Effects of passive ankle exoskeletons on neuromuscular function during exaggerated standing sway.

Wearable robotic exoskeletons designed to assist human movement should integrate with the neuromusculoskeletal system. This means assisting movement while not perturbing motor control. We sought to test if passive ankle exoskeletons, which have been shown to successfully assist human gait, affect neuromuscular control of an exaggerated anterior-posterior standing sway task. Participants actively swayed while wearing an ankle exoskeleton that provided 0, 42 or 85 Nm rad-1 of additional stiffness to the ankle joint in resistance to dorsiflexion. Sway amplitude was controlled via biofeedback to elicit similar ankle angle displacements across conditions. With greater exoskeleton stiffness, participants swayed at lower sway-cycle frequencies and slower centre of pressure speeds. Furthermore, increasing exoskeleton stiffness resulted in longer operating lengths of the medial gastrocnemius and overall reduced plantar flexor muscle activation. For the soleus, there was also a temporal shift in the cross-correlation of its electromyogram with the centre of pressure displacement, meaning that muscle activation peaked later than anterior sway displacement. Together, these data suggest that assistive ankle exoskeletons influence neuromuscular control of ankle-based sway tasks. Changes in fascicle lengths could influence afferent feedback signals and the short-range stiffness of ankle muscles, while shifts in muscle activation timing suggest changes in neural control. The observed neuromuscular adaptations to exoskeleton assistance demonstrate the potential implications for standing balance and overall movement control, prompting future investigations.

Domain consolidation in Bacterial 50S assembly revealed by Anti-Sense Oligonucleotide Probing.

Investigating the intricate and rapid folding kinetics of large RNA-protein complexes (RNPs), like the bacterial ribosome, remains a formidable challenge in structural biology. Previous genetic approaches to probe assembly have focused on modulating the expression of either r-proteins or assembly factors. Here, anti-sense oligonucleotides (ASOs) were used to disrupt native RNA/RNA and RNA/protein interactions, in order to generate novel folding intermediates. In an in vitro co-transcriptional assembly assay, 8 assembly inhibitor ASOs were identified. Using cryo-electron microscopy, 38 new intermediate structures were determined resulting from the specific inhibitions. In particular a novel intermediate class provided compelling evidence of independent rRNA domain folding before proper interdomain docking. Three PNAs targeting domain-I of 23S- rRNA further subdivided the previously identified assembly core into smaller blocks representing the earliest steps in assembly. The resulting assembly graph reveals template-directed RNA foldon docking and domain consolidation, which provides a hierarchical view of the RNP assembly process.

Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly.

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. B. subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis . While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after E. coli and T. thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

Proteome partitioning constraints in long-term laboratory evolution.

Adaptive laboratory evolution experiments provide a controlled context in which the dynamics of selection and adaptation can be followed in real-time at the single-nucleotide level. And yet this precision introduces hundreds of degrees-of-freedom as genetic changes accrue in parallel lineages over generations. On short timescales, physiological constraints have been leveraged to provide a coarse-grained view of bacterial gene expression characterized by a small set of phenomenological parameters. Here, we ask whether this same framework, operating at a level between genotype and fitness, informs physiological changes that occur on evolutionary timescales. Using a strain adapted to growth in glucose minimal medium, we find that the proteome is substantially remodeled over 40 000 generations. The most striking change is an apparent increase in enzyme efficiency, particularly in the enzymes of lower-glycolysis. We propose that deletion of metabolic flux-sensing regulation early in the adaptation results in increased enzyme saturation and can account for the observed proteome remodeling.

Assembly of the Bacterial Ribosome with Circularly Permuted rRNA.

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of twenty-three resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit, and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

Tandem Mass Spectrometry across Platforms.

PubChem serves as a comprehensive repository, housing over 100 million unique chemical structures representing the breadth of our chemical knowledge across numerous fields including metabolism, pharmaceuticals, toxicology, cosmetics, agriculture, and many more. Rapid identification of these small molecules increasingly relies on electrospray ionization (ESI) paired with tandem mass spectrometry (MS/MS), particularly by comparison to genuine standard MS/MS data sets. Despite its widespread application, achieving consistency in MS/MS data across various analytical platforms remains an unaddressed concern. This study evaluated MS/MS data derived from one hundred molecular standards utilizing instruments from five manufacturers, inclusive of quadrupole time-of-flight (QTOF) and quadrupole orbitrap "exactive" (QE) mass spectrometers by Agilent (QTOF), Bruker (QTOF), SCIEX (QTOF), Waters (QTOF), and Thermo QE. We assessed fragment ion variations at multiple collisional energies (0, 10, 20, and 40 eV) using the cosine scoring algorithm for comparisons and the number of fragments observed. A parallel visual analysis of the MS/MS spectra across instruments was conducted, consistent with a standard procedure that is used to circumvent the still prevalent issue of mischaracterizations as shown for dimethyl sphingosine and C20 sphingosine. Our analysis revealed a notable consistency in MS/MS data and identifications, with fragment ions' m/z values exhibiting the highest concordance between instrument platforms at 20 eV, the other collisional energies (0, 10, and 40 eV) were significantly lower. While moving toward a standardized ESI MS/MS protocol is required for dependable molecular characterization, our results also underscore the continued importance of corroborating MS/MS data against standards to ensure accurate identifications. Our findings suggest that ESI MS/MS manufacturers, akin to the established norms for gas chromatography mass spectrometry instruments, should standardize the collision energy at 20 eV across different instrument platforms.

Elastic ankle exoskeletons influence soleus fascicle dynamics during unexpected perturbations.

Spring-based passive ankle exoskeletons have been designed to emulate the energy conservation and power amplification roles of biological muscle-tendon units during locomotion. Yet, it remains unknown if similar assistive devices can serve the other elastomechanical role of biological muscle-tendon units - power attenuation. Here we explored the effect of bilateral passive ankle exoskeletons on neuromuscular control and muscle fascicle dynamics in the ankle plantarflexors during rapid, unexpected vertical perturbations. We recorded muscle activation and soleus fascicle length changes during hopping with and without exoskeleton assistance (0 and 76 Nm rad-1) on elevated platforms (20 cm), which were removed at an unknown time. Our results demonstrate that exoskeleton assistance leads to a reduction in soleus muscle activation, increases in fascicle length change and decreases in muscle forces during perturbed hopping. These changes have competing effects on the mechanics and energetics of lower limb muscles, likely limiting the capacity for series elastic tissues to absorb energy. As we strive towards the design of wearable assistive devices for everyday locomotion, information regarding real-time muscle-tendon behavior may enable tunable assistance that adapts to both the user and the environment.

Assembly landscape for the bacterial large ribosomal subunit.

Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.

Inherited chitinases enable sustained growth and rapid dispersal of bacteria from chitin particles.

Many biogeochemical functions involve bacteria utilizing solid substrates. However, little is known about the coordination of bacterial growth with the kinetics of attachment to and detachment from such substrates. In this quantitative study of Vibrio sp. 1A01 growing on chitin particles, we reveal the heterogeneous nature of the exponentially growing culture comprising two co-existing subpopulations: a minority replicating on chitin particles and a non-replicating majority which was planktonic. This partition resulted from a high rate of cell detachment from particles. Despite high detachment, sustained exponential growth of cells on particles was enabled by the enrichment of extracellular chitinases excreted and left behind by detached cells. The 'inheritance' of these chitinases sustains the colonizing subpopulation despite its reduced density. This simple mechanism helps to circumvent a trade-off between growth and dispersal, allowing particle-associated marine heterotrophs to explore new habitats without compromising their fitness on the habitat they have already colonized.

Early HIV-1 Gag Assembly on Lipid Membrane with vRNA.

HIV-1 capsid assembly is an essential process in the virus infection cycle. Initiation of capsid assembly involves viral proteins, genomic RNA, and the inner leaflet of the plasma membrane, facilitated by a number of cellular factors1. The viral structural protein Gag plays a number of central roles in this process, including association with the membrane, selective binding of genomic RNA, and oligomerization and packaging to ultimately produce an immature budded pro-viral particle2. While there have been intensive studies regarding the early stages of Gag assembly, there is a lack of consensus on the mechanism for nucleation and growth of Gag complexes3-7. Here we show that myristoylated Gag forms a trimer nucleus in a model membrane that can selectively bind a dimeric RNA containing the packaging signal. Subsequent growth of myristoyl-Gag oligomers requires vRNA, and occurs by addition of 1 or 2 Gag monomers at a time from solution. These data support a model where the immature capsid lattice formation occurs by a gradual lattice edge expansion, following a trimeric nucleation event. The dynamic single molecule data that support this model were recorded using mass photometry, involving full length myristoylated protein, RNA, and lipid together. These data are the first to support a lattice edge expansion model of Gag during early stages of assembly in a biological-relevant setting, providing insights to the fundamental models of virus structural protein assembly process.

Proteomic analysis of circulating immune cells identifies cellular phenotypes associated with COVID-19 severity.

Certain serum proteins, including C-reactive protein (CRP) and D-dimer, have prognostic value in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonetheless, these factors are non-specific, providing limited mechanistic insight into the peripheral blood mononuclear cell (PBMC) populations that drive the pathogenesis of severe COVID-19. To identify cellular phenotypes associated with disease, we performed a comprehensive, unbiased analysis of total and plasma-membrane PBMC proteomes from 40 unvaccinated individuals with SARS-CoV-2, spanning the whole disease spectrum. Combined with RNA sequencing (RNA-seq) and flow cytometry from the same donors, we define a comprehensive multi-omic profile for each severity level, revealing that immune-cell dysregulation progresses with increasing disease. The cell-surface proteins CEACAMs1, 6, and 8, CD177, CD63, and CD89 are strongly associated with severe COVID-19, corresponding to the emergence of atypical CD3+CD4+CEACAM1/6/8+CD177+CD63+CD89+ and CD16+CEACAM1/6/8+ mononuclear cells. Utilization of these markers may facilitate real-time patient assessment by flow cytometry and identify immune populations that could be targeted to ameliorate immunopathology.

Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.

Altered plasma membrane abundance of the sulfatide-binding protein NF155 links glycosphingolipid imbalances to demyelination.

Myelin is a multilayered membrane that tightly wraps neuronal axons, enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signaling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule neurofascin (NFASC), a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the NFASC isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.

The influence of elastic ankle exoskeletons on lower limb mechanical energetics during unexpected perturbations.

Passive elastic ankle exoskeletons have been used to augment locomotor performance during walking, running and hopping. In this study, we aimed to determine how these passive devices influence lower limb joint and whole-body mechanical energetics to maintain stable upright hopping during rapid, unexpected perturbations. We recorded lower limb kinematics and kinetics while participants hopped with exoskeleton assistance (0, 76 and 91 Nm rad-1) on elevated platforms (15 and 20 cm) which were rapidly removed at an unknown time. Given that springs cannot generate nor dissipate energy, we hypothesized that passive ankle exoskeletons would reduce stability during an unexpected perturbation. Our results demonstrate that passive exoskeletons lead to a brief period of instability during unexpected perturbations - characterized by increased hop height. However, users rapidly stabilize via a distal-to-proximal redistribution of joint work such that the knee performs an increased energy dissipation role and stability is regained within one hop cycle. Together, these results demonstrate that despite the inability of elastic exoskeletons to directly dissipate mechanical energy, humans can still effectively dissipate the additional energy of a perturbation, regain stability and recover from a rapid unexpected vertical perturbation to maintain upright hopping.

Early HIV-1 Gag Assembly on Lipid Membrane with vRNA.

Mass photometry (MP) was used to investigate the assembly of myristoylated full-length HIV-1 Gag (myr-Gag) and vRNA 5’ UTR fragment in a supported lipid bilayer (SLB) model system. The MP trajectories demonstrated that Gag trimerization on the membrane is a key step of early Gag assembly in the presence of vRNA. Growth of myr-Gag oligomers requires vRNA, occuring by addition of 1 or 2 monomers at a time from solution. These data support a model where formation of the Gag hexamers characteristic of the immature capsid lattice occurs by a gradual edge expansion, following a trimeric nucleation event. These dynamic single molecule data involving protein, RNA, and lipid components together, provide novel and fundamental insights into the initiation of virus capsid assembly.

Achalasia: investigation and management.

Achalasia, characterised by the absence of peristalsis and failure of relaxation of the lower oesophageal sphincter, is an uncommon degenerative condition that results in dysphagia. If left untreated it can lead to aspiration, oesophageal perforation, oesophagitis and malnutrition. It has a range of immune, allergic, viral and genetic aetiological causes. Successful diagnosis relies on the use of oesophagogastroduodenoscopy, barium swallow and oesophageal manometry to characterise the severity of the disease and to rule out underlying malignancy. Although no treatment can reverse the degenerative process, therapeutic strategies including lifestyle modification, medication, endoscopic and operative intervention can help to reduce symptoms. This article reviews the latest methods used to investigate and manage achalasia.

Quantitative proteomic analysis of SARS-CoV-2 infection of primary human airway ciliated cells and lung epithelial cells demonstrates the effectiveness of SARS-CoV-2 innate immune evasion.

Background: Quantitative proteomics is able to provide a comprehensive, unbiased description of changes to cells caused by viral infection, but interpretation may be complicated by differential changes in infected and uninfected 'bystander' cells, or the use of non-physiological cellular models. Methods: In this paper, we use fluorescence-activated cell sorting (FACS) and quantitative proteomics to analyse cell-autonomous changes caused by authentic SARS-CoV-2 infection of respiratory epithelial cells, the main target of viral infection in vivo. First, we determine the relative abundance of proteins in primary human airway epithelial cells differentiated at the air-liquid interface (basal, secretory and ciliated cells). Next, we specifically characterise changes caused by SARS-CoV-2 infection of ciliated cells. Finally, we compare temporal proteomic changes in infected and uninfected 'bystander' Calu-3 lung epithelial cells and compare infection with B.29 and B.1.1.7 (Alpha) variants. Results: Amongst 5,709 quantified proteins in primary human airway ciliated cells, the abundance of 226 changed significantly in the presence of SARS-CoV-2 infection (q <0.05 and >1.5-fold). Notably, viral replication proceeded without inducing a type-I interferon response. Amongst 6,996 quantified proteins in Calu-3 cells, the abundance of 645 proteins changed significantly in the presence of SARS-CoV-2 infection (q < 0.05 and > 1.5-fold). In contrast to the primary cell model, a clear type I interferon (IFN) response was observed. Nonetheless, induction of IFN-inducible proteins was markedly attenuated in infected cells, compared with uninfected 'bystander' cells. Infection with B.29 and B.1.1.7 (Alpha) variants gave similar results. Conclusions: Taken together, our data provide a detailed proteomic map of changes in SARS-CoV-2-infected respiratory epithelial cells in two widely used, physiologically relevant models of infection. As well as identifying dysregulated cellular proteins and processes, the effectiveness of strategies employed by SARS-CoV-2 to avoid the type I IFN response is illustrated in both models.

EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling.

EROS (essential for reactive oxygen species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease, but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58 kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions, and P2X7 is almost absent in EROS-deficient mouse and human primary cells. Accordingly, lack of murine EROS results in markedly abnormal P2X7 signalling, inflammasome activation, and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection in mice. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation, and possibly gene therapy.

Using Body-worn Accelerometers to Detect Physiological Changes During Periods of Blast Overpressure Exposure.

Repetitive exposure to non-concussive blast expo-sure may result in sub-clinical neurological symptoms. These changes may be reflected in the neural control gait and balance. In this study, we collected body-worn accelerometry data on individuals who were exposed to repetitive blast overpressures as part of their occupation. Accelerometry features were gener-ated within periods of low-movement and gait. These features were the eigenvalues of high-dimensional correlation matrices, which were constructed with time-delay embedding at multiple delay scales. When focusing on the gait windows, there were significant correlations of the changes in features with the cumulative dose of blast exposure. When focusing on the low-movement frames, the correlation with exposure were lower than that of the gait frames and statistically insignificant. In a cross-validated model, the overpressure exposure was predicted from gait features alone. The model was statistically significant and yielded an RMSE of 1.27 dB. With continued development, the model may be used to assess the physiological effects of repetitive blast exposure and guide training procedures to minimize impact on the individual.

Impact of routine reporting of estimated glomerular filtration rate using the European Kidney Function Consortium and Chronic Kidney Disease Epidemiology Collaboration equations in a Western Australian community population.

AIM: In 2020, the European Kidney Function Consortium (EKFC) published a new creatinine-based equation to estimate glomerular filtration rate (eGFR) to overcome known limitations in existing equations. The aim of this study is to model the potential impact on service referral and health expenditure of routine reporting of eGFR using the EKFC equation as compared to the CKD-EPI equation in a Western Australian population. METHODS: eGFR was calculated for 760 614 patients with 2 368 234 creatinine results using the CKD-EPI and EKFC formulas. Patients were grouped into a CKD cohort if they had at least two eGFR results of <60 ml/min/1.73 m2 from tests at least 90 days apart. The impact of each equation on the reclassification of CKD stages, CKD cohort classification, the rate of change in eGFR and direct health costs were assessed. RESULTS: About 90.66% of patients had a lower eGFR when calculated using the EKFC equation. About 12.6% of individuals were classified into a different CKD stage using the EKFC equation with 97.43% of these patients classified into a higher (more advanced) stage. There was a 25.9% increase in the number of patients identified as having CKD when calculated using the EKFC equation. Direct health costs also increased with the use of EKFC reporting. CONCLUSION: Use of the EKFC equation will increase population prevalence of CKD and will result in a shift to higher stages of CKD. This has implications for monitoring and referral of patients within specialist services and has the potential to increase the need for multidisciplinary care.