In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment.

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.

Visualizing chaperonin function in situ by cryo-electron tomography.

Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1-3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4-6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.

Serial Lift-Out: sampling the molecular anatomy of whole organisms.

Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically <300 nm. Here we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to Caenorhabditis elegans L1 larvae, yielding a cryo-ET dataset sampling the worm's anterior-posterior axis, and resolve its ribosome structure to 7 Å and a subregion of the 11-protofilament microtubule to 13 Å, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.

In situ structural analysis reveals membrane shape transitions during autophagosome formation.

Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.

Impact of enhanced recovery program implementation on postoperative outcomes after liver surgery: a monocentric retrospective study.

INTRODUCTION: It is still unclear whether enhanced recovery programs (ERPs) reduce postoperative morbidity after liver surgery. This study investigated the effect on liver surgery outcomes of labeling as a reference center for ERP. MATERIALS AND METHODS: Perioperative data from 75 consecutive patients who underwent hepatectomy in our institution after implementation and labeling of our ERP were retrospectively compared to 75 patients managed before ERP. Length of hospital stay, postoperative complications, and adherence to protocol were examined. RESULTS: Patient demographics, comorbidities, and intraoperative data were similar in the two groups. Our ERP resulted in shorter length of stay (3 days [1-6] vs. 4 days [2-7.5], p = 0.03) and fewer postoperative complications (24% vs. 45.3%, p = 0.0067). This reduction in postoperative morbidity can be attributed exclusively to a lower rate of minor complications (Clavien-dindo grade < IIIa), and in particular to a lower rate of postoperative ileus, after labeling. (5.3% vs. 25.3%, p = 0.0019). Other medical and surgical complications were not significantly reduced. Adherence to protocol improved after labeling (17 [16-18] vs. 14 [13-16] items, p < 0.001). CONCLUSIONS: The application of a labeled enhanced recovery program for liver surgery was associated with a significant shortening of hospital stay and a halving of postoperative morbidity, mainly ileus.

A transformation clustering algorithm and its application in polyribosomes structural profiling.

Improvements in cryo-electron tomography sample preparation, electron-microscopy instrumentations, and image processing algorithms have advanced the structural analysis of macromolecules in situ. Beyond such analyses of individual macromolecules, the study of their interactions with functionally related neighbors in crowded cellular habitats, i.e. 'molecular sociology', is of fundamental importance in biology. Here we present a NEighboring Molecule TOpology Clustering (NEMO-TOC) algorithm. We optimized this algorithm for the detection and profiling of polyribosomes, which play both constitutive and regulatory roles in gene expression. Our results suggest a model where polysomes are formed by connecting multiple nonstochastic blocks, in which translation is likely synchronized.

Improvements in cryo-electron tomography sample preparation, electron-microscopy instrumentations, and image processing algorithms have advanced the structural analysis of macromolecules in situ. Beyond such analyses of individual macromolecules, the study of their interactions with functionally related neighbors in crowded cellular habitats, i.e. "molecular sociology", is of fundamental importance in biology. Here we present a NEighboring Molecule TOpology Clustering (NEMO-TOC) algorithm. We optimized this algorithm for the detection and profiling of polyribosomes, which play both constitutive and regulatory roles in gene expression. Our results suggest a model where polysomes are formed by connecting multiple nonstochastic blocks, in which translation is likely synchronized.

Oral as compared to intravenous tranexamic acid to limit peri-operative blood loss associated with primary total hip arthroplasty: A randomised noninferiority trial.

BACKGROUND: Oral as compared to intravenous tranexamic acid (TXA) is an attractive option, in terms of cost and safety, to reduce blood loss and transfusion in total hip arthroplasty. Exclusion criteria applied in the most recent randomised trials may have limited the generalisability of oral tranexamic acid in this indication. Larger and more inclusive studies are needed to definitively establish oral administration as a credible alternative to intravenous administration. OBJECTIVES: To assess the noninferiority of oral to intravenous TXA at reducing intra-operative and postoperative total blood loss (TBL) in primary posterolateral approached total hip arthroplasty (PLTHA). DESIGN: Noninferiority, single centre, randomised, double-blind controlled study. SETTING: Patients scheduled for primary PLTHA. Data acquisition occurred between May 2021 and November 2022 at the University Hospital of Liège, Belgium. PATIENTS: Two hundred and twenty-eight patients, randomised in a 1 : 1 ratio from a computer-generated list, completed the trial. INTERVENTIONS: Administration of 2 g of oral TXA 2 h before total hip arthroplasty and 4 h after incision (Group oral) was compared to the intravenous administration of 1 g of TXA 30 min before surgery and 4 h after incision (Group i.v.). MAIN OUTCOME MEASURES: TBL (measured intra-operative and drainage blood loss up to 48 h after surgery, primary outcome), decrease in haemoglobin concentration, D-Dimer at day 1 and day 3, transfusion rate (secondary outcomes). RESULTS: Analyses were performed on 108 out of 114 participants (Group i.v.) and 104 out of 114 participants (Group oral). Group oral was noninferior to Group i.v. with regard to TBL, with a difference between medians (95% CI) of 35 ml (-103.77 to 33.77) within the noninferiority margins. Median [IQR] of estimated TBL was 480 ml [350 to 565] and 445 ml [323 to 558], respectively. No significant interaction between group and time was observed regarding the evolution of TBL and haemoglobin over time. CONCLUSIONS: TXA as an oral premedication before PLTHA is noninferior to its intravenous administration regarding peri-operative TBL. TRIAL REGISTRATION: European Clinical Trial Register under EudraCT-number 2020-004167-29 ( https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-004167-29/BE ).

HADS and SOMS-2 brief score evaluation can prevent unnecessary minimal invasive spine interventions - a prospective blinded observational clinical trial.

INTRODUCTION: To test the hypothesis, whether HADS/SOMS is practical in a spine surgery consultation setting and that patients with CLBP, but a high-risk of psychic comorbidities using above screenings will not improve after minimal-invasive spine interventions (MIS). METHODS: n = 150 completed HADS and SOMS prior to the acquisition of history and examination. Primary outcome was improvement by numeric rating scale (NRS), Pain disability index (PDI) and oswestry disability index (ODI) at baseline and 6 months after intervention. In case of sciatica due to disc herniation epidural neurolysis, for facet or SI-joint-syndrome, radiofrequency and for discogenic pain intradiscal electrothermal therapy (IDET) was performed. RESULTS: 6 months after interventions, pat. with a high-risk of anxiety or depression showed no clinically important improvements in NRS, PDI and ODI, whereas in the low-risk group all 3 parameters were significantly reduced. We found a statistically significant difference in the improvement of NRS (p < 0·05), PDI (p < 0·001), ODI (p < 0·001) between high- and low-risk HADS-anxiety and depression groups and in the improvement of NRS and PDI (p < 0·05) between high- and low-risk SOMS-2-subgroups. CONCLUSIONS: In this group of CLBP patients, the easy-to-administer HADS/SOMS reliably predicted outcome after MIS due to the detection of somatoform comorbidities. Thus, 30 out of 150 patients were invasively treated without improvement. This is alarming not only because of unnecessary MIS being performed on these individuals, but also because it represents an inefficient allocation of increasingly limited healthcare funds. LEVEL OF EVIDENCE: II.

[Sphenopalatine ganglion block : a promising approach for headache and supratentorial intracranial surgery ?] / Le bloc du ganglion sphénopalatin : un traitement prometteur pour les céphalées et la chirurgie intracrânienne supratentorielle ?

We aimed to determine whether sphenopalatine ganglion block (SPGB) had a place as a treatment for headache, as well as its place as an anterior component of scalp block for supratentorial intracranial surgery. Using the keywords «sphenopalatine ganglion¼, «sphenopalatine ganglion block¼, «pain¼, «surgery¼ and «craniotomy¼ in PubMed and Google Scholar, we identified 14 relevant articles. The methods used to perform the block and the treatment of control groups differed between studies. The main outcomes studied were reduction in pain scores and hemodynamic variations during supratentorial intracranial surgery. All articles concluded that SPGB was equal or superior compared with other procedures. Although there are still several points to be studied, considering the ease of application, the low number of complications and the results of the studies, the use of SPGB for the headache treatment and in neurosurgery as an anterior component of scalp block seems appropriate.

Cette revue de la littérature narrative vise à déterminer la place du bloc du ganglion sphénopalatin (BGSP) comme traitement des céphalées ainsi que sa place comme composante antérieure du bloc de scalp pour la chirurgie intracrânienne supratentorielle. Avec les mots-clés «ganglion sphénopalatin¼, «bloc du ganglion sphénopalatin¼, «douleur¼, «chirurgie¼ et «crâniotomie dans PubMed et Google Scholar, nous avons identifié 14 articles pertinents. Les méthodes de réalisation du bloc ainsi que les traitements des groupes témoins différaient selon les études. Les résultats principaux étudiés étaient la réduction des scores de douleur ainsi que les variations hémodynamiques au cours de la chirurgie intracrânienne supratentorielle. Tous les articles ont conclu à la non-infériorité ou la supériorité du BGSP en comparaison à d'autres procédures. Bien que plusieurs points restent à étudier, compte tenu de la facilité d'application, du peu de complications et des résultats, l'utilisation du BGSP pour le traitement des céphalées et en neurochirurgie comme composante antérieure du bloc de scalp semble appropriée.

Direct visualization of degradation microcompartments at the ER membrane.

To promote the biochemical reactions of life, cells can compartmentalize molecular interaction partners together within separated non-membrane-bound regions. It is unknown whether this strategy is used to facilitate protein degradation at specific locations within the cell. Leveraging in situ cryo-electron tomography to image the native molecular landscape of the unicellular alga Chlamydomonas reinhardtii, we discovered that the cytosolic protein degradation machinery is concentrated within ∼200-nm foci that contact specialized patches of endoplasmic reticulum (ER) membrane away from the ER-Golgi interface. These non-membrane-bound microcompartments exclude ribosomes and consist of a core of densely clustered 26S proteasomes surrounded by a loose cloud of Cdc48. Active proteasomes in the microcompartments directly engage with putative substrate at the ER membrane, a function canonically assigned to Cdc48. Live-cell fluorescence microscopy revealed that the proteasome clusters are dynamic, with frequent assembly and fusion events. We propose that the microcompartments perform ER-associated degradation, colocalizing the degradation machinery at specific ER hot spots to enable efficient protein quality control.

Comparison between supra-inguinal fascia iliaca and pericapsular nerve group blocks on postoperative pain and functional recovery after total hip arthroplasty: A noninferiority randomised clinical trial.

BACKGROUND: Pain after a posterolateral approach for total hip arthroplasty (THA) may affect early functional recovery. Supra-inguinal fascia iliaca (SFIB) and pericapsular nerve group (PENG) blocks have been proposed as promising analgesia techniques. OBJECTIVES: This trial was conducted to compare a PENG with a SFIB for controlling postoperative pain and for providing functional recovery. DESIGN: Noninferiority monocentric randomised controlled study. SETTING: One hundred and two patients scheduled for a total hip arthroplasty via the posterolateral approach under spinal anaesthesia were prospectively allocated to two groups. Data acquisition occurred between October 2021 and July 2022 at the University Hospital of Liege. PATIENTS: One hundred and two patients completed the trial. INTERVENTIONS: Group SFIB received supra-inguinal fascia iliaca block (SFIB) (40 ml ropivacaine 0.375%), whereas group PENG received a PENG block (20 ml ropivacaine 0.75%). MAIN OUTCOME MEASURES: Rest and mobilisation pain on a 0 to 10 numeric rating scale at fixed time points: 1 and 6 h after surgery, on day-1 and day-2 at 8 a.m.,1 p.m. and 6 p.m. On day-1 and day-2, evolution of quality-of-recovery-15 score was assessed, and timed-up-and-go, 2 and 6 min-walking tests. The noninferiority margin was set as 1 numeric rating scale point 6 h after surgery. RESULTS: Six hours after surgery, pain scores in group PENG were noninferior to those of group SFIB, with a difference between medians at 0 (95% CI -0.93 to 0.93). There were no significant differences between the groups regarding rest and dynamic pain trajectories during the first 48 postoperative hours, with no significant effects of group (rest P  = 0.800; dynamic P  = 0.708) or interaction between group and time (rest P  = 0.803; dynamic P  = 0.187). Similarly, no significant differences were observed regarding motor and functional recovery as assessed by timed-up-and-go ( P  = 0.197), 2 min ( P  = 0.364), and 6 min walking ( P  = 0.347) tests and quality-of-recovery-15 ( P  = 0.417) score. CONCLUSION: Following a total hip arthroplasty via the posterolateral approach, a PENG block is noninferior to SFIB regarding postoperative pain control 6 h after surgery, and functional recovery. TRIAL REGISTRATION: European Clinical Trial Register under EudraCT-number 2020-005126-28 ( https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-005126-28/BE ).

[Plasma biomarkers in traumatic brain injury : current status]. / Biomarqueurs plasmatiques dans le traumatisme crânien : état des lieux.

Traumatic Brain Injury (TBI) is a common trauma and a worldwide major cause of mortality or disability in both civilian and military populations. TBI is not a pathological entity in its own, but rather a pattern of heterogeneous traumas with diverse and varied mechanisms and clinical expressions. Therefore, no universal medical response can be settled to it. Instead, medical management must be tailored to each individual's specific needs. If the current identification and prognosis of TBI is basically based on neurological examination and computerized tomography, specific biomarkers could represent a valuable aid in this diagnostic and therapeutic challenge. The potential implications of these markers are broad, encompassing among others the detection of unsuspected TBI, the monitoring of trauma severity, short-, intermediate- and long-term prognosis and apprehension of the extent of the sequelae. This topic has received broad and growing interest over the past decade, and the current literature is extensive. This short narrative review summarizes the latest advances in the field of plasma biomarkers in TBI patients.

Le traumatisme crânien (TC) est une pathologie courante et une cause majeure de mortalité ou d'invalidité dans le monde, tant chez les civils que chez les militaires. Le TC ne constitue pas une entité pathologique à part entière, mais plutôt un ensemble de lésions hétérogènes dont les mécanismes et les expressions cliniques sont divers et variés. Par conséquent, aucune prise en charge universelle ne peut être établie et les traitements médicaux doivent être adaptés et ajustés à l'individu. Si, actuellement, l'identification et le pronostic des TC reposent essentiellement sur l'examen clinique neurologique et la tomodensitométrie, des biomarqueurs spécifiques pourraient représenter une aide précieuse dans ce défi diagnostique et thérapeutique. L'implication potentielle de ces marqueurs est vaste, avec un rôle à jouer, entre autres, dans la détection de la présence d'un TC insoupçonné, le suivi de la sévérité du traumatisme, le pronostic de morbi-mortalité à court, moyen et long termes, et la prédiction de la gravité et de l'étendue des séquelles. Ce sujet a suscité un intérêt large et croissant au cours de la dernière décennie, et la littérature actuelle est abondante. Cette brève revue narrative résume les dernières avancées dans le domaine des biomarqueurs plasmatiques lors d'un TC.

Volumetric macromolecule identification in cryo-electron tomograms using capsule networks.

BACKGROUND: Despite recent advances in cellular cryo-electron tomography (CET), developing automated tools for macromolecule identification in submolecular resolution remains challenging due to the lack of annotated data and high structural complexities. To date, the extent of the deep learning methods constructed for this problem is limited to conventional Convolutional Neural Networks (CNNs). Identifying macromolecules of different types and sizes is a tedious and time-consuming task. In this paper, we employ a capsule-based architecture to automate the task of macromolecule identification, that we refer to as 3D-UCaps. In particular, the architecture is composed of three components: feature extractor, capsule encoder, and CNN decoder. The feature extractor converts voxel intensities of input sub-tomograms to activities of local features. The encoder is a 3D Capsule Network (CapsNet) that takes local features to generate a low-dimensional representation of the input. Then, a 3D CNN decoder reconstructs the sub-tomograms from the given representation by upsampling. RESULTS: We performed binary and multi-class localization and identification tasks on synthetic and experimental data. We observed that the 3D-UNet and the 3D-UCaps had an [Formula: see text]score mostly above 60% and 70%, respectively, on the test data. In both network architectures, we observed degradation of at least 40% in the [Formula: see text]-score when identifying very small particles (PDB entry 3GL1) compared to a large particle (PDB entry 4D8Q). In the multi-class identification task of experimental data, 3D-UCaps had an [Formula: see text]-score of 91% on the test data in contrast to 64% of the 3D-UNet. The better [Formula: see text]-score of 3D-UCaps compared to 3D-UNet is obtained by a higher precision score. We speculate this to be due to the capsule network employed in the encoder. To study the effect of the CapsNet-based encoder architecture further, we performed an ablation study and perceived that the [Formula: see text]-score is boosted as network depth is increased which is in contrast to the previously reported results for the 3D-UNet. To present a reproducible work, source code, trained models, data as well as visualization results are made publicly available. CONCLUSION: Quantitative and qualitative results show that 3D-UCaps successfully perform various downstream tasks including identification and localization of macromolecules and can at least compete with CNN architectures for this task. Given that the capsule layers extract both the existence probability and the orientation of the molecules, this architecture has the potential to lead to representations of the data that are better interpretable than those of 3D-UNet.

A feature-guided, focused 3D signal permutation method for subtomogram averaging.

Advances in electron microscope instrumentation, cryo-electron tomography data collection, and subtomogram averaging have allowed for the in-situ visualization of molecules and their complexes in their native environment. Current data processing pipelines commonly extract subtomograms as a cubic subvolume with the key assumption that the selected object of interest is discrete from its surroundings. However, in instances when the object is in its native environment, surrounding densities may negatively affect the subsequent alignment and refinement processes, leading to loss of information due to misalignment. For example, the strong densities from surrounding membranes may dominate the alignment process for membrane proteins. Here, we developed methods for feature-guided subtomogram alignment and 3D signal permutation for subtomogram averaging. Our 3D signal permutation method randomizes and filters voxels outside a mask of any shape and blurs the boundary of the mask that encapsulates the object of interest. The randomization preserves global statistical properties such as mean density and standard deviation of voxel density values, effectively producing a featureless background surrounding the object of interest. This signal permutation process can be repeatedly applied with intervening alignments of the 3D signal-permuted subvolumes, recentering of the mask, and optional adjustments of the shape of the mask. We have implemented these methods in a new processing pipeline which starts from tomograms, contains feature-guided subtomogram extraction and alignment, 3D signal-permutation, and subtomogram visualization tools. As an example, feature-guided alignment and 3D signal permutation leads to improved subtomogram average maps for a dataset of synaptic protein complexes in their native environment.

Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle.

Proteasomes occur in all three domains of life, and are the principal molecular machines for the regulated degradation of intracellular proteins. They play key roles in the maintenance of protein homeostasis, and control vital cellular processes. While the eukaryotic 26S proteasome is extensively characterized, its putative evolutionary precursor, the archaeal proteasome, remains poorly understood. The primordial archaeal proteasome consists of a 20S proteolytic core particle (CP), and an AAA-ATPase module. This minimal complex degrades protein unassisted by non-ATPase subunits that are present in a 26S proteasome regulatory particle (RP). Using cryo-EM single-particle analysis, we determined structures of the archaeal CP in complex with the AAA-ATPase PAN (proteasome-activating nucleotidase). Five conformational states were identified, elucidating the functional cycle of PAN, and its interaction with the CP. Coexisting nucleotide states, and correlated intersubunit signaling features, coordinate rotation of the PAN-ATPase staircase, and allosterically regulate N-domain motions and CP gate opening. These findings reveal the structural basis for a sequential around-the-ring ATPase cycle, which is likely conserved in AAA-ATPases.

Cryo-EM structure of the cetacean morbillivirus nucleoprotein-RNA complex.

Cetacean morbillivirus (CeMV) is an emerging and highly infectious paramyxovirus that causes outbreaks in cetaceans and occasionally in pinnipeds, representing a major threat to biodiversity and conservation of endangered marine mammal populations in both hemispheres. As for all non-segmented, negative-sense, single-stranded RNA (ssRNA) viruses, the morbilliviral genome is enwrapped by thousands of nucleoprotein (N) protomers. Each bound to six ribonucleotides, N protomers assemble to form a helical ribonucleoprotein (RNP) complex that serves as scaffold for nucleocapsid formation and as template for viral replication and transcription. While the molecular details on RNP complexes elucidated in human measles virus (MeV) served as paradigm model for these processes in all members of the Morbillivirus genus, no structural information has been obtained from other morbilliviruses, nor has any CeMV structure been solved so far. We report the structure of the CeMV RNP complex, reconstituted in vitro upon binding of recombinant CeMV N to poly-adenine ssRNA hexamers and solved to 4.0 Å resolution by cryo-electron microscopy. In spite of the amino acid sequence similarity and consequently similar folding of the N protomer, the CeMV RNP complex exhibits different helical parameters as compared to previously reported MeV orthologs. The CeMV structure reveals exclusive interactions leading to more extensive protomer-RNA and protomer-protomer interfaces. We identified twelve residues, among those varying between CeMV strains, as putatively important for the stabilization of the RNP complex, which highlights the need to study the potential of CeMV N mutations that modulate nucleocapsid assembly to also affect viral phenotype and host adaptation.

High-resolution structure and biophysical characterization of the nucleocapsid phosphoprotein dimerization domain from the Covid-19 severe acute respiratory syndrome coronavirus 2.

Unprecedented by number of casualties and socio-economic burden occurring worldwide, the coronavirus disease 2019 (Covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the worst health crisis of this century. In order to develop adequate countermeasures against Covid-19, identification and structural characterization of suitable antiviral targets within the SARS-CoV-2 protein repertoire is urgently needed. The nucleocapsid phosphoprotein (N) is a multifunctional and highly immunogenic determinant of virulence and pathogenicity, whose main functions consist in oligomerizing and packaging the single-stranded RNA (ssRNA) viral genome. Here we report the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD), on which both N homo-oligomerization and ssRNA binding depend. Crystal structures solved at 1.44 Å and 1.36 Å resolution describe a rhombus-shape N CTD dimer, which stably exists in solution as validated by size-exclusion chromatography coupled to multi-angle light scattering and analytical ultracentrifugation. Differential scanning fluorimetry revealed moderate thermal stability and a tendency towards conformational change. Microscale thermophoresis demonstrated binding to a 7-bp SARS-CoV-2 genomic ssRNA fragment at micromolar affinity. Furthermore, a low-resolution preliminary model of the full-length SARS-CoV N in complex with ssRNA, obtained by cryo-electron microscopy, provides an initial understanding of self-associating and RNA binding functions exerted by the SARS-CoV-2 N.

Is MRI a viable alternative to CT/CBCT to identify the course of the inferior alveolar nerve in relation to the roots of the third molars?

OBJECTIVES: To assess the reliability of judging the spatial relation between the inferior alveolar nerve (IAN) and mandibular third molar (MTM) based on MRI or CT/CBCT images. METHODS: Altogether, CT/CBCT and MRI images of 87 MTMs were examined twice by 3 examiners with different degrees of experience. The course of the IAN in relation to the MTM, the presence/absence of a direct contact between IAN and MTM, and the presence of accessory IAN were determined. RESULTS: The IAN was in > 40% of the cases judged as inferior, while an interradicular position was diagnosed in < 5% of the cases. The overall agreement was good (κ = 0.72) and any disagreement between the imaging modalities was primarily among the adjacent regions, i.e., buccal/lingual/interradicular vs. inferior. CT/CBCT judgements presented a very good agreement for the inter- and intrarater comparison (κ > 0.80), while MRI judgements showed a slightly lower, but good agreement (κ = 0.74). A direct contact between IAN and MTM was diagnosed in about 65%, but in almost 20% a disagreement between the judgements based on MRI and CT/CBCT was present resulting in a moderate overall agreement (κ = 0.60). The agreement between the judgements based on MRI and CT/CBCT appeared independent of the examiner's experience and accessory IAN were described in 10 cases in MRI compared to 3 cases in CT/CBCT images. CONCLUSIONS: A good inter- and intrarater agreement has been observed for the assessment of the spatial relation between the IAN and MTM based on MRI images. Further, MRI images might provide advantages in the detection of accessory IAN compared to CT/CBCT. CLINICAL RELEVANCE: MRI appears as viable alternative to CT/CBCT for preoperative assessment of the IAN in relation to the MTM.

In situ structural studies of tripeptidyl peptidase II (TPPII) reveal spatial association with proteasomes.

Tripeptidyl peptidase II (TPPII) is a eukaryotic protease acting downstream of the 26S proteasome; it removes tripeptides from the degradation products released by the proteasome. Structural studies in vitro have revealed the basic architecture of TPPII, a two-stranded linear polymer that assembles to form a spindle-shaped complex of ∼6 MDa. Dependent on protein concentration, TPPII has a distinct tendency for polymorphism. Therefore, its structure in vivo has remained unclear. To resolve this issue, we have scrutinized cryo-electron tomograms of rat hippocampal neurons for the occurrence and spatial distribution of TPPII by template matching. The quality of the tomograms recorded with the Volta phase plate enabled a detailed structural analysis of TPPII despite its low abundance. Two different assembly states (36-mers and 32-mers) coexist as well as occasional extended forms with longer strands. A distance analysis of the relative locations of TPPII and 26S proteasomes confirmed the visual impression that these two complexes spatially associate in agreement with TPPII's role in postproteasomal degradation.

Structural insights into the functional cycle of the ATPase module of the 26S proteasome.

In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA+ ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA+ ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.