A service-based approach to cryoEM facility processing pipelines at eBIC.

Electron cryo-microscopy image-processing workflows are typically composed of elements that may, broadly speaking, be categorized as high-throughput workloads which transition to high-performance workloads as preprocessed data are aggregated. The high-throughput elements are of particular importance in the context of live processing, where an optimal response is highly coupled to the temporal profile of the data collection. In other words, each movie should be processed as quickly as possible at the earliest opportunity. The high level of disconnected parallelization in the high-throughput problem directly allows a completely scalable solution across a distributed computer system, with the only technical obstacle being an efficient and reliable implementation. The cloud computing frameworks primarily developed for the deployment of high-availability web applications provide an environment with a number of appealing features for such high-throughput processing tasks. Here, an implementation of an early-stage processing pipeline for electron cryotomography experiments using a service-based architecture deployed on a Kubernetes cluster is discussed in order to demonstrate the benefits of this approach and how it may be extended to scenarios of considerably increased complexity.

ChAdOx1 COVID vaccines express RBD open prefusion SARS-CoV-2 spikes on the cell surface.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1-vectored HexaPro-stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.

Unconscious Communications by the Body in Catatonia-A Jungian Perspective.

Outside of specific motor conditions, bodily movements are rarely considered in contemporary psychiatry. Stereotypies and mannerisms in clinical cases of catatonia are seen as having no deeper meaning in contemporary psychiatry. Perhaps we are missing something that could be important for us and our patients. The psychiatrist and analyst Carl Jung suggested there was an unconscious communication, and therefore a meaning in psychotic symptoms, including the movements in catatonia. The unconscious is rarely considered in psychotic presentations, yet psychosis is a prevalent condition in clinical settings. In this article Jung's ideas are presented along with case examples that invite the reader to consider them in their own future practice.

Molecular architecture and conservation of an immature human endogenous retrovirus.

The human endogenous retrovirus K (HERV-K) is the most recently acquired endogenous retrovirus in the human genome and is activated and expressed in many cancers and amyotrophic lateral sclerosis. We present the immature HERV-K capsid structure at 3.2 Å resolution determined from native virus-like particles using cryo-electron tomography and subtomogram averaging. The structure shows a hexamer unit oligomerized through a 6-helix bundle, which is stabilized by a small molecule analogous to IP6 in immature HIV-1 capsid. The HERV-K immature lattice is assembled via highly conserved dimer and trimer interfaces, as detailed through all-atom molecular dynamics simulations and supported by mutational studies. A large conformational change mediated by the linker between the N-terminal and the C-terminal domains of CA occurs during HERV-K maturation. Comparison between HERV-K and other retroviral immature capsid structures reveals a highly conserved mechanism for the assembly and maturation of retroviruses across genera and evolutionary time.

Selective Activation of D3 Dopamine Receptors Ameliorates DOI-Induced Head Twitching Accompanied by Changes in Corticostriatal Processing.

D3 receptors, a key component of the dopamine system, have emerged as a potential target of therapies to improve motor symptoms across neurodegenerative and neuropsychiatric conditions. In the present work, we evaluated the effect of D3 receptor activation on the involuntary head twitches induced by 2,5-dimethoxy-4-iodoamphetamine (DOI) at behavioral and electrophysiological levels. Mice received an intraperitoneal injection of either a full D3 agonist, WC 44 [4-(2-fluoroethyl)-N-[4-[4-(2-methoxyphenyl)piperazin 1-yl]butyl]benzamide] or a partial D3 agonist, WW-III-55 [N-(4-(4-(4-methoxyphenyl)piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide] five minutes before the intraperitoneal administration of DOI. Compared to the control group, both D3 agonists delayed the onset of the DOI-induced head-twitch response and reduced the total number and frequency of the head twitches. Moreover, the simultaneous recording of neuronal activity in the motor cortex (M1) and dorsal striatum (DS) indicated that D3 activation led to slight changes in a single unit activity, mainly in DS, and increased its correlated firing in DS or between presumed cortical pyramidal neurons (CPNs) and striatal medium spiny neurons (MSNs). Our results confirm the role of D3 receptor activation in controlling DOI-induced involuntary movements and suggest that this effect involves, at least in part, an increase in correlated corticostriatal activity. A further understanding of the underlying mechanisms may provide a suitable target for treating neuropathologies in which involuntary movements occur.

Molecular architecture and conservation of an immature human endogenous retrovirus.

A significant part of the human genome consists of endogenous retroviruses sequences. Human endogenous retrovirus K (HERV-K) is the most recently acquired endogenous retrovirus, is activated and expressed in many cancers and amyotrophic lateral sclerosis and possibly contributes to the aging process. To understand the molecular architecture of endogenous retroviruses, we determined the structure of immature HERV-K from native virus-like particles (VLPs) using cryo-electron tomography and subtomogram averaging (cryoET STA). The HERV-K VLPs show a greater distance between the viral membrane and immature capsid lattice, correlating with the presence of additional peptides, SP1 and p15, between the capsid (CA) and matrix (MA) proteins compared to the other retroviruses. The resulting cryoET STA map of the immature HERV-K capsid at 3.2 Å resolution shows a hexamer unit oligomerized through a 6-helix bundle which is further stabilized by a small molecule in the same way as the IP6 in immature HIV-1 capsid. The HERV-K immature CA hexamer assembles into the immature lattice via highly conserved dimmer and trimer interfaces, whose interactions were further detailed through all-atom molecular dynamics simulations and supported by mutational studies. A large conformational change mediated by the flexible linker between the N-terminal and the C-terminal domains of CA occurs between the immature and the mature HERV-K capsid protein, analogous to HIV-1. Comparison between HERV-K and other retroviral immature capsid structures reveals a highly conserved mechanism for the assembly and maturation of retroviruses across genera and evolutionary time.

Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography.

Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.

Jung and Working with Acute Psychosis.

Carl Jung is one of the founders of depth psychology. What is less known is his career as a psychiatrist before the creation of his analytical psychology. Jung made significant contributions to the understanding of psychotic illness. However, Jung's contributions go largely unrecognized. This is a loss for the psychiatrist who wants to incorporate psychodynamic ways of working into their daily practice. In this short essay, I describe an overview of Jung's suggestions on treating acute psychosis. A comment follows on use in contemporary practice based on personal accounts and relevant literature.

Broad-Spectrum Extracellular Antiviral Properties of Cucurbit[n]urils.

Viruses are microscopic pathogens capable of causing disease and are responsible for a range of human mortalities and morbidities worldwide. They can be rendered harmless or destroyed with a range of antiviral chemical compounds. Cucurbit[n]urils (CB[n]s) are a family of macrocycle chemical compounds existing as a range of homologues; due to their structure, they can bind to biological materials, acting as supramolecular "hosts" to "guests", such as amino acids. Due to the increasing need for a nontoxic antiviral compound, we investigated whether cucurbit[n]urils could act in an antiviral manner. We have found that certain cucurbit[n]uril homologues do indeed have an antiviral effect against a range of viruses, including herpes simplex virus 2 (HSV-2), respiratory syncytial virus (RSV) and SARS-CoV-2. In particular, we demonstrate that CB[7] is the active homologue of CB[n], having an antiviral effect against enveloped and nonenveloped species. High levels of efficacy were observed with 5 min contact times across different viruses. We also demonstrate that CB[7] acts with an extracellular virucidal mode of action via host-guest supramolecular interactions between viral surface proteins and the CB[n] cavity, rather than via cell internalization or a virustatic mechanism. This finding demonstrates that CB[7] acts as a supramolecular virucidal antiviral (a mechanism distinct from other current extracellular antivirals), demonstrating the potential of supramolecular interactions for future antiviral disinfectants.

Electrocatalytic water oxidation from a mixed linker MOF based on NU-1000 with an integrated ruthenium-based metallo-linker.

A novel tetratopic metallo-linker, [Ru(tda)(py(PhCOOH)2)2], 1, (tda = 2,2':6',2''-terpyridine-6,6''-dicarboxylate; py(PhCOOH)2 = (4,4'-(pyridine-3,5-diyl)dibenzoic acid), that is structurally based on one of the most active molecular water oxidation catalysts has been prepared and fully characterized, including single crystal X-ray diffraction. 1 bears geometric similarities to H4TBAPy (H4TBAPy = 4,4',4'',4'''-(pyrene-1,3,6,8-tetrayl)tetrabenzoic acid), i.e. the native linker in NU-1000, which offers the possibility to synthesize NU-1000-Ru mixed linker MOFs solvothermally. Mixed linker MOF formation was demonstrated by powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM), and Ru linker incorporation confirmed by FT-IR, energy-dispersive X-ray (EDX) spectroscopy and inductively coupled plasma optical emission spectroscopy (ICP-OES). It was found that the Ru contents in the final mixed linker MOFs correlate with the amount of Ru linker present during solvothermal synthesis, albeit not in a linear fashion. The cyclic voltammograms (CV) of the mixed linker MOFs are largely dominated by TBAPy-based oxidations with features attributed to 1. Interestingly, Ru linkers near the crystal surface are oxidized directly by interfacial hole transfer form the electrode, while those in the crystal interior can be oxidized indirectly from oxidized TBAPy linkers at more anodic potential. Upon repeated scanning, the CVs show the appearance of new waves that arise from irreversible TBAPy oxidation, as well as from the activation of the Ru-based water oxidation catalyst. Of the materials prepared, the one with the highest Ru content, NU-1000-Ruhigh, was shown to catalyze the electrochemical oxidation of water to dioxygen. The Faradaic efficiency (FE) of the construct is 37%, due to water oxidation being accompanied by oxidative transformations of the TBAPy linkers. Despite the low FE, NU-1000-Ruhigh is still among the best MOF-based water oxidation catalysts, operating by a unique co-linker mediated hole-transport mechanism to supply oxidizing equivalents also to catalysts in the crystal interior.

A Case Study Using a Behavioural Contract in Alcohol Dependence within a Crisis Home Treatment Team.

In this case study, we present a novel approach to care within a Home Treatment Team, using a behavioural contract. This is a signed, written agreement that targets specific behaviours for change. The concept draws on social learning theory in that it requires social interaction and a relationship to work. In psychiatric settings, the behavioural contract often finds use in Democratic Therapeutic Communities but rarely in crisis or acute services. In this case study, we attempted to use a behavioural contract within our Home Treatment Team to help a patient address his alcohol dependence and its subsequent effect on his daily living activities. The behavioural contract provided an alternative way to manage a crisis episode. We hope that other crisis service staff reading this case study may use a behavioural contract in their work to a similar beneficial effect.

Cryo-ET detects bundled triple helices but not ladders in meiotic budding yeast.

In meiosis, cells undergo two sequential rounds of cell division, termed meiosis I and meiosis II. Textbook models of the meiosis I substage called pachytene show that nuclei have conspicuous 100-nm-wide, ladder-like synaptonemal complexes and ordered chromatin loops. It remains unknown if these cells have any other large, meiosis-related intranuclear structures. Here we present cryo-ET analysis of frozen-hydrated budding yeast cells before, during, and after pachytene. We found no cryo-ET densities that resemble dense ladder-like structures or ordered chromatin loops. Instead, we found large numbers of 12-nm-wide triple-helices that pack into ordered bundles. These structures, herein called meiotic triple helices (MTHs), are present in meiotic cells, but not in interphase cells. MTHs are enriched in the nucleus but not enriched in the cytoplasm. Bundles of MTHs form at the same timeframe as synaptonemal complexes (SCs) in wild-type cells and in mutant cells that are unable to form SCs. These results suggest that in yeast, SCs coexist with previously unreported large, ordered assemblies.

The antibody response to SARS-CoV-2 Beta underscores the antigenic distance to other variants.

Alpha-B.1.1.7, Beta-B.1.351, Gamma-P.1, and Delta-B.1.617.2 variants of SARS-CoV-2 express multiple mutations in the spike protein (S). These may alter the antigenic structure of S, causing escape from natural or vaccine-induced immunity. Beta is particularly difficult to neutralize using serum induced by early pandemic SARS-CoV-2 strains and is most antigenically separated from Delta. To understand this, we generated 674 mAbs from Beta-infected individuals and performed a detailed structure-function analysis of the 27 most potent mAbs: one binding the spike N-terminal domain (NTD), the rest the receptor-binding domain (RBD). Two of these RBD-binding mAbs recognize a neutralizing epitope conserved between SARS-CoV-1 and -2, while 18 target mutated residues in Beta: K417N, E484K, and N501Y. There is a major response to N501Y, including a public IgVH4-39 sequence, with E484K and K417N also targeted. Recognition of these key residues underscores why serum from Beta cases poorly neutralizes early pandemic and Delta viruses.

Modulation of lateral septal and dorsomedial striatal neurons by hippocampal sharp-wave ripples, theta rhythm, and running speed.

Single units were recorded in hippocampus, lateral septum (LS), and dorsomedial striatum (DMS) while freely behaving rats (n = 3) ran trials in a T-maze task and rested in a holding bucket between trials. In LS, 28% (64/226) of recorded neurons were excited and 14% (31/226) were inhibited during sharp wave ripples (SWRs). LS neurons that were excited during SWRs fired preferentially on the downslope of hippocampal theta rhythm and had firing rates that were positively correlated with running speed; LS neurons that were inhibited during SWRs fired preferentially on the upslope of hippocampal theta rhythm and had firing rates that were negatively correlated with running speed. In DMS, only 3.3% (12/366) of recorded neurons were excited and 5.7% (21/366) were inhibited during SWRs. As in LS, DMS neurons that were excited by SWRs tended to have firing rates that were positively modulated by running speed, whereas DMS neurons that were inhibited by SWRs tended to have firing rates that were negatively modulated by running speed. But in contrast with LS, these two DMS subpopulations did not clearly segregate their spikes to different phases of the theta cycle. Based on these results and a review of prior findings, we discuss how concurrent activation of spatial trajectories in hippocampus and motor representations in LS and DMS may contribute to neural computations that support reinforcement learning and value-based decision making.

Revealing the contribution of astrocytes to glutamatergic neuronal transmission.

Research on glutamatergic neurotransmission has focused mainly on the function of presynaptic and postsynaptic neurons, leaving astrocytes with a secondary role only to ensure successful neurotransmission. However, recent evidence indicates that astrocytes contribute actively and even regulate neuronal transmission at different levels. This review establishes a framework by comparing glutamatergic components between neurons and astrocytes to examine how astrocytes modulate or otherwise influence neuronal transmission. We have included the most recent findings about the role of astrocytes in neurotransmission, allowing us to understand the complex network of neuron-astrocyte interactions. However, despite the knowledge of synaptic modulation by astrocytes, their contribution to specific physiological and pathological conditions remains to be elucidated. A full understanding of the astrocyte's role in neuronal processing could open fruitful new frontiers in the development of therapeutic applications.

The effect of phenytoin on embryonic heart rate in Vivo.

Phenytoin is a known human teratogen with unknown etiology. Several mechanisms have been proposed including disturbances in folate metabolism, induction of embryonic hypoxia following phenytoin-induced bradycardia, free radical formation following re-oxygenation and phenytoin-induced maternal hyperglycemia. Using high frequency ultrasound, we demonstrated that phenytoin induced a dramatic decrease in the heart rate of embryos. This coincided with a moderate transient decrease in maternal heart rate and blood glucose levels. Embryonic heart rate had not fully recovered 24 h later in some embryos despite normal maternal physiological parameters. In a separate study, extent of hypoxia was measured using the marker pimonidazole. Phenytoin-exposed embryos did not demonstrate increased hypoxia compared to control embryos at 2, 4, 8 or 24 h dosing. Together our results show that phenytoin induces malformations as a result of a combination of insults: embryonic bradycardia, maternal bradycardia and maternal hyperglycemia. However, this does not appear to result in measurable embryonic hypoxia in our animal model.

Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Single Particle Cryo-Electron Microscopy: From Sample to Structure.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Native-like SARS-CoV-2 Spike Glycoprotein Expressed by ChAdOx1 nCoV-19/AZD1222 Vaccine.

Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation, and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirm the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.