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• Protocol for extracting and analyzing pollen grains from fossil insects
• Individual fossil grains can be analyzed using a combined approach
• Simple and fast TEM embedding and sectioning protocol
• Protocol enables a taxonomic assignment of pollen
Summary
This protocol explains how to extract pollen from fossil insects with subsequent descriptions of pollen treatment. We also describe how to document morphological and ultrastructural features with light-microscopy and electron microscopy. It enables a taxonomic assignment of pollen that can be used to interpret flower-insect interactions, foraging and feeding behavior of insects, and the paleoenvironment. The protocol is limited by the state of the fossil, the presence/absence of pollen on fossil specimens, and the availability of extant pollen for comparison.
Parallel multisite recordings in the visual cortex of trained monkeys revealed that the responses of spatially distributed neurons to natural scenes are ordered in sequences. The rank order of these sequences is stimulus-specific and maintained even if the absolute timing of the responses is modified by manipulating stimulus parameters. The stimulus specificity of these sequences was highest when they were evoked by natural stimuli and deteriorated for stimulus versions in which certain statistical regularities were removed. This suggests that the response sequences result from a matching operation between sensory evidence and priors stored in the cortical network. Decoders trained on sequence order performed as well as decoders trained on rate vectors but the former could decode stimulus identity from considerably shorter response intervals than the latter. A simulated recurrent network reproduced similarly structured stimulus-specific response sequences, particularly once it was familiarized with the stimuli through non-supervised Hebbian learning. We propose that recurrent processing transforms signals from stationary visual scenes into sequential responses whose rank order is the result of a Bayesian matching operation. If this temporal code were used by the visual system it would allow for ultrafast processing of visual scenes.
Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot, J. L., and Engelman, D. M. (1990) Biochemistry 29, 4031–4037) is postulated to proceed in 2 steps: partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two, and the first three transmembrane (TM) segments, as well as a construct comprising TM1–TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin, A., Cohen, L. S., Arshava, B., Tantry, S., Becker, J. M., Zerbe, O., and Naider, F. (2009) Biophys. J. 96, 3187–3196), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123, and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior.
Lantibiotics are peptide-derived antibiotics that inhibit the growth of Gram-positive bacteria via interactions with lipid II and lipid II-dependent pore formation in the bacterial membrane. Due to their general mode of action the Gram-positive producer strains need to express immunity proteins (LanI proteins) for protection against their own lantibiotics. Little is known about the immunity mechanism protecting the producer strain against its own lantibiotic on the molecular level. So far, no structures have been reported for any LanI protein. We solved the structure of SpaI, a LanI protein from the subtilin producing strain Bacillus subtilis ATCC 6633. SpaI is a 16.8-kDa lipoprotein that is attached to the outside of the cytoplasmic membrane via a covalent diacylglycerol anchor. SpaI together with the ABC transporter SpaFEG protects the B. subtilis membrane from subtilin insertion. The solution-NMR structure of a 15-kDa biologically active C-terminal fragment reveals a novel fold. We also demonstrate that the first 20 N-terminal amino acids not present in this C-terminal fragment are unstructured in solution and are required for interactions with lipid membranes. Additionally, growth tests reveal that these 20 N-terminal residues are important for the immunity mediated by SpaI but most likely are not part of a possible subtilin binding site. Our findings are the first step on the way of understanding the immunity mechanism of B. subtilis in particular and of other lantibiotic producing strains in general.
The solution structure of the lantibiotic immunity protein NisI and its interactions with nisin
(2015)
Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity.
Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.
Background: The causative agent of Chagas disease, Trypanosoma cruzi, and its nonpathogenic relative, Trypanosoma rangeli, are transmitted by haematophagous triatomines and undergo a crucial ontogenetic phase in the insect’s intestine. In the process, the parasites interfere with the host immune system as well as the microbiome present in the digestive tract potentially establishing an environment advantageous for development. However, the coherent interactions between host, pathogen and microbiota have not yet been elucidated in detail. We applied a metagenome shotgun sequencing approach to study the alterations in the microbiota of Rhodnius prolixus, a major vector of Chagas disease, after exposure to T. cruzi and T. rangeli focusing also on the functional capacities present in the intestinal microbiome of the insect.
Results: The intestinal microbiota of R. prolixus was dominated by the bacterial orders Enterobacterales, Corynebacteriales, Lactobacillales, Clostridiales and Chlamydiales, whereas the latter conceivably originated from the blood used for pathogen exposure. The anterior and posterior midgut samples of the exposed insects showed a reduced overall number of organisms compared to the control group. However, we also found enriched bacterial groups after exposure to T. cruzi as well as T rangeli. While the relative abundance of Enterobacterales and Corynebacteriales decreased considerably, the Lactobacillales, mainly composed of the genus Enterococcus, developed as the most abundant taxonomic group. This applies in particular to vectors challenged with T. rangeli and at early timepoints after exposure to vectors challenged with T. cruzi. Furthermore, we were able to reconstruct four metagenome-assembled genomes from the intestinal samples and elucidate their unique metabolic functionalities within the triatomine microbiome, including the genome of a recently described insect symbiont, Candidatus Symbiopectobacterium, and the secondary metabolites producing bacteria Kocuria spp.
Conclusions: Our results facilitate a deeper understanding of the processes that take place in the intestinal tract of triatomine vectors during colonisation by trypanosomal parasites and highlight the influential aspects of pathogen-microbiota interactions. In particular, the mostly unexplored metabolic capacities of the insect vector’s microbiome are clearer, underlining its role in the transmission of Chagas disease.
Understanding how species relate mechanistically to their environment via traits is a central goal in ecology. Many macroecological rules were found for macroorganisms, however, whether they can explain microorganismal macroecological patterns still requires investigation. Further, whether macroecological rules are also applicable in microclimates is largely unexplored. Here we use fruit body-forming fungi to understand both aspects better. A recent study showed first evidence for the thermal-melanism hypothesis (Bogert’s rule) in fruit body-forming fungi and relied on a continental spatial scale with large grid size. At large spatial extent and grid sizes, other factors like dispersal limitation or local microclimatic variability might influence observed patterns besides the rule of interest. Therefore, we test fungal assemblage fruit body color lightness along a local elevational gradient (mean annual temperature gradient of 7°C) while considering the vegetation cover as a proxy for local variability in microclimate. Using multivariate linear modeling, we found that fungal fruiting assemblages are significantly darker at lower mean annual temperatures supporting the thermal-melanism hypothesis. Further, we found a non-significant trend of assemblage color lightness with vegetation cover. Our results support Bogert’s rule for microorganisms with macroclimate, which was also found for macroorganisms.
Out-of-school laboratories, also called student labs, are an advantageous opportunity to teach biological subjects. Particularly in the case of complex fields such as neurobiology, student labs offer the opportunity to learn about difficult topics in a practical way. Due to numerous advantages, digital student labs are becoming increasingly popular nowadays. In this study, we investigated the effect of an electrophysiological setup for a virtual experiment with and without hands-on elements on participant motivation and technology acceptance. For this purpose, 235 students were questioned during a student laboratory day. The surveyed students showed high motivation and technology acceptance for the virtual experiment. In the comparison, the electrophysiological setup with hands-on elements performs better in the intrinsic components than the setup without hands-on elements: Thus, the hands-on approach is rated as more interesting and the perceived enjoyment scores higher. Nevertheless, both experimental groups show high values, so that the results of the study support the positive influence of digital laboratory as well as a positive influence of hands-on elements.
Determining the structure and mechanisms of all individual functional modules of cells at high molecular detail has often been seen as equal to understanding how cells work. Recent technical advances have led to a flush of high-resolution structures of various macromolecular machines, but despite this wealth of detailed information, our understanding of cellular function remains incomplete. Here, we discuss present-day limitations of structural biology and highlight novel technologies that may enable us to analyze molecular functions directly inside cells. We predict that the progression toward structural cell biology will involve a shift toward conceptualizing a 4D virtual reality of cells using digital twins. These will capture cellular segments in a highly enriched molecular detail, include dynamic changes, and facilitate simulations of molecular processes, leading to novel and experimentally testable predictions. Transferring biological questions into algorithms that learn from the existing wealth of data and explore novel solutions may ultimately unveil how cells work.