Structural analysis of membrane protein biogenesis and ribosome stalling by cryo-electron microscopy

To study the mechanisms of membrane protein insertion we established a protocol that allows isolation of in vivo assembled ribosome nascent chain complexes (RNCs) from E. coli in high yield and quality. To investigate the interaction of SecY with a translating ribosome, model membrane proteins of different length and topology were over-expressed and the respective RNCs were isolated under mild conditions to allow co-purification of the SecY complex. Analysis of the interaction of RNCs with SecY in vivo suggested that, as expected, a tight engagement of the ribosome and SecY is only established for nascent chains that are translocated co-translationally. We observed that SecY and the RNC do not form a stable complex at the moment of hydrophobic transmembrane segments inserting in the translocon. However, a stable engagement of the RNC with SecY was observed, when inserting a transmembrane segment with a type II topology into SecY followed by a hydrophilic loop of a certain length which allows the isolation of this complex. That suggested a dual binding mode of tight and loose coupling of SecY to the translating ribosome dependent on the nature of the nascent substrate. We present the first three dimensional structure of an in vivo assembled, tightly coupled polytopic RNC-SecYE complex at 7.2 Å solved by cryo-EM and single particle reconstruction. A molecular model based on the cryo-EM structure reveals that SecYE could be trapped in a post-insertion state, with the two substrate helices interacting with the periphery of SecY, while still translocating the hydrophilic loop. The lateral gate of SecY remains in a ‘pre-opened’ conformation during the translocation of the hydrophilic loop. The interaction sites of SecY with the ribosome were found as described. Remarkably, we could also reveal an interaction of helix 59 in the ribosome with nascent membrane protein via positively charged residues in the first cytoplasmic loop of the substrate. It is tempting to speculate that this interaction contributes to the positive inside rule. Though, we provided an unprecedented snapshot of an inserting polytopic membrane protein, the exact path of the nascent chain and the molecular mechanism of the actual insertion could not be solved so far. Expression of the E. coli tryptophanase (TnaA) operon is triggered by ribosome stalling during translation of the upstream TnaC leader peptide. Notably, this stalling is strictly dependent on the presence of tryptophan that acts in a hitherto unknown way. Here, we present a cryo-EM reconstruction of the stalled nascent TnaC leader peptide in the ribosomal exit tunnel. The structure of the TnaC-stalled ribosome was solved to an average resolution of 3.8 Å by cryo-EM and single particle analysis. It reveals the conformation of the silenced peptidyl-transferase center as well as the exact path of the stalled nascent peptide and its contacts in detail. Furthermore, we clearly resolve not a single but two free tryptophan molecules in the ribosomal exit tunnel. The nascent TnaC peptide chain together with distinct rRNA bases in the ribosomal exit tunnel creates two hydrophobic binding pockets for the tryptophan coordination. One tryptophan molecule is coordinated by V20 and I19 of TnaC and interacts with U2586 of the rRNA, the second tryptophan is bound between I19 and I15 in the area of A2058 and A2059 of the rRNA. Interestingly, the latter is also the binding platform for macrolide antibiotics. Engagement of L-Trp in these composite binding pockets leads to subtle conformational changes in residues of the ribosomal tunnel wall that are translated to the PTC eventually resulting in silencing by stabilizing the conformations of the conserved nucleotides A2602 and U2585. These conformations of the two nucleotides in the PTC are incompatible with the correct accommodation of the GGQ motive of release factor 2, thus inhibiting the peptide release

Structural analysis of membrane protein biogenesis and ribosome stalling by cryo-electron microscopy

To study the mechanisms of membrane protein insertion we established a protocol that allows isolation of in vivo assembled ribosome nascent chain complexes (RNCs) from E. coli in high yield and quality. To investigate the interaction of SecY with a translating ribosome, model membrane proteins of different length and topology were over-expressed and the respective RNCs were isolated under mild conditions to allow co-purification of the SecY complex. Analysis of the interaction of RNCs with SecY in vivo suggested that, as expected, a tight engagement of the ribosome and SecY is only established for nascent chains that are translocated co-translationally. We observed that SecY and the RNC do not form a stable complex at the moment of hydrophobic transmembrane segments inserting in the translocon. However, a stable engagement of the RNC with SecY was observed, when inserting a transmembrane segment with a type II topology into SecY followed by a hydrophilic loop of a certain length which allows the isolation of this complex. That suggested a dual binding mode of tight and loose coupling of SecY to the translating ribosome dependent on the nature of the nascent substrate. We present the first three dimensional structure of an in vivo assembled, tightly coupled polytopic RNC-SecYE complex at 7.2 Å solved by cryo-EM and single particle reconstruction. A molecular model based on the cryo-EM structure reveals that SecYE could be trapped in a post-insertion state, with the two substrate helices interacting with the periphery of SecY, while still translocating the hydrophilic loop. The lateral gate of SecY remains in a ‘pre-opened’ conformation during the translocation of the hydrophilic loop. The interaction sites of SecY with the ribosome were found as described. Remarkably, we could also reveal an interaction of helix 59 in the ribosome with nascent membrane protein via positively charged residues in the first cytoplasmic loop of the substrate. It is tempting to speculate that this interaction contributes to the positive inside rule. Though, we provided an unprecedented snapshot of an inserting polytopic membrane protein, the exact path of the nascent chain and the molecular mechanism of the actual insertion could not be solved so far. Expression of the E. coli tryptophanase (TnaA) operon is triggered by ribosome stalling during translation of the upstream TnaC leader peptide. Notably, this stalling is strictly dependent on the presence of tryptophan that acts in a hitherto unknown way. Here, we present a cryo-EM reconstruction of the stalled nascent TnaC leader peptide in the ribosomal exit tunnel. The structure of the TnaC-stalled ribosome was solved to an average resolution of 3.8 Å by cryo-EM and single particle analysis. It reveals the conformation of the silenced peptidyl-transferase center as well as the exact path of the stalled nascent peptide and its contacts in detail. Furthermore, we clearly resolve not a single but two free tryptophan molecules in the ribosomal exit tunnel. The nascent TnaC peptide chain together with distinct rRNA bases in the ribosomal exit tunnel creates two hydrophobic binding pockets for the tryptophan coordination. One tryptophan molecule is coordinated by V20 and I19 of TnaC and interacts with U2586 of the rRNA, the second tryptophan is bound between I19 and I15 in the area of A2058 and A2059 of the rRNA. Interestingly, the latter is also the binding platform for macrolide antibiotics. Engagement of L-Trp in these composite binding pockets leads to subtle conformational changes in residues of the ribosomal tunnel wall that are translated to the PTC eventually resulting in silencing by stabilizing the conformations of the conserved nucleotides A2602 and U2585. These conformations of the two nucleotides in the PTC are incompatible with the correct accommodation of the GGQ motive of release factor 2, thus inhibiting the peptide release

Targeting new ways for large-scale, high-speed surface functionalization using direct laser interference patterning in a roll-to-roll process

Direct Laser Interference Patterning (DLIP) is used to texture current collector foils in a roll-to-roll process using a high-power picosecond pulsed laser system operating at either fundamental wavelength of 1064 nm or 2nd harmonic of 532 nm. The raw beam having a diameter of 3 mm @ 1/e$^2$ is shaped into an elongated top-hat intensity profile using a diffractive so-called FBS®-L element and cylindrical telescopes. The shaped beam is split into its diffraction orders, where the two first orders are parallelized and guided into a galvanometer scanner. The deflected beams inside the scan head are recombined with an F-theta objective on the working position generating the interference pattern. The DLIP spot has a line-like interference pattern with about 15 μm spatial period. Laser fluences of up to 8 J cm$^{−2}$ were achieved using a maximum pulse energy of 0.6 mJ. Furthermore, an in-house built roll-to-roll machine was developed. Using this setup, aluminum and copper foil of 20 μm and 9 μm thickness, respectively, could be processed. Subsequently to current collector structuring coating of composite electrode material took place. In case of lithium nickel manganese cobalt oxide (NMC 622) cathode deposited onto textured aluminum current collector, an increased specific discharge capacity could be achieved at a C-rate of 1 °C. For the silicon/graphite anode material deposited onto textured copper current collector, an improved rate capability at all C-rates between C/10 and 5 °C was achieved. The rate capability was increased up to 100% compared to reference material. At C-rates between C/2 and 2 °C, the specific discharge capacity was increased to 200 mAh g$^{−1}$, while the reference electrodes with untextured current collector foils provided a specific discharge capacity of 100 mAh g$^{−1}$, showing the potential of the DLIP technology for cost-effective production of battery cells with increased cycle lifetime

Novel methods for in vitro modeling of pancreatic cancer reveal important aspects for successful primary cell culture

Background: Pancreatic cancer remains a fatal disease. Experimental systems are needed for personalized treatment strategies, drug testing and to further understand tumor biology. Cell cultures can serve as an excellent preclinical platform, but their generation remains challenging. Methods: Tumor cells from surgically removed pancreatic ductal adenocarcinoma (PDAC) specimens were cultured under novel protocols. Cellular growth and composition were analyzed and culture conditions were continuously optimized. Characterization of cell cultures and primary tumors was performed via hematoxylin and eosin (HE) and immunofluorescence (IF) staining. Results: Protocols for two- and three-dimensional PDAC primary cell cultures could successfully be established. Primary cell culture depended on dissociation techniques, growth factor supplementation and extracellular matrix components containing Matrigel being crucial for the transformation to three-dimensional PDAC organoids. The generated cultures showed to be highly resemblant to established PDAC primary cell cultures. HE and IF staining for cell culture and corresponding primary tumor characterization could successfully be performed. Conclusions: The work presented herein shows novel and effective methods to successfully establish primary PDAC cell cultures in a distinct time frame. Factors contributing to cell growth and differentiation could be identified with important implications for further primary cell culture protocols. The established protocols might serve as novel tools in personalized tumor therapy

Asteroid 2008 TC3, not a polymict ureilitic but a polymict C1 chondrite parent body? Survey of 249 Almahata Sitta fragments

On October 7, 2008, the asteroid 2008 TC3 exploded as it entered the Earth’s atmosphere, producing significant dust (in the atmosphere) and delivering thousands of stones in a strewn field in Sudan, collectively known as the Almahata Sitta (AhS) stones. About 600 fragments were officially recovered in 2008 and 2009. Further rocks were collected since the fall event by local people. From these stones, 249 were classified at the Institut für Planetologie in Münster (MS) known as MS‐xxx or MS‐MU‐xxx AhS subsamples. Most of these rocks are ureilitic in origin (168; 67%): 87 coarse‐grained ureilites, 60 fine‐grained ureilites, 15 ureilites with variable texture/mineralogy, four trachyandesites, and two polymict breccias. We identified 81 non‐ureilitic fragments, corresponding to 33% of the recovered samples studied in Münster. These included chondrites, namely 65 enstatite chondrites (43 EL; 22 EH), 11 ordinary chondrites (OC), one carbonaceous chondrite, and one unique R‐like chondrite. Furthermore, three samples represent a unique type of enstatite achondrite. Since all AhS stones must be regarded as individual specimens independent from each other, the number of fresh ureilite and enstatite chondrite falls in our meteorite collections has been increased by several hundred percent. Overall, the samples weigh between <1 and 250 g and have a mean mass of ~15 g. If we consider—almost 15 years after the fall—the mass calculations, observations during and after the asteroid entered the atmosphere, the mineralogy of the C1 stones AhS 91A and AhS 671, and the experimental work on fitting the asteroid spectrum (e.g., Goodrich et al., 2019; Jenniskens et al., 2010; Shaddad et al., 2010), the main portion of the meteoroid was likely made of the fine‐grained (carbonaceous) dust and was mostly lost in the atmosphere. In particular, the fact that C1 materials were found has important implications for interpreting asteroid 2008 TC3's early spectroscopic results. Goodrich et al. (2019) correctly suggested that if scientists had not recovered the “water‐free” samples (e.g., ureilites, enstatites, and OC) from the AhS strewn field, 2008 TC3 would have been assumed to be a carbonaceous chondrite meteoroid. Considering that the dominating mass of the exploding meteoroid consisted of carbonaceous materials, asteroid 2008 TC3 cannot be classified as a polymict ureilite; consequently, we state that the asteroid was a polymict carbonaceous chondrite breccia, specifically a polymict C1 object that may have formed by late accretion at least 50–100 Ma after calcium–aluminum‐rich inclusions

Dietary protein intake and health-related outcomes: a methodological protocol for the evidence evaluation and the outline of an evidence to decision framework underlying the evidence-based guideline of the German Nutrition Society

Purpose The present work aimed to delineate (i) a revised protocol according to recent methodological developments in evidence generation, to (ii) describe its interpretation, the assessment of the overall certainty of evidence and to (iii) outline an Evidence to Decision framework for deriving an evidence-based guideline on quantitative and qualitative aspects of dietary protein intake. Methods A methodological protocol to systematically investigate the association between dietary protein intake and several health outcomes and for deriving dietary protein intake recommendations for the primary prevention of various non-communicable diseases in the general adult population was developed. Results The developed methodological protocol relies on umbrella reviews including systematic reviews with or without meta-analyses. Systematic literature searches in three databases will be performed for each health-related outcome. The methodological quality of all selected systematic reviews will be evaluated using a modified version of AMSTAR 2, and the outcome-specific certainty of evidence for systematic reviews with or without meta-analysis will be assessed with NutriGrade. The general outline of the Evidence to Decision framework foresees that recommendations in the derived guideline will be given based on the overall certainty of evidence as well as on additional criteria such as sustainability. Conclusion The methodological protocol permits a systematic evaluation of published systematic reviews on dietary protein intake and its association with selected health-related outcomes. An Evidence to Decision framework will be the basis for the overall conclusions and the resulting recommendations for dietary protein intake.Open Access funding enabled and organized by Projekt DEAL.Bundesministerium für Ernährung und Landwirtschaft http://dx.doi.org/10.13039/501100005908Hochschule Fulda (3361

The role of prediction and outcomes in adaptive cognitive control

Humans adaptively perform actions to achieve their goals. This flexible behaviour requires two core abilities: the ability to anticipate the outcomes of candidate actions and the ability to select and implement actions in a goal-directed manner. The ability to predict outcomes has been extensively researched in reinforcement learning paradigms, but this work has often focused on simple actions that are not embedded in hierarchical and sequential structures that are characteristic of goal-directed human behaviour. On the other hand, the ability to select actions in accordance with high-level task goals, particularly in the presence of alternative responses and salient distractors, has been widely researched in cognitive control paradigms. Cognitive control research, however, has often paid less attention to the role of action outcomes. The present review attempts to bridge these accounts by proposing an outcome-guided mechanism for selection of extended actions. Our proposal builds on constructs from the hierarchical reinforcement learning literature, which emphasises the concept of reaching and evaluating informative states, i.e., states that constitute subgoals in complex actions. We develop an account of the neural mechanisms that allow outcome-guided action selection to be achieved in a network that relies on projections from cortical areas to the basal ganglia and back-projections from the basal ganglia to the cortex. These cortico-basal ganglia-thalamo-cortical ‘loops’ allow convergence – and thus integration – of information from non-adjacent cortical areas (for example between sensory and motor representations). This integration is essential in action sequences, for which achieving an anticipated sensory state signals the successful completion of an action. We further describe how projection pathways within the basal ganglia allow selection between representations, which may pertain to movements, actions, or extended action plans. The model lastly envisages a role for hierarchical projections from the striatum to dopaminergic midbrain areas that enable more rostral frontal areas to bias the selection of inputs from more posterior frontal areas via their respective representations in the basal ganglia.This work is supported by the Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/I019847/1, awarded to NY and FW

Preliminary Results for the Multi-Robot, Multi-Partner, Multi-Mission, Planetary Exploration Analogue Campaign on Mount Etna

This paper was initially intended to report on the outcome of the twice postponed demonstration mission of the ARCHES project. Due to the global COVID pandemic, it has been postponed from 2020, then 2021, to 2022. Nevertheless, the development of our concepts and integration has progressed rapidly, and some of the preliminary results are worthwhile to share with the community to drive the dialog on robotics planetary exploration strategies. This paper includes an overview of the planned 4-week campaign, as well as the vision and relevance of the missiontowards the planned official space missions. Furthermore, the cooperative aspect of the robotic teams, the scientific motivation, the sub task achievements are summarised