570 Biowissenschaften; Biologie
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Bacteria play key roles in the function and diversity of aquatic systems, but aside from study of specific bloom systems, little is known about the diversity or biogeography of bacteria associated with harmful cyanobacterial blooms (cyanoHABs). CyanoHAB species are known to shape bacterial community composition and to rely on functions provided by the associated bacteria, leading to the hypothesized cyanoHAB interactome, a coevolved community of synergistic and interacting bacteria species, each necessary for the success of the others. Here, we surveyed the microbiome associated with Microcystis aeruginosa during blooms in 12 lakes spanning four continents as an initial test of the hypothesized Microcystis interactome. We predicted that microbiome composition and functional potential would be similar across blooms globally. Our results, as revealed by 16S rRNA sequence similarity, indicate that M. aeruginosa is cosmopolitan in lakes across a 280 degrees longitudinal and 90 degrees latitudinal gradient. The microbiome communities were represented by a wide range of operational taxonomic units and relative abundances. Highly abundant taxa were more related and shared across most sites and did not vary with geographic distance, thus, like Microcystis, revealing no evidence for dispersal limitation. High phylogenetic relatedness, both within and across lakes, indicates that microbiome bacteria with similar functional potential were associated with all blooms. While Microcystis and the microbiome bacteria shared many genes, whole-community metagenomic analysis revealed a suite of biochemical pathways that could be considered complementary. Our results demonstrate a high degree of similarity across global Microcystis blooms, thereby providing initial support for the hypothesized Microcystis interactome.
Parasites, such as bacterial viruses (phages), can have large effects on host populations both at the ecological and evolutionary levels. In the case of cyanobacteria, phages can reduce primary production and infected hosts release intracellular nutrients influencing planktonic food web structure, community dynamics, and biogeochemical cycles. Cyanophages may be of great importance in aquatic food webs during large cyanobacterial blooms unless the host population becomes resistant to phage infection. The consequences on plankton community dynamics of the evolution of phage resistance in bloom forming cyanobacterial populations are still poorly studied. Here, we examined the effect of different frequencies of a phage-resistant genotype within a filamentous nitrogen-fixing Nodularia spumigena population on an experimental plankton community. Three Nodularia populations with different initial frequencies (0%, 5%, and 50%) of phage-resistant genotypes were inoculated in separate treatments with the phage 2AV2, the green alga Chlorella vulgaris, and the rotifer Brachionus plicatilis, which formed the experimental plankton community subjected to either nitrogen-limited or nitrogen-rich conditions. We found that the frequency of the phage-resistant Nodularia genotype determined experimental community dynamics. Cyanobacterial populations with a high frequency (50%) of the phage-resistant genotype dominated the cultures despite the presence of phages, retaining most of the intracellular nitrogen in the plankton community. In contrast, populations with low frequencies (0% and 5%) of the phage-resistant genotype were lysed and reduced to extinction by the phage, transferring the intracellular nitrogen held by Nodularia to Chlorella and rotifers, and allowing Chlorella to dominate the communities and rotifers to survive. This study shows that even though phages represent minuscule biomass, they can have key effects on community composition and eco-evolutionary feedbacks in plankton communities.
Negative phototactic response to UVR in three cosmopolitan rotifers: a video analysis approach
(2019)
The movement of microswimmers is often described by active Brownian particle models. Here we introduce a variant of these models with several internal states of the swimmer to describe stochastic strategies for directional swimming such as run and tumble or run and reverse that are used by microorganisms for chemotaxis. The model includes a mechanism to generate a directional bias for chemotaxis and interactions with external fields (e.g., gravity, magnetic field, fluid flow) that impose forces or torques on the swimmer. We show how this modified model can be applied to various scenarios: First, the run and tumble motion of E. coli is used to establish a paradigm for chemotaxis and investigate how it is affected by external forces. Then, we study magneto-aerotaxis in magnetotactic bacteria, which is biased not only by an oxygen gradient towards a preferred concentration, but also by magnetic fields, which exert a torque on an intracellular chain of magnets. We study the competition of magnetic alignment with active reorientation and show that the magnetic orientation can improve chemotaxis and thereby provide an advantage to the bacteria, even at rather large inclination angles of the magnetic field relative to the oxygen gradient, a case reminiscent of what is expected for the bacteria at or close to the equator. The highest gain in chemotactic velocity is obtained for run and tumble with a magnetic field parallel to the gradient, but in general a mechanism for reverse motion is necessary to swim against the magnetic field and a run and reverse strategy is more advantageous in the presence of a magnetic torque. This finding is consistent with observations that the dominant mode of directional changes in magnetotactic bacteria is reversal rather than tumbles. Moreover, it provides guidance for the design of future magnetic biohybrid swimmers. Author summary In this paper, we propose a modified Active Brownian particle model to describe bacterial swimming behavior under the influence of external forces and torques, in particular of a magnetic torque. This type of interaction is particularly important for magnetic biohybrids (i.e. motile bacteria coupled to a synthetic magnetic component) and for magnetotactic bacteria (i.e. bacteria with a natural intracellular magnetic chain), which perform chemotaxis to swim along chemical gradients, but are also directed by an external magnetic field. The model allows us to investigate the benefits and disadvantages of such coupling between two different directionality mechanisms. In particular we show that the magnetic torque can speed chemotaxis up in some conditions, while it can hinder it in other cases. In addition to an understanding of the swimming strategies of naturally magnetotactic organisms, the results may guide the design of future biomedical devices.
Domestic Bactrian camel (Camelus bactrianus) used to be one of the most important livestock species in Chinese history, as well as the major transport carrier on the ancient Silk Road. However, archeological studies on Chinese C. bactrianus are still limited, and molecular biology research on this species is mainly focused on modern specimens. In this study, we retrieved the complete mitochondrial genome from a C. bactrianus specimen, which was excavated from northwestern China and dated at 1290-1180 cal. Phylogenetic analyses using 18 mitochondrial genomes indicated that the C. bactrianus clade was divided into two maternal lineages. The majority of samples originating from Iran to Japan and Mongolia belong to subclade A1, while our sample together with two Mongolian individuals formed the much smaller subclade A2. Furthermore, the divergence time of these two maternal lineages was estimated as 165 Kya (95% credibility interval 117-222 Kya), this might indicate that several different evolutionary lineages were incorporated into the domestic gene pool during the initial domestication process. Bayesian skyline plot (BSP) analysis a slow increase in female effective population size of C. bactrianus from 5000 years ago, which to the beginning of domestication of C. bactrianus. The present study also revealed that there were extensive exchanges of genetic information among C. bactrianus populations in regions along the Silk Road.
Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1(-/-) zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.
Aging is accompanied by the accumulation of oxidized proteins. To remove them, cells employ the proteasomal and autophagy-lysosomal systems; however, if the clearance rate is inferior to its formation, protein aggregates form as a hallmark of proteostasis loss. In cells, during stress conditions, actin aggregates accumulate leading to impaired proliferation and reduced proteasomal activity, as observed in cellular senescence. The heat shock protein 90 (Hsp90) is a molecular chaperone that binds and protects the proteasome from oxidative inactivation. We hypothesized that in oxidative stress conditions a malfunction of Hsp90 occurs resulting in the aforementioned protein aggregates. Here, we demonstrate that upon oxidative stress Hsp90 loses its function in a highly specific non-enzymatic iron-catalyzed oxidation event and its breakdown product, a cleaved form of Hsp90 (Hsp90cl), acquires a new function in mediating the accumulation of actin aggregates. Moreover, the prevention of Hsp90 cleavage reduces oxidized actin accumulation, whereas transfection of the cleaved form of Hsp90 leads to an enhanced accumulation of oxidized actin. This indicates a clear role of the Hsp90cl in the aggregation of oxidized proteins.
The activity of neutral sphingomyelinase-2 (NSM2) to catalyze the conversion of sphingomyelin (SM) to ceramide and phosphocholine at the cytosolic leaflet of plasma membrane (PM) is important in T cell receptor (TCR) signaling. We recently identified PKC zeta as a major NSM2 downstream effector which regulates microtubular polarization. It remained, however, unclear to what extent NSM2 activity affected overall composition of PM lipids and downstream effector lipids in antigen stimulated T cells. Here, we provide a detailed lipidomics analyses on PM fractions isolated from TCR stimulated wild type and NSM2 deficient (Delta NSM) Jurkat T cells. This revealed that in addition to that of sphingolipids, NSM2 depletion also affected concentrations of many other lipids. In particular, NSM2 ablation resulted in increase of lyso-phosphatidylcholine (LPC) and lyso-phosphatidylethanolamine (LPE) which both govern PM biophysical properties. Crucially, TCR dependent upregulation of the important T cell signaling lipid diacylglycerol (DAG), which is fundamental for activation of conventional and novel PKCs, was abolished in Delta NSM cells. Moreover, NSM2 activity was found to play an important role in PM cholesterol transport to the endoplasmic reticulum (ER) and production of cholesteryl esters (CE) there. Most importantly, CE accumulation was essential to sustain human T cell proliferation. Accordingly, inhibition of CE generating enzymes, the cholesterol acetyltransferases ACAT1/SOAT1 and ACAT2/SOAT2, impaired TCR driven expansion of both CD4(+) and CD8(+) T cells. In summary, our study reveals an important role of NSM2 in regulating T cell functions by its multiple effects on PM lipids and cholesterol homeostasis.
Species of rust fungi of the genus Milesina (Pucciiastraceae, Pucciniales) are distributed mainly in northern temperate regions. They host-alternate between needles of fir (Abies spp.) and fronds of ferns (species of Polypodiales). Milesina species are distinguished based on host taxonomy and urediniospore morphology. In this study, 12 species of Milesina from Europe were revised. Specimens were examined by light and scanning electron microscopy for urediniospore morphology with a focus on visualising germ pores (number, size and position) and echinulation. In addition, barcode loci (ITS, nad6, 28S) were used for species delimitation and for molecular phylogenetic analyses. Barcodes of 72 Milesina specimens were provided, including 11 of the 12 species. Whereas urediniospore morphology features were sufficient to distinguish all 12 Milesina species except for 2 (M. blechni and M. kriegeriana), ITS sequences separated only 4 of 11 species. Sequencing with 28S and nad6 did not improve species resolution. Phylogenetic analysis, however, revealed four phylogenetic groups within Milesina that also correlate with specific urediniospore characters (germ pore number and position and echinulation). These groups are proposed as new sections within Milesina (sections Milesina, Vogesiacae M. Scholler & Bubner, sect. nov., Scolopendriorum M. Scholler & Bubner, sect. nov. and Carpaticae M. Scholler & Bubner, sect. nov.). In addition, Milesina woodwardiana Buchheit & M. Scholler, sp. nov. on Woodwardia radicans, a member of the type section Milesina, is newly described. An identification key for European Milesina species, based on urediniospore features, is provided.
NK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells ex vivo, e.g., by using specific cytokines to upregulate both their proliferation and surface expression of distinct activating receptors. Apart from an enhanced activity, application of NK cells as immunotherapeutic agent further requires sufficient cell numbers and a high purity. All these parameters depend on a variety of different factors including the starting material, additives like cytokines as well as the culture system. Here we analyzed PBMC-derived NK cells of five anonymized healthy donors expanded under specific conditions in an innovative perfusion bioreactor system with respect to their phenotype, IFN gamma production, and cytotoxicity in vitro. Important features of the meander type bioreactors used here are a directed laminar flow of medium and control of relevant process parameters. Cells are cultivated under "steady state" conditions in perfusion mode. Our data demonstrate that expansion of CD3(+) T cell depleted PBMCs in our standardized system generates massive amounts of highly pure (>85%) and potent anticancer active NK cells. These cells express a variety of important receptors driving NK cell recruitment, adhesion as well as activation. More specifically, they express the chemokine receptors CXCR3, CXCR4, and CCR7, the adhesion molecules L-selectin, LFA-1, and VLA-4, the activating receptors NKp30, NKp44, NKp46, NKG2D, DNAM1, and CD16 as well as the death ligands TRAIL and Fas-L. Moreover, the generated NK cells show a strong IFN gamma expression upon cultivation with K562 tumor cells and demonstrate a high cytotoxicity toward leukemic as well as solid tumor cell lines in vitro. Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation.
Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 angstrom resolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an E15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.
Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo
(2019)
We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce similar to 100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.
The short- and long-term thrombogenicity of implant materials is still unpredictable, which is a significant challenge for the treatment of cardiovascular diseases. A knowledge-based approach for implementing biofunctions in materials requires a detailed understanding of the medical device in the biological system. In particular, the interplay between material and blood components/cells as well as standardized and commonly acknowledged in vitro test methods allowing a reproducible categorization of the material thrombogenicity requires further attention. Here, the status of in vitro thrombogenicity testing methods for biomaterials is reviewed, particularly taking in view the preparation of test materials and references, the selection and characterization of donors and blood samples, the prerequisites for reproducible approaches and applied test systems. Recent joint approaches in finding common standards for a reproducible testing are summarized and perspectives for a more disease oriented in vitro thrombogenicity testing are discussed.
The human intestinal anaerobe Eubacterium ramulus is known for its ability to degrade various dietary flavonoids. In the present study, we demonstrate the cleavage of the heterocyclic C-ring of flavanones and flavanonols by an oxygen-sensitive NADH-dependent reductase, previously described as enoate reductase, from E. ramulus. This flavanone- and flavanonol-cleaving reductase (Fcr) was purified following its heterologous expression in Escherichia coli and further characterized. Fcr cleaved the flavanones naringenin, eriodictyol, liquiritigenin, and homoeriodictyol. Moreover, the flavanonols taxifolin and dihydrokaempferol served as substrates. The catalyzed reactions were stereospecific for the (2R)-enantiomers of the flavanone substrates and for the (25,35)-configured flavanonols. The enantioenrichment of the nonconverted stereoisomers allowed for the determination of hitherto unknown flavanone racemization rates. Fcr formed the corresponding dihydrochalcones and hydroxydihydrochalcones in the course of an unusual reductive cleavage of cyclic ether bonds. Fcr did not convert members of other flavonoid subclasses, including flavones, flavonols, and chalcones, the latter indicating that the reaction does not involve a chalcone intermediate. This view is strongly supported by the observed enantiospecificity of Fcr. Cinnamic acids, which are typical substrates of bacterial enoate reductases, were also not reduced by Fcr. Based on the presence of binding motifs for dinucleotide cofactors and a 4Fe-4S cluster in the amino acid sequence of Fcr, a cofactor-mediated hydride transfer from NADH onto C-2 of the respective substrate is proposed. IMPORTANCE Gut bacteria play a crucial role in the metabolism of dietary flavonoids, thereby contributing to their activation or inactivation after ingestion by the human host. Thus, bacterial activities in the intestine may influence the beneficial health effects of these polyphenolic plant compounds. While an increasing number of flavonoid-converting gut bacterial species have been identified, knowledge of the responsible enzymes is still limited. Here, we characterized Fcr as a key enzyme involved in the conversion of flavonoids of several subclasses by Eubacterium ramulus, a prevalent human gut bacterium. Sequence similarity of this enzyme to hypothetical proteins from other flavonoid-degrading intestinal bacteria in databases suggests a more widespread occurrence of this enzyme. Functional characterization of gene products of human intestinal microbiota enables the assignment of metagenomic sequences to specific bacteria and, more importantly, to certain activities, which is a prerequisite for targeted modulation of gut microbial functionality.
Intra-organ communication guides morphogenetic processes that are essential for an organ to carry out complex physiological functions. In the heart, the growth of the myocardium is tightly coupled to that of the endocardium, a specialized endothelial tissue that lines its interior. Several molecular pathways have been implicated in the communication between these tissues including secreted factors, components of the extracellular matrix, or proteins involved in cell-cell communication. Yet, it is unknown how the growth of the endocardium is coordinated with that of the myocardium. Here, we show that an increased expansion of the myocardial atrial chamber volume generates higher junctional forces within endocardial cells. This leads to biomechanical signaling involving VE-cadherin, triggering nuclear localization of the Hippo pathway transcriptional regulator Yap1 and endocardial proliferation. Our work suggests that the growth of the endocardium results from myocardial chamber volume expansion and ends when the tension on the tissue is relaxed.
The relationships between sedentary lifestyle, sitting behaviour, and low back pain (LBP) remain controversial. In this study, we investigated the relationship between back pain and occupational sitting habits in 64 call-centre employees. A textile pressure mat was used to evaluate and parameterise sitting behaviour over a total of 400 h, while pain questionnaires evaluated acute and chronic LBP. Seventy-five percent of the participants reported some level of either chronic or acute back pain. Individuals with chronic LBP demonstrated a possible trend (t-test not significant) towards more static sitting behaviour compared to their pain-free counterparts. Furthermore, a greater association was found between sitting behaviour and chronic LBP than for acute pain/disability, which is plausibly due to a greater awareness of pain-free sitting positions in individuals with chronic pain compared to those affected by acute pain.
Winter is an important season for many limnological processes, which can range from biogeochemical transformations to ecological interactions. Interest in the structure and function of lake ecosystems under ice is on the rise. Although limnologists working at polar latitudes have a long history of winter work, the required knowledge to successfully sample under winter conditions is not widely available and relatively few limnologists receive formal training. In particular, the deployment and operation of equipment in below 0 degrees C temperatures pose considerable logistical and methodological challenges, as do the safety risks of sampling during the ice-covered period. Here, we consolidate information on winter lake sampling and describe effective methods to measure physical, chemical, and biological variables in and under ice. We describe variation in snow and ice conditions and discuss implications for sampling logistics and safety. We outline commonly encountered methodological challenges and make recommendations for best practices to maximize safety and efficiency when sampling through ice or deploying instruments in ice-covered lakes. Application of such practices over a broad range of ice-covered lakes will contribute to a better understanding of the factors that regulate lakes during winter and how winter conditions affect the subsequent ice-free period.
Predator-prey cycles rank among the most fundamental concepts in ecology, are predicted by the simplest ecological models and enable, theoretically, the indefinite persistence of predator and prey(1-4). However, it remains an open question for how long cyclic dynamics can be self-sustained in real communities. Field observations have been restricted to a few cycle periods(5-8) and experimental studies indicate that oscillations may be short-lived without external stabilizing factors(9-19). Here we performed microcosm experiments with a planktonic predator-prey system and repeatedly observed oscillatory time series of unprecedented length that persisted for up to around 50 cycles or approximately 300 predator generations. The dominant type of dynamics was characterized by regular, coherent oscillations with a nearly constant predator-prey phase difference. Despite constant experimental conditions, we also observed shorter episodes of irregular, non-coherent oscillations without any significant phase relationship. However, the predator-prey system showed a strong tendency to return to the dominant dynamical regime with a defined phase relationship. A mathematical model suggests that stochasticity is probably responsible for the reversible shift from coherent to non-coherent oscillations, a notion that was supported by experiments with external forcing by pulsed nutrient supply. Our findings empirically demonstrate the potential for infinite persistence of predator and prey populations in a cyclic dynamic regime that shows resilience in the presence of stochastic events.
In biomaterial development, the design of material surfaces that mimic the extra-cellular matrix (ECM) in order to achieve favorable cellular instruction is rather challenging. Collagen-type IV (Col-IV), the major scaffolding component of Basement Membranes (BM), a specialized ECM with multiple biological functions, has the propensity to form networks by self-assembly and supports adhesion of cells such as endothelial cells or stem cells. The preparation of biomimetic Col-IV network-like layers to direct cell responses is difficult. We hypothesize that the morphology of the layer, and especially the density of the available adhesion sites, regulates the cellular adhesion to the layer. The Langmuir monolayer technique allows for preparation of thin layers with precisely controlled packing density at the air-water (A-W) interface. Transferring these layers onto cell culture substrates using the Langmuir-Schafer (LS) technique should therefore provide a pathway for preparation of BM mimicking layers with controlled cell adherence properties. In situ characterization using ellipsometry and polarization modulation-infrared reflection absorption spectroscopy of Col-IV layer during compression at the A-W interface reveal that there is linear increase of surface molecule concentration with negligible orientational changes up to a surface pressure of 25 mN m(-1). Smooth and homogeneous Col-IV network-like layers are successfully transferred by LS method at 15 mN m(-1) onto poly(ethylene terephthalate) (PET), which is a common substrate for cell culture. In contrast, the organization of Col-IV on PET prepared by the traditionally employed solution deposition method results in rather inhomogeneous layers with the appearance of aggregates and multilayers. Progressive increase in the number of early adherent mesenchymal stem cells (MSCs) after 24 h by controlling the areal Col-IV density by LS transfer at 10, 15 and 20 mN m(-1) on PET is shown. The LS method offers the possibility to control protein characteristics on biomaterial surfaces such as molecular density and thereby, modulate cell responses.
Farber disease is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments for Farber disease are clinically available, and affected patients have a severely shortened lifespan. We have recently reported a novel acid ceramidase deficiency model that mirrors the human disease closely. Acid sphingomyelinase is the enzyme that generates ceramide upstream of acid ceramidase in the lysosomes. Using our acid ceramidase deficiency model, we tested if acid sphingomyelinase could be a potential novel therapeutic target for the treatment of Farber disease. A number of functional acid sphingomyelinase inhibitors are clinically available and have been used for decades to treat major depression. Using these as a therapeutic for Farber disease, thus, has the potential to improve central nervous symptoms of the disease as well, something all other treatment options for Farber disease can’t achieve so far. As a proof-of-concept study, we first cross-bred acid ceramidase deficient mice with acid sphingomyelinase deficient mice in order to prevent ceramide accumulation. Double-deficient mice had reduced ceramide accumulation, fewer disease manifestations, and prolonged survival. We next targeted acid sphingomyelinase pharmacologically, to test if these findings would translate to a setting with clinical applicability. Surprisingly, the treatment of acid ceramidase deficient mice with the acid sphingomyelinase inhibitor amitriptyline was toxic to acid ceramidase deficient mice and killed them within a few days of treatment. In conclusion, our study provides the first proof-of-concept that acid sphingomyelinase could be a potential new therapeutic target for Farber disease to reduce disease manifestations and prolong survival. However, we also identified previously unknown toxicity of the functional acid sphingomyelinase inhibitor amitriptyline in the context of Farber disease, strongly cautioning against the use of this substance class for Farber disease patients.
The highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometric analysis, we identified four arginylation sites in the most abundant actin isoform of Dictyostelium, in addition to arginylation sites in other actin isoforms and several actin-binding proteins. In vitro polymerization assays with actin purified from ate1-null cells revealed a diminished polymerization capacity in comparison to wild-type actin. Our data indicate that arginylation plays a crucial role in the regulation of cytoskeletal activities.
The nuclear envelope consists of the outer and the inner nuclear membrane, the nuclear lamina and the nuclear pore complexes, which regulate nuclear import and export.The major constituent of the nuclear lamina of Dictyostelium is the lamin NE81. It can form filaments like B-type lamins and it interacts with Sun 1, as well as with the LEM/HeH-family protein Src1. Sun 1 and Src1 are nuclear envelope transmembrane proteins involved in the centrosome-nucleus connection and nuclear envelope stability at the nucleolar regions, respectively. In conjunction with a KASH-domain protein, Sun 1 usually forms a so-called LINC complex.Two proteins with functions reminiscent of KASH-domain proteins at the outer nuclear membrane of Dictyostelium are known; interaptin which serves as an actin connector and the kinesin Kif9 which plays a role in the microtubule-centrosome connector. However, both of these lack the conserved KASH-domain. The link of the centrosome to the nuclear envelope is essential for the insertion of the centrosome into the nuclear envelope and the appropriate spindle formation. Moreover, centrosome insertion is involved in perm eabilization of the mitotic nucleus, which ensures access of tubulin dimers and spindle assembly factors. Our recent progress in identifying key molecular players at the nuclear envelope of Dictyostelium promises further insights into the mechanisms of nuclear envelope dynamics.
The endosperm is an ephemeral tissue that nourishes the developing embryo, similar to the placenta in mammals. In most angiosperms, endosperm development starts as a syncytium, in which nuclear divisions are not followed by cytokinesis. The timing of endosperm cellularization largely varies between species, and the event triggering this transition remains unknown. Here we show that increased auxin biosynthesis in the endosperm prevents its cellularization, leading to seed arrest. Auxin-overproducing seeds phenocopy paternal-excess triploid seeds derived from hybridizations of diploid maternal plants with tetraploid fathers. Concurrently, auxin-related genes are strongly overexpressed in triploid seeds, correlating with increased auxin activity. Reducing auxin biosynthesis and signaling reestablishes endosperm cellularization in triploid seeds and restores their viability, highlighting a causal role of increased auxin in preventing endosperm cellularization. We propose that auxin determines the time of endosperm cellularization, and thereby uncovered a central role of auxin in establishing hybridization barriers in plants.
In self-incompatible plants the female style rejects self pollen, yet the extent to which the female style in the many self-compatible species can still select between different pollen genotypes and thus bias fertilization success is unclear. A new study identifies the molecular basis for how styles of the self-compatible coyote tobacco bias the fertilization success of pollen genotypes using matching gene expression patterns in a manner analogous to cryptic female choice in animals.
The frequent production of the hepatotoxin microcystin (MC) and its impact on the lifestyle of bloom-forming cyanobacteria are poorly understood. Here, we report that MC interferes with the assembly and the subcellular localization of RubisCO, in Microcystis aeruginosa PCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes in M. aeruginosa and relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of MC to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2 fixation product 3-phosphoglycerate are significantly higher in the MC-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction of M. aeruginosa with its heterotrophic microbial community.
While the underlying mechanisms of Parkinson’s disease (PD) are still insufficiently studied, a complex interaction between genetic and environmental factors is emphasized. Nevertheless, the role of the essential trace element zinc (Zn) in this regard remains controversial. In this study we altered Zn balance within PD models of the versatile model organism Caenorhabditis elegans (C. elegans) in order to examine whether a genetic predisposition in selected genes with relevance for PD affects Zn homeostasis. Protein-bound and labile Zn species act in various areas, such as enzymatic catalysis, protein stabilization pathways and cell signaling. Therefore, total Zn and labile Zn were quantitatively determined in living nematodes as individual biomarkers of Zn uptake and bioavailability with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) or a multi-well method using the fluorescent probe ZinPyr-1. Young and middle-aged deletion mutants of catp-6 and pdr-1, which are orthologues of mammalian ATP13A2 (PARK9) and parkin (PARK2), showed altered Zn homeostasis following Zn exposure compared to wildtype worms. Furthermore, age-specific differences in Zn uptake were observed in wildtype worms for total as well as labile Zn species. These data emphasize the importance of differentiation between Zn species as meaningful biomarkers of Zn uptake as well as the need for further studies investigating the role of dysregulated Zn homeostasis in the etiology of PD.
Multidrug resistant (MDR) Pseudomonas aeruginosa having strong biofilm potential and virulence factors are a serious threat for hospitalized patients having compromised immunity In this study, 34 P. aeruginosa isolates of human origin (17 MDR and 17 non-MDR clinical isolates) were checked for biofilm formation potential in enriched and minimal media. The biofilms were detected using crystal violet method and a modified software package of the automated VideoScan screening method. Cytotoxic potential of the isolates was also investigated on HepG2, LoVo and T24 cell lines using automated VideoScan technology. Pulse field gel electrophoresis revealed 10 PFGE types in MDR and 8 in non-MDR isolates. Although all isolates showed biofilm formation potential, strong biofilm formation was found more in enriched media than in minimal media. Eight MDR isolates showed strong biofilm potential in both enriched and minimal media by both detection methods. Strong direct correlation between crystal violet and VideoScan methods was observed in identifying strong biofilm forming isolates. High cytotoxic effect was observed by 4 isolates in all cell lines used while 6 other isolates showed high cytotoxic effect on T24 cell line only. Strong association of multidrug resistance was found with biofilm formation as strong biofilms were observed significantly higher in MDR isolates (p-value < 0.05) than non-MDR isolates. No significant association of cytotoxic potential with multidrug resistance or biofilm formation was found (p-value > 0.05). The MDR isolates showing significant cytotoxic effects and strong biofilm formation impose a serious threat for hospitalized patients with weak immune system.
Coherent network partitions
(2019)
Graph clustering is widely applied in the analysis of cellular networks reconstructed from large-scale data or obtained from experimental evidence. Here we introduce a new type of graph clustering based on the concept of coherent partition. A coherent partition of a graph G is a partition of the vertices of G that yields only disconnected subgraphs in the complement of G. The coherence number of G is then the size of the smallest edge cut inducing a coherent partition. A coherent partition of G is optimal if the size of the inducing edge cut is the coherence number of G. Given a graph G, we study coherent partitions and the coherence number in connection to (bi)clique partitions and the (bi)clique cover number. We show that the problem of finding the coherence number is NP-hard, but is of polynomial time complexity for trees. We also discuss the relation between coherent partitions and prominent graph clustering quality measures.
The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.
Widely used diagnostic tools make use of antibodies recognizing targeted molecules, but additional techniques are required in order to alleviate the disadvantages of antibodies. Herein, molecular dynamic calculations are performed for the design of high affinity artificial protein binding surfaces for the recognition of neuron specific enolase (NSE), a known cancer biomarker. Computational simulations are employed to identify particularly stabile secondary structure elements. These epitopes are used for the subsequent molecular imprinting, where surface imprinting approach is applied. The molecular imprints generated with the calculated epitopes of greater stability (Cys-Ep1) show better binding properties than those of lower stability (Cys-Ep5). The average binding strength of imprints created with stabile epitopes is found to be around twofold and fourfold higher for the NSE derived peptide and NSE protein, respectively. The recognition of NSE is investigated in a wide concentration range, where high sensitivity (limit of detection (LOD) = 0.5 ng mL(-1)) and affinity (dissociation constant (K-d) = 5.3 x 10(-11)m) are achieved using Cys-Ep1 imprints reflecting the stable structure of the template molecules. This integrated approach employing stability calculations for the identification of stabile epitopes is expected to have a major impact on the future development of high affinity protein capturing binders.
Zinc is an essential trace element, making it crucial to have a reliable biomarker for evaluating an individual’s zinc status. The total serum zinc concentration, which is presently the most commonly used biomarker, is not ideal for this purpose, but a superior alternative is still missing. The free zinc concentration, which describes the fraction of zinc that is only loosely bound and easily exchangeable, has been proposed for this purpose, as it reflects the highly bioavailable part of serum zinc. This report presents a fluorescence-based method for determining the free zinc concentration in human serum samples, using the fluorescent probe Zinpyr-1. The assay has been applied on 154 commercially obtained human serum samples. Measured free zinc concentrations ranged from 0.09 to 0.42 nM with a mean of 0.22 ± 0.05 nM. It did not correlate with age or the total serum concentrations of zinc, manganese, iron or selenium. A negative correlation between the concentration of free zinc and total copper has been seen for sera from females. In addition, the free zinc concentration in sera from females (0.21 ± 0.05 nM) was significantly lower than in males (0.23 ± 0.06 nM). The assay uses a sample volume of less than 10 µL, is rapid and cost-effective and allows us to address questions regarding factors influencing the free serum zinc concentration, its connection with the body’s zinc status, and its suitability as a future biomarker for an individual’s zinc status.
The plasma membrane (PM) is at the interface of plant-pathogen interactions and, thus, many bacterial type-III effector (T3E) proteins target membrane-associated processes to interfere with immunity. The Pseudomonas syringae T3E HopZ1a is a host cell PM-localized effector protein that has several immunity-associated host targets but also activates effector-triggered immunity in resistant backgrounds. Although HopZ1a has been shown to interfere with early defense signaling at the PM, no dedicated PM-associated HopZ1a target protein has been identified until now. Here, we show that HopZ1a interacts with the PM-associated remorin protein NbREM4 from Nicotiana benthamiana in several independent assays. NbREM4 relocalizes to membrane nanodomains after treatment with the bacterial elicitor flg22 and transient overexpression of NbREM4 in N. benthamiana induces the expression of a subset of defense-related genes. We can further show that NbREM4 interacts with the immune-related receptor-like cytoplasmic kinase avrPphB-susceptible 1 (PBS1) and is phosphorylated by PBS1 on several residues in vitro. Thus, we conclude that NbREM4 is associated with early defense signaling at the PM. The possible relevance of the HopZ1a-NbREM4 interaction for HopZ1a virulence and avirulence functions is discussed.
Vielfalt in der Uckermark
(2019)