that are overexpressing the mutant forms of APP which are unable to bind Aβ or to activate Go protein, we have found that treatment with aggregated Aβ fails to increase colocalization of APP with BACE1 indicating that Aβ-APP/Go signaling is involved in this process. Moreover, inhibition of Gβγ subunit signaling with βARKct or gallein prevents Aβ-dependent interaction of APP and BACE1 in endosomes, β-processing of APP, and intracellular accumulation of Aβ42. Collectively, our findings uncover a signaling mechanism leading to a feed-forward loop of amyloidogenesis that might contribute to Aβ pathology in the early stages of AD and suggest that gallein could have therapeutic potential.
In recent years, the number and type of treatment options in advanced bladder cancer (BC) have been rapidly evolving. To select an effective therapy and spare unnecessary side effects, predictive biomarkers are urgently needed. As the host’s anti-cancer immune response is by far the most effective system to impede malignant tumor growth, immune system-based biomarkers are promising. We have recently described altered proteasomal epitope processing as an effective immune escape mechanism to impair cytotoxic T-cell activity. By altering the neoantigens’ characteristics through different proteasomal peptide cleavage induced by non-synonymous somatic mutations, the ability for T-cell activation was decreased (“processing escapes”). In the present study, we analyzed primary chemo-naïve tissue samples of 26 adjuvant platinum-treated urothelial BC patients using a targeted next-generation sequencing panel followed by the epitope determination of affected genes, a machine-learning based prediction of epitope processing and proteasomal cleavage and of HLA-affinity as well as immune activation. Immune infiltration (immunohistochemistries for CD8, granzyme B, CD45/LCA) was digitally quantified by a pathologist and clinico-pathological and survival data were collected. We detected 145 epitopes with characteristics of a processing escape associated with a higher number of CD8-positive but lower number of granzyme B-positive cells and no association with PD-L1-expression. In addition, a high prevalence of processing escapes was associated with unfavorable overall survival. Our data indicate the presence of processing escapes in advanced BC, potentially creating a tumor-promoting pro-inflammatory environment with lowered anti-cancerous activity and independence from PD-L1-expression. The data also need to be prospectively validated in BC treated with immune therapy.
Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.
In recent decades, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quantification of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review actual strategies and problems of the methods for the quantitative analysis of peptides, proteins, and finally proteomes by mass spectrometry. The common themes, the differences, and the potential pitfalls of the main approaches are presented in order to provide a survey of the emerging field of quantitative, mass spectrometry-based proteomics.
Biallelic pathogenic variants in CLPP, encoding mitochondrial matrix peptidase ClpP, cause a rare autosomal recessive condition, Perrault syndrome type 3 (PRLTS3). It is characterized by primary ovarian insufficiency and early sensorineural hearing loss, often associated with progressive neurological deficits. Mouse models showed that accumulations of (i) its main protein interactor, the substrate-selecting AAA+ ATPase ClpX, (ii) mitoribosomes, and (iii) mtDNA nucleoids are the main cellular consequences of ClpP absence. However, the sequence of these events and their validity in human remain unclear. Here, we studied global proteome profiles to define ClpP substrates among mitochondrial ClpX interactors, which accumulated consistently in ClpP-null mouse embryonal fibroblasts and brains. Validation work included novel ClpP-mutant patient fibroblast proteomics. ClpX co-accumulated in mitochondria with the nucleoid component POLDIP2, the mitochondrial poly(A) mRNA granule element LRPPRC, and tRNA processing factor GFM1 (in mouse, also GRSF1). Only in mouse did accumulated ClpX, GFM1, and GRSF1 appear in nuclear fractions. Mitoribosomal accumulation was minor. Consistent accumulations in murine and human fibroblasts also affected multimerizing factors not known as ClpX interactors, namely, OAT, ASS1, ACADVL, STOM, PRDX3, PC, MUT, ALDH2, PMPCB, UQCRC2, and ACADSB, but the impact on downstream metabolites was marginal. Our data demonstrate the primary impact of ClpXP on the assembly of proteins with nucleic acids and show nucleoid enlargement in human as a key consequence.
The maintenance of cellular homeostasis over time is essential to avoid the degeneration of biological systems leading to aging and disease. Several interconnected pathways are active in this kind of quality control. One of them is autophagy, the vacuolar degradation of cellular components. The absence of the sorting nexin PaATG24 (SNX4 in other organisms) has been demonstrated to result in impairments in different types of autophagy and lead to a shortened lifespan. In addition, the growth rate and the size of vacuoles are strongly reduced. Here, we report how an oleic acid diet leads to longevity of the wild type and a PaAtg24 deletion mutant (ΔPaAtg24). The lifespan extension is linked to altered membrane trafficking, which abrogates the observed autophagy defects in ΔPaAtg24 by restoring vacuole size and the proper localization of SNARE protein PaSNC1. In addition, an oleic acid diet leads to an altered use of the mitochondrial respiratory chain: complex I and II are bypassed, leading to reduced reactive oxygen species (ROS) production. Overall, our study uncovers multiple effects of an oleic acid diet, which extends the lifespan of P. anserina and provides perspectives to explain the positive nutritional effects on human aging.
The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.
Spaceflight affects the body on every level. Reports on astronaut health identify bone marrow remodelling and dysfunction of the innate immune system as significant health risks of long-term habitation in space. Microgravity-induced alterations of the bone marrow induce physical changes to the bone marrow stem cell niche. Downstream effects on innate immunity are expected due to impaired hematopoiesis and myelopoiesis. To date, few studies have investigated these effects in real microgravity and the sparsely available literature often reports contrasting results. This emphasizes a need for the development of physiologically relevant in vitro models of the bone marrow stem cell niche, capable of delivering appropriate sample sizes for robust statistics. Here, we review recent findings on the impact of spaceflight conditions on innate immunity in in vitro and animal models and discusses the latest in vitro models of the bone marrow stem cell niche and their potential translatability to gravitational biology research.
Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal.
The nucleus reuniens drives hippocampal goal‑directed trajectory sequences for route planning
(2023)
Goal-directed spatial navigation requires accurate estimates of one’s position and destination, as well as careful planning of a route between them to avoid known obstacles in the environment. Despite its general importance across species, the neural circuitry supporting the ability for route planning remains largely unclear. Previous studies described that place cells in the hippocampal CA1 encode the animal's next movement direction (Wood et al., 2000; Ito et al., 2015) and upcoming navigational routes (Pfeiffer & Foster, 2013). However, it has been shown that part of the CA1 activity representing the animal’s future behaviors is not necessarily generated in the hippocampus, but is derived from the medial prefrontal cortex (PFC) via the nucleus reuniens of the thalamus (RE) (Ito et al., 2015). Notably, the importance of the PFC in navigation has been demonstrated in several studies, including the recent finding of a goal map in the orbitofrontal cortex (Basu et al., 2021). Therefore, I hypothesized that information flow from the PFC to CA1 via the RE plays a key role in route planning.
To assess the animals' route planning ability, I designed a new navigation task in which a rat has to navigate to a fixed target location from various starting positions in an arena. Furthermore, by adding an L-shaped wall in the maze and removing all light sources in the experimental room, this task forced the animals to plan a wall-avoiding route without relying on direct sensory perceptions. I confirmed that rats could learn this task successfully, memorizing the wall location and taking a smooth wall-avoidance route. To test the role of the RE, I inactivated RE neurons by expressing the inhibitory opsin SwiChR++, which resulted in a significant deficit in the animal’s route planning ability, taking a longer non-smooth path to the destination. By contrast, this manipulation did not affect navigation performance when a straight goal-directed route was available, suggesting a specific role of the RE in route planning. I further found that DREADDs-mediated inactivation of neurons in the bilateral hippocampi resulted in a similar deficit in route planning ability, implying cooperation between the RE and the hippocampus.
I finally examined the activity of hippocampal CA1 neurons with and without RE inactivation. While neurons in the hippocampus exhibited brief trajectory sequences corresponding to the animal’s subsequent goal-directed journey, I found that this goal-directed bias of trajectory events was significantly reduced by RE inactivation, likely associated with route-planning deficits in these animals.
Altogether, this dissertation demonstrates the role of the RE from both behavioral and neural coding perspectives, identifying a pivotal circuit element supporting the animal’s route-planning ability.
Fungi belonging to the Rhytismatales (Ascomycota) are parasites or endophytes of plants, some are saprophytes. Their fruiting bodies are localized in different organs of the host plants belonging to many different families of gymnosperms and angiosperms. Many species of Rhytismatales are known on species of Pinaceae, Ericaceae, and Poaceae. These fungi usually have ascomata that are more or less embedded in host tissue and open by longitudinal or radial splits. They have a more or less carbonized covering stroma, thin-walled, iodine negative asci, and ascospores usually covered by gelatinous sheaths.
In the present study, two lists of species of Rhytismatales in China are presented. One is based on literature and includes 103 species in 15 genera. The second one contains the names of the species in the present study, 57 species in 20 genera based on 90 specimens I collected in the Yunnan and Anhui province in China during July to August in 2001. 31 species in the second list are new species or new records for China, so we presently know 134 species in 22 genera of Rhytismatales for China. 28 new species of Rhytismatales are proposed, 21 species from the Yunnan province and seven from the Anhui province. Among them, three new species are proposed in three new genera, Nematococcomyces, New Genus 1, and New Genus 2, respectively. The 28 new species are Cerion sp., Coccomyces spp. 1-2, Colpoma spp. 1-2, Hypoderma spp. 1-6, Lirula sp., Lophodermella sp., Lophodermium spp. 1-5, Nematococcomyces rhododendri C.-L. Hou, M. Piepenbr. & Oberw., Neococcomyces sp., New Genus 1 sp., New Genus 2 sp., Rhytisma spp. 1-2, Soleella sp., Terriera spp. 1-2, and Therrya sp. The genus Davisomycella is proposed as a synonym of Lophodermella based on observations of the morphology, ecology, and the infected organ. The four genera Cerion, Naemacyclus, Terriera, and Therrya, and three species, Hypoderma rubi, Lophodermium uncinatum, and Naemacyclus pinastri, are reported for the first time for China. All the new taxa, the newly recorded ones, as well as six species which had not been illustrated in detail before, are carefully described and illustrated by line drawings in the present study.
The results show that species of Rhytismatales are highly diverse especially in the natural vegetation in high mountainous areas in China. Most species of Rhytismatales are conspicuously host specific. The diversity of Rhytismatales is closely related to that of the preferred hosts, which are members of Pinaceae, Ericaceae, and Cupressaceae. Based on the detailed morphological observations, the significance of different morphological characteristics for a natural classification of Rhytismatales is discussed. Genera are traditionally defined by character states of a few characteristics, namely the opening patterns of ascomata, the depth of ascomata in the host tissue, and asci and ascospore shape. Data from collections in the field, detailed morphological investigation, and molecular data show, however, that the ecology, the infected organ, the host relationship, and many other characteristics have to be combined to circumscribe natural groups.
The discussion of the systematic significance of morphological characteristics is complemented by molecular data. In the present study, partial nuclear large subunit rDNA sequences of 52 specimens representing 38 species are used to analyse phylogenetic relationships for members of Rhytismatales.
Most species of Rhytismatales are placed in a monophyletic group corresponding to the Rhytismatales in the Maximum Parsimony analysis. The delimitation of the Rhytismatales from the Helotiales is, however, difficult. Cyclaneusma minus should be transferred from the Rhytismatales to the Helotiales, and Cudonia circinans and Spathularia flavida from the Helotiales to the Rhytismatales. These tranfers have previously been proposed based on SSU rDNA analysis by other authors. New Genus 1 sp. has morphological characteristics typical for species of Rhytismatales. In the LSU rDNA analysis, however, it is more closely related to Helotiales rather than toRhytismatales. Therefore New Genus 1 sp. is placed in the Helotiales.
Tryblidiopsis pinastri is morphologically intermediate between members of Rhytismataceae and Cudoniaceae. LSU rDNA sequences in the present study show that T. pinastri is more closely related to species of Cudoniaceae. Therefore, this species is removed from the Rhytismataceae to the Cudoniaceae. The delimitation of further families could not be resolved in the present analysis.
Though many new morphological, ecological, and molecular phylogenetic findings are contributed for the first time, the systematic conclusions at generic, family, and order level can only be fragmentary in the present study. With more collections and more molecular data of the worldwide 450 known and many more unknown species of Rhytismatales at hand, a natural system combining morphological and molecular analysis can be elaborated.
Cytochrome P450 (CYP) enzymes oxidize, peroxidize and/or reduce cholesterol, vitamins, steroids, xenobiotics and numerous pharmacological substances in an oxygen- and NADPHdependent manner. Since many CYP isozymes are also capable of metabolizing arachidonic acid to biologically active products, CYP enzymes are often described as the third pathway of arachidonic acid metabolism i.e., in addition to cyclooxygenases and lipoxygenases. CYP enzymes are predominantly expressed in the liver while others, such as members of the CYP 2J, CYP 2C and CYP 4A subfamilies, can be detected in extrahepatic tissues, particularly in the cardiovascular system. Recent data suggest that a CYP 2C enzyme(s) expressed in coronary artery endothelial cells generate epoxyeicosatrienoic acids (5,6-; 8,9-; 11,12- and 14,15-EET) which contribute to the acute control of vascular tone and the longterm regulation of vascular homeostasis.
The expression of CYP 2C in coronary artery endothelial cells is regulated by a number of stimuli, such as cyclic stretch and fluid shear stress as well as by the corticosteroid cortisol and a number of CYP substrates (nifedipine, cerivastatin and -naphthoflavone). However, the signalling pathways and the transcription factors involved in regulating the expression of the gene are unknown.
Since most of the CYP 2C enzymes are transcriptionally regulated, we were interested in identifying the CYP 2C isoform(s) expressed in porcine coronary artery endothelial cells (PCAEC) as well as determining its/their promoter sequence(s). The overall goal was to study the involvement of different transcription factor binding elements in the regulation of the CYP 2C gene(s). Porcine coronary arteries were used given the possibility of analysing the results obtained at the cellular level with alterations in vascular function. Comparison of the porcine CYP 2C and the human CYP 2C8 and 2C9 promoters was also a major goal of this study.
To identify the relevant porcine CYP 2C isoform nested RT-PCR was performed using total RNA from porcine coronary artery endothelial cells. Comparison of the sequence of the product of this reaction with the NCBI database suggested that the CYP 2C expressed in PCAEC was approximately 85% homologous with the human CYP 2C9 enzyme. To obtain the full length CYP 2C isoform 5´ rapid amplification of cDNA end (5´ RACE) was performed using a downstream reverse gene specific primer which is conserved in all of the porcine CYP 2C isoforms. The intention behind using such a primer was to amplify all the possible CYP cDNAs expressed in PCAEC. With the 5´ RACE technology it was possible not only to identify the exact isoform (CYP 2C34) expressed in PCAEC, but it was also possible to amplify 550 bp of the 5´ upstream region. This result was authenticated by comparing the protein/nucleotide sequence with other human CYP 2C genes such as CYP 2C8 and CYP 2C9 as well as different porcine CYP 2C genes (CYP 2C34, CYP 2C49). Multiple protein/nucleotide sequence alignment revealed approximately 85-90% sequence identity. An exon1-2 specific radio-labelled probe of the CYP 2C34 gene was then used to screen a porcine genomic library for positive genomic clones containing the promoter region of the CYP 2C34 gene.
For the isolation of 5´ flanking region of CYP 2C34 gene a PCR-based directional genome walking strategy was used in which the positive porcine genomic BAC clones were taken as a DNA template. Four arbitrarily designed universal walking primers and a gene-specific primer derived from the CYP 2C34 gene sequence were employed and led to the identification and isolation of 1.4 kb of the 5´ flanking region.
The 1.4 kb 5´ flanking region of CYP 2C34 gene contains multiple transcription factor binding sites including glucocorticoid-responsive element (GRE), hypoxia-responsive element (HRE), CAAT-enhancer binding protein (C/EBP), stress responsive element (STRE) consensus sequences. CYP 2C34 promoter constructs were generated and reporter gene activity (luciferase) activity was compared with that of a promoterless vector (pGL3-Basic) at first in HEK cells and then in PCAEC. After using cortisol as a positive control to demonstrate that the promoter constructs generated were functional we determined the effects of physiologically relevant stimuli i.e., hypoxia and cyclic stretch. Additional experiments with zinc sulphate were performed in a preliminary analysis of the role of Zn2+ inducible transcription factors and might be cooperative heterodimerization formation with these transcription factor with C/EBP in the regulation of CYP 2C34 expression. With all these stimuli, reporter gene activity of CYP 2C34 promoter was significantly (3-8 fold) increased over values obtained in unstimulated cells.
Analysis of the regions that are essential for the induction of promoter activity in response to the different stimuli of interest have to be performed in combination with gel shift assays, siRNA experiments as well as site-directed mutagenesis experiments. Comparison of the regulation of the CYP 2C34 gene and correlation with changes in vascular function (in isolated porcine coronary arteries) should deliver information relevant to the regulation of the CYP 2C enzyme expressed in human coronary artery endothelial cells. The recent demonstration of a clinically relevant role for CYP 2C9 in coronary heart disease underlines the importance of such a study.
Determination of the structure of complex I of Yarrowia lipolytica by single particle analysis
(2004)
Komplex I enthält ein Flavinmononukleotid sowie mindestens acht Eisen- Schwefel Zentren als redoxaktive Cofaktoren. Da ein wesentlicher Teil des mitochondrialen Genoms für Untereinheiten von Komplex I codiert, betrifft eine Vielzahl von mitochondrialen Erkrankungen diesen Enzymkomplex.
Komplex I wurde bisher aus Mitochondrien, Chloroplasten und Bakterien isoliert. Die Minimalform von Komplex I wird in Bakterien gefunden, wo er aus 14 (bzw 13 im Falle einer Genfusion) Untereinheiten besteht und eine Masse von etwa 550 kDa aufweist. Generell werden sieben hydrophile und sieben hydrophobe Untereinheiten mit über 50 vorhergesagten Transmembranhelices gefunden. Im Komplex I aus Eukaryoten wurde eine grössere Anzahl zusätzlicher, akzessorischer Untereinheiten nachgewiesen. Hier werden die sieben hydrophoben Untereinheiten vom mitochondrialen Genom codiert, während alle anderen Untereinheiten kerncodiert sind und in das Mitochondrium importiert werden müssen.
Die obligat aerobe Hefe Yarrowia lipolytica wurde als Modellsystem zur Untersuchung von eukaryotischem Komplex I etabliert. Die bisher am besten untersuchte Hefe Saccharomyces cerevisiae enthält keinen Komplex I. Hier wird die Oxidation von NADH durch eine andere Klasse von sogenannten alternativen NADH Dehydrogenasen durchgeführt. Auch Y. lipolytica enthält ein solches alternatives Enzym, das allerdings mit seiner Substratbindungsstelle zur Aussenseite der inneren Mitochondrienmembran orientiert ist. Durch molekularbiologische Manipulation konnte eine interne Version dieses Enzymes exprimiert werden, wodurch es möglich ist, letale Defekte in Komplex I Deletionsmutanten zu kompensieren. Mittlerweile wurden alle Voraussetzungen geschaffen, um kerncodierte Untereinheiten von Komplex I aus Y. lipolytica gezielt genetisch zu verändern. Die Proteinreinigung wird durch die Verwendung einer auf einem His-tag basierenden Affinitätsreinigung erheblich erleichtert...
The transcriptional regulator RcsB controls the expression of a minimum of 20 different genes having diverse functionalities and biosynthetic operons in the family of Enterobacteriaceae. While in the heterodimeric complex with the co activator RcsA, the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various co activators, resulting in recognition of different DNA targets. In this study the interaction of RcsB with some of these DNA targets have been analysed by a diverse array of techniques including gel shift assay and SPR. The solution structure of the C-terminal DNA-binding domain of RcsB from Erwinia amylovora spanning amino acid residues 129-215 has been solved in this study by heteronuclear NMR spectroscopy. The C-terminal domain is composed of four α-helices where the two central helices of the H-T-H motif are similar to the structures of the regulatory proteins GerE, NarL and TraR. The DNA-binding activity of the C-terminal domain alone is established for the first time in this study and was specified by fluorescence spectroscopy, SPR and NMR titration experiments. The molecular interaction between the individual RcsB domains was analysed by cross-linking experiments and heteronuclear NMR spectroscopy and the amino acid residues of the C-terminal domain involved in this interaction were identified precisely. Another important part of this project was the cell-free production of different Trp analogue labelled RcsB protein. RcsB protein was produced in quite a good yield with different Trp analogue having spectrally enhanced properties. The isolated RcsB alloproteins proved to be ideal for protein interaction studies by fluorescence spectroscopy and the very first evidence of an oligomerization of RcsB due to molecular association has been put forth from these studies. The phosphorylated state of the RcsB protein was mimicked by a beryllofluoride complex in order to study its role in transcriptional regulation. It was found that RcsB alone could bind to DNA targets upon this modification by the beryllofluoride complex. Thus the phosphorylation of the protein that involves the Asp 56 residue induces a structural change of the protein followed probably by a domain movement also, so that the C-terminal domain having the H-T-H DNA binding motif that was previously eclipsed by the N-terminal domain is relieved of this constraint.
Highlights
• Cryo-EM structure of a yeast F1Fo-ATP synthase dimer
• Inhibitor-free X-ray structure of the F1 head and rotor complex
• Mechanism of ATP generation by rotary catalysis
• Structural basis of cristae formation in the inner mitochondrial membrane
Summary
We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing.
Understanding the complexity of transcriptional regulation is a major goal of computational biology. Because experimental linkage of regulatory sites to genes is challenging, computational methods considering epigenomics data have been proposed to create tissue-specific regulatory maps. However, we showed that these approaches are not well suited to account for the variations of the regulatory landscape between cell-types. To overcome these drawbacks, we developed a new method called STITCHIT, that identifies and links putative regulatory sites to genes. Within STITCHIT, we consider the chromatin accessibility signal of all samples jointly to identify regions exhibiting a signal variation related to the expression of a distinct gene. STITCHIT outperforms previous approaches in various validation experiments and was used with a genome-wide CRISPR-Cas9 screen to prioritize novel doxorubicin-resistance genes and their associated non-coding regulatory regions. We believe that our work paves the way for a more refined understanding of transcriptional regulation at the gene-level.
In fungi, the mitochondrial respiratory chain complexes (complexes I–IV) are responsible for oxidative phosphorylation, as in higher eukaryotes. Cryo-EM was used to identify a 200 kDa membrane protein from Neurospora crassa in lipid nanodiscs as cytochrome c oxidase (complex IV) and its structure was determined at 5.5 Å resolution. The map closely resembles the cryo-EM structure of complex IV from Saccharomyces cerevisiae. Its ten subunits are conserved in S. cerevisiae and Bos taurus, but other transmembrane subunits are missing. The different structure of the Cox5a subunit is typical for fungal complex IV and may affect the interaction with complex III in a respiratory supercomplex. Additional density was found between the matrix domains of the Cox4 and Cox5a subunits that appears to be specific to N. crassa.
CryoEM at IUCRJ: a new era
(2016)
Highlights
• Cryo-EM structures of the yeast low-affinity phosphate importer ScPho90
• Complementary structures reveal insights into the substrate translocation mechanism
• Comparisons with homologous transporters highlight the conserved transport mechanism
• Regulation by the SPX domain is discussed
Summary
Phosphate homeostasis is essential for all living organisms. Low-affinity phosphate transporters are involved in phosphate import and regulation in a range of eukaryotic organisms. We have determined the structures of the Saccharomyces cerevisiae phosphate importer Pho90 by electron cryomicroscopy in two complementary states at 2.3 and 3.1 Å resolution. The symmetrical, outward-open structure in the presence of phosphate indicates bound substrate ions in the binding pocket. In the absence of phosphate, Pho90 assumes an asymmetric structure with one monomer facing inward and one monomer facing outward, providing insights into the transport mechanism. The Pho90 transport domain binds phosphate ions on one side of the membrane, then flips to the other side where the substrate is released. Together with functional experiments, these complementary structures illustrate the transport mechanism of eukaryotic low-affinity phosphate transporters.
MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3' untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T-cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.
In this special issue of Matrix Biology centered on proteoglycan biology we have assembled a blend of articles focused on the state-of-the-art of proteoglycanology. The field has greatly expanded in the past three decades and now encompasses all the areas of biology. This special issue is divided into five chapters describing hyaluronan metabolism, biosynthetic and catabolic pathways of proteoglycans and their roles in inflammation, cancer, repair and development. We hope that the new original work and the reviews from recognized leaders will stimulate investigations in this exciting and fertile field of research.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals with convolutional neural networks to achieve high single-molecule accuracy and outperforms other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions, using signals from nanopore direct RNA sequencing. CHEUI processes observed and expected signals with convolutional neural networks to achieve high single-molecule accuracy and outperform other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI’s capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.
The expanding field of epitranscriptomics might rival the epigenome in the diversity of the biological processes impacted. However, the identification of modifications in individual RNA molecules remains challenging. We present CHEUI, a new method that detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) at single-nucleotide and single-molecule resolution from Nanopore signals. CHEUI predicts methylation in Nanopore reads and transcriptomic sites in a single condition, and differential m6A and m5C methylation between any two conditions. Using extensive benchmarking with Nanopore data derived from synthetic and natural RNA, CHEUI showed higher accuracy than other existing methods in detecting m6A and m5C sites and quantifying the site stoichiometry levels, while maintaining a lower proportion of false positives. CHEUI provides a new capability to detect RNA modifications with high accuracy and resolution that can be cost-effectively expanded to other modifications to unveil the full span of the epitranscriptome in normal and disease conditions.
The translation eukaryotic elongation factor 1alpha (eEF1A) is a monomeric GTPase involved in protein synthesis. In addition, this protein is thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis. Here we show that eEF1A is associated with the alpha2 subunit of the inhibitory glycine receptor in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Glycine receptor activation in spinal cord neurons cultured for 1 week resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites, and at synapses of cultured hippocampal and spinal cord neurons. Consistent with our biochemical data, immunoreactivities of both proteins were partially overlapping with glycine receptor immunoreactivity in cultured spinal cord and hippocampal neurons. After 5 weeks in culture, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons. Interestingly, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or glycine receptors and simultaneous N-methyl-D-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway.
Mutations in the clk-1 gene result in slower development and increased life span in Caenorhabditis elegans. The Saccharomyces cerevisiae homologue COQ7/CAT5 is essential for several metabolic pathways including ubiquinone biosynthesis, respiration, and gluconeogenic gene activation. We show here that Coq7p/Cat5p is a mitochondrial inner membrane protein directly involved in ubiquinone biosynthesis, and that the defect in gluconeogenic gene activation in coq7/cat5 null mutants is a general consequence of a defect in respiration. These results obtained in the yeast model suggest that the effects on development and life span in C. elegans clk-1 mutants may relate to changes in the amount of ubiquinone, an essential electron transport component and a lipid soluble antioxidant.
Calreticulin is a Ca2+ -binding chaperone that resides in the lumen of the endoplasmic reticulum and is involved in the regulation of intracellular Ca2+ homeostasis and in the folding of newly synthesized glycoproteins. In this study, we have used site-specific mutagenesis to map amino acid residues that are critical in calreticulin function. We have focused on two cysteine residues (Cys(88) and Cys(120)), which form a disulfide bridge in the N-terminal domain of calreticulin, on a tryptophan residue located in the carbohydrate binding site (Trp(302)), and on certain residues located at the tip of the "hairpin-like" P-domain of the protein (Glu(238), Glu(239), Asp(241), Glu(243), and Trp(244)). Calreticulin mutants were expressed in crt(-/-) fibroblasts, and bradykinin-dependent Ca2+ release was measured as a marker of calreticulin function. Bradykinin-dependent Ca2+ release from the endoplasmic reticulum was rescued by wild-type calreticulin and by the Glu(238), Glu(239), Asp(241), and Glu(243) mutants. The Cys(88) and Cys(120) mutants rescued the calreticulin-deficient phenotype only partially ( approximately 40%), and the Trp(244) and Trp(302) mutants did not rescue it at all. We identified four amino acid residues (Glu(239), Asp(241), Glu(243), and Trp(244)) at the hairpin tip of the P-domain that are critical in the formation of a complex between ERp57 and calreticulin. Although the Glu(239), Asp(241), and Glu(243) mutants did not bind ERp57 efficiently, they fully restored bradykinin-dependent Ca2+ release in crt(-/-) cells. This indicates that binding of ERp57 to calreticulin may not be critical for the chaperone function of calreticulin with respect to the bradykinin receptor.
Antigen presentation to cytotoxic T lymphocytes via major histocompatibility complex class I (MHC I) molecules depends on the heterodimeric transporter associated with antigen processing (TAP). For efficient antigen supply to MHC I molecules in the ER, TAP assembles a macromolecular peptide-loading complex (PLC) by recruiting tapasin. In evolution, TAP appeared together with effector cells of adaptive immunity at the transition from jawless to jawed vertebrates and diversified further within the jawed vertebrates. Here, we compared TAP function and interaction with tapasin of a range of species within two classes of jawed vertebrates. We found that avian and mammalian TAP1 and TAP2 form heterodimeric complexes across taxa. Moreover, the extra N-terminal domain TMD0 of mammalian TAP1 and TAP2 as well as avian TAP2 recruits tapasin. Strikingly, however, only TAP1 and TAP2 from the same taxon can form a functional heterodimeric translocation complex. These data demonstrate that the dimerization interface between TAP1 and TAP2 and the tapasin docking sites for PLC assembly are conserved in evolution, whereas elements of antigen translocation diverged later in evolution and are thus taxon specific.
GTPase-activating proteins are required to terminate signaling by Rap1, a small guanine nucleotide-binding protein that controls integrin activity and cell adhesion. Recently, we identified Rap1GAP2, a GTPase-activating protein of Rap1 in platelets. Here we show that 14-3-3 proteins interact with phosphorylated serine 9 at the N terminus of Rap1GAP2. Platelet activation by ADP and thrombin enhances serine 9 phosphorylation and increases 14-3-3 binding to endogenous Rap1GAP2. Conversely, inhibition of platelets by endothelium-derived factors nitric oxide and prostacyclin disrupts 14-3-3 binding. These effects are mediated by cGMP- and cAMP-dependent protein kinases that phosphorylate Rap1GAP2 at serine 7, adjacent to the 14-3-3 binding site. 14-3-3 binding does not change the GTPase-activating function of Rap1GAP2 in vitro. However, 14-3-3 binding attenuates Rap1GAP2 mediated inhibition of cell adhesion. Our findings define a novel crossover point of activatory and inhibitory signaling pathways in platelets.
Interest is an important factor for successful learning that has been the subject of intensive research for decades. Although interest in nature is of great importance for environmental education, to date there is no valid and reliable measurement tool. Therefore, the purpose of this study was to develop and test a scale for interest in nature, the Nature Interest Scale (NIS). In study 1, nine items were selected based on the three dimensions of the psychological interest construct to represent interest in nature. The factor structure of this new measurement instrument, was tested using confirmatory factor analyses. The results show that the instrument represents the three dimensions of the interest construct well. In study 2 the validity (discriminant and convergent validity) as well as the reliability (internal consistency, composite reliability, test-retest reliability) of the NIS were demonstrated. In study 3, the applicability of the NIS was tested with a different target group, students with learning disabilities. The results of this factor analysis also confirm the factor structure of the scale. Thus, this study provides a valid and reliable measurement tool for individual interest in nature that can be used for future research.
Nitric oxide (NO) is a potent mediator with pleiotropic functions such as inhibition of platelet aggregation, smooth muscle relaxation and regulation of neuronal transmission. These effects are mostly mediated by intracellular NO-sensitive guanylyl cyclases (GCs) which convert GTP into the second messenger, cGMP. This messenger in turn activates multiple downstream effectors such as cGMP-dependent protein kinases, cGMP-regulated ion channels and cGMPdependent phosphodiesterases. Mammalian NO-sensitive GCs are obligate heterodimers of an α and β subunit each. Given that these enzymes play a key role in cGMP-mediated pathways, one may anticipate that mechanisms other than allosteric activation via NO may exist to regulate the production and turnover of cGMP. In this thesis, novel aspects of the regulation of the most abundantly expressed GC heterodimer α1β1 are presented.
A possible mechanism of regulation that was tested here, is tyrosine phosphorylation. Using anti-phosphotyrosine antibodies, the phosphorylation of the β1 subunit was detected after incubation of β1-overexpressing COS-1 cells with protein tyrosine phosphatase (PTP) inhibitors such as pervanadate and bpV(phen). β1 phosphorylation on tyrosines was also observed in PC-12 cells which endogenously express GC and in rat aorta after inhibition of PTPs. Furthermore, hydrogen peroxide was found to be a physiological stimulus for the induction of reversible β1 tyrosine phosphorylation in intact cells. Using phenylalanine mutants of different tyrosines, residue 192 (Y192) of β1 was identified as the major phosphorylation site. Consistent with this finding, sequence analyses showed that Y192 forms part of a motif that resembles a preferential target site for Src-like kinases. When tyrosine-phosphorylated, this motif exposes a typical SH2 docking site for members of the Src kinase family.
Experiments with inhibitors of Src kinases, PP1 and PP2, clearly showed that phosphorylation of Y192 is Src-dependent. Preincubation of β1-expressing cells with these inhibitors significantly reduced the level of phosphorylated β1 after bpV(phen) treatment. Furthermore, co-expression of β1 with Src led to a strong phosphorylation of this subunit. Co-precipitation experiments showed that Src interacts with GC. Interestingly, kinases of the Src family are recruited to β1 via the SH2 domain upon phosphorylation of Y192. Together, these results indicate that Src kinases phosphorylate tyrosine 192 thereby creating a docking site for their own SH2 domains. Kinase bound to GC may then catalyze phosphorylation of GC or other downstream effectors. Inhibition of PTPs altered GC activity in two ways: it increased both the basal activity and the YC-1- and BAY 41-2272-stimulated activity two-fold, and it reduced the sensitivity of the enzyme towards NO. The detailed mechanism of action is still unknown, but experiments using the mutant β1[Y192F] demonstrated that residue 192 is not responsible for these effects.
Another major focus of this thesis was the identification of novel GC binding proteins. Using the yeast two-hybrid approach, the carboxy-terminal portion of a protein named AGAP1 (amino acid (aa) 399-804) was found to interact with the catalytic domain of α1 (aa 466-690) and with the regulatory domain of β1 (aa 1-348). Human AGAP1 is a multidomain protein of 804 amino acids with a calculated molecular mass of 89,1 kDa comprising an Arf-GAP (GAP:GTPase activating protein), a putative GTPase domain, two Ankyrin repeats and a PHdomain. Co-precipitation experiments using lysates from mammalian cells overexpressing both binding partners confirmed the interaction of AGAP1 with the GC subunits. Immunofluorescence analyses demonstrated that AGAP1 co-localizes with GC in the cytoplasm of COS-1 cells.
In Northern blots, AGAP1 mRNA was detected in various human and murine tissues showing a comparable expression pattern described for the mRNA of α1 and β1. Using an AGAP1-specific antibody, endogenous protein was precipitated from lysates of HEK-293 cells derived from human embryonic kidney. The same antibody efficiently cross-reacted with the rat homologue (rAGAP1) and immunoprecipitated endogenous rAGAP1 from lysates of PC-12 cells, aorta and heart. The molecular mass of rAGAP1 is larger than that of the human protein, possibly due to an additional exon present in the rat genome. Like β1, AGAP1 is a substrate for tyrosine kinases. Phosphorylation of AGAP1 was detected after inhibition of PTPs or by coexpression of Src. Furthermore, the kinase inhibitor PP2 strongly impaired phosphorylation of AGAP1 after pervanadate treatment suggesting that tyrosine kinases of the Src family are involved. Measurements of cGMP production showed that AGAP1 has no influence on the activity of NO-sensitive GC. Interestingly, inhibition of PTPs potently increased the complex formation between AGAP1 and GC indicating that the interaction between these two proteins is modulated by reversible tyrosine phosphorylation. Whether this effect is due to the phosphorylation of AGAP1 or GC is still unknown. AGAP1 associates with endosomes and exposes Arf-GAP activity towards Arf1 and Arf5 which are involved in vesicular transport. Thus, one may hypothesize that binding of α1β1 to AGAP1 targets GC to distinct subcellular compartments in close proximity to cGMP-dependent effectors, thereby optimizing cGMP generation and fostering cGMP-driven actions.
Taken together, these results demonstrate that beside the modulation of GC by NO the enzyme is regulated by tyrosine phosphorylation and interaction with AGAP1.
Nuclear pore complexes (NPCs) constitute giant channels within the nuclear envelope that mediate nucleocytoplasmic exchange. NPC diameter is thought to be regulated by nuclear envelope tension, but how such diameter changes are physiologically linked to cell differentiation, where mechanical properties of nuclei are remodeled and nuclear mechanosensing occurs, remains unstudied. Here we used cryo-electron tomography to show that NPCs dilate during differentiation of mouse embryonic stem cells into neural progenitors. In Nup133-deficient cells, which are known to display impaired neural differentiation, NPCs however fail to dilate. By analyzing the architectures of individual NPCs with template matching, we revealed that the Nup133-deficient NPCs are structurally heterogeneous and frequently disintegrate, resulting in the formation of large nuclear envelope openings. We propose that the elasticity of the NPC scaffold mechanically safeguards the nuclear envelope. Our studies provide a molecular explanation for how genetic perturbation of scaffolding components of macromolecular complexes causes tissue-specific phenotypes.
Upon infection, human immunodeficiency virus (HIV-1) releases its cone-shaped capsid into the cytoplasm of infected T-cells and macrophages. As its largest known cargo, the capsid enters the nuclear pore complex (NPC), driven by interactions with numerous FG-repeat nucleoporins (FG-Nups). Whether NPCs structurally adapt to capsid passage and whether capsids are modified during passage remains unknown, however. Here, we combined super-resolution and correlative microscopy with cryo electron tomography and molecular simulations to study nuclear entry of HIV-1 capsids in primary human macrophages. We found that cytosolically bound cyclophilin A is stripped off capsids entering the NPC, and the capsid hexagonal lattice remains largely intact inside and beyond the central channel. Strikingly, the NPC scaffold rings frequently crack during capsid passage, consistent with computer simulations indicating the need for NPC widening. The unique cone shape of the HIV-1 capsid facilitates its entry into NPCs and helps to crack their rings.
Tree-related microhabitats (TreMs) describe the microhabitats that a tree can provide for a multitude of other taxonomic groups and have been proposed as an important indicator for forest biodiversity (Asbeck et al., 2021). So far, the focus of TreM studies has been on temperate forests, although many trees in the tropics harbour exceptionally high numbers of TreMs. In this study, TreMs in the lowland tropical forests of the Choco (Ecuador) and in the mountain tropical forests of Mount Kilimanjaro (Tanzania) were surveyed. Our results extend the existing typology of TreMs of Larrieu et al. (2018) to include tropical forests and enabled a comparison of the relative recordings and diversity of TreMs between tropical and temperate forests. A new TreM form, Root formations, and three new TreM groups, concavities build by fruits or leaves, dendrotelms, and root formations, were established. In total, 15 new TreM types in five different TreM groups were specified. The relative recordings of most TreMs were similar between tropical and temperate forests. However, ivy and lianas, and ferns were more common in the lowland rainforest than in temperate forests, and bark microsoil, limb breakage, and foliose and fruticose lichens in tropical montane forest than in lowland rainforest. Mountain tropical forests hosted the highest diversity for common and dominant TreM types, and lowland tropical forest the highest diversity for rare TreMs. Our extended typology of tree-related microhabitats can support studies of forest-dwelling biodiversity in tropical forests. Specifically, given the ongoing threat to tropical forests, TreMs can serve as an additional tool allowing rapid assessments of biodiversity in these hyperdiverse ecosystems.
NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22(phox), Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22(phox), Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.