Cost-benefit analysis of cryogenic electron tomography subtomogram averaging of chaperonin MmCpn at near atomic resolution.

Cryogenic electron microscopy single particle analysis (cryoEM-SPA) has evolved into a routine approach for determining macromolecule structures to near-atomic resolution. Cryogenic electron tomography subtomogram averaging (cryoET-STA) toward a similar resolution, in contrast, is still under active development. Here, we use the archeal chaperonin MmCpn as a model macromolecule to quantitatively investigate the resolution limiting factors of cryoET-STA in terms of cumulative electron dose, ice thickness, subtomogram numbers, and tilt angle ranges.

Visualizing nucleation, condensation and propagation of β-tubulin folding in chaperonin TRiC.

The folding nucleus (FN) initiates protein folding and enables an efficient folding pathway. Here we directly visualize the tubulin FN consisting of a nonnative, partially assembled Rossmann fold, in the closed chamber of human chaperonin TRiC. Chaperonin TRiC interacts with non-natively folded secondary structural elements, stabilizing the nucleus for transition into its first native domain.

Imaging high-frequency voltage dynamics in multiple neuron classes of behaving mammals.

Fluorescent genetically encoded voltage indicators report transmembrane potentials of targeted cell-types. However, voltage-imaging instrumentation has lacked the sensitivity to track spontaneous or evoked high-frequency voltage oscillations in neural populations. Here we describe two complementary TEMPO voltage-sensing technologies that capture neural oscillations up to ~100 Hz.

Neural circuit basis of placebo pain relief.

Placebo effects are notable demonstrations of mind-body interactions. During pain perception, in the absence of any treatment, an expectation of pain relief can reduce the experience of pain-a phenomenon known as placebo analgesia. However, despite the strength of placebo effects and their impact on everyday human experience and the failure of clinical trials for new therapeutics, the neural circuit basis of placebo effects has remained unclear.

Hub stability in the calcium calmodulin-dependent protein kinase II.

The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within CaMKII isoforms, but the link between polymorphism and subunit exchange has not been resolved. Here, we present near-atomic resolution cryogenic electron microscopy (cryo-EM) structures revealing that hubs from the α and β isoforms, either standalone or within an β holoenzyme, coexist as 12 and 14 subunit assemblies.

The advent of preventive high-resolution structural histopathology by artificial-intelligence-powered cryogenic electron tomography.

Advances in cryogenic electron microscopy (cryoEM) single particle analysis have revolutionized structural biology by facilitating the determination of atomic- and near-atomic-resolution structures for fully hydrated macromolecular complexes exhibiting compositional and conformational heterogeneity across a wide range of sizes. Cryogenic electron tomography (cryoET) and subtomogram averaging are rapidly progressing toward delivering similar insights for macromolecular complexes , without requiring tags or harsh biochemical purification. Furthermore, cryoET enables the visualization of cellular and tissue phenotypes directly at molecular, nanometric resolution without chemical fixation or staining artifacts.

Cryogenic electron tomography reveals novel structures in the apical complex of .

Unlabelled: Intracellular infectious agents, like the malaria parasite, , face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion.

Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses.

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses.

Tertiary folds of the SL5 RNA from the 5' proximal region of SARS-CoV-2 and related coronaviruses.

Coronavirus genomes sequester their start codons within stem-loop 5 (SL5), a structured, 5' genomic RNA element. In most alpha- and betacoronaviruses, the secondary structure of SL5 is predicted to contain a four-way junction of helical stems, some of which are capped with UUYYGU hexaloops. Here, using cryogenic electron microscopy (cryo-EM) and computational modeling with biochemically-determined secondary structures, we present three-dimensional structures of SL5 from six coronaviruses.

RNA target highlights in CASP15: Evaluation of predicted models by structure providers.

The first RNA category of the Critical Assessment of Techniques for Structure Prediction competition was only made possible because of the scientists who provided experimental structures to challenge the predictors. In this article, these scientists offer a unique and valuable analysis of both the successes and areas for improvement in the predicted models. All 10 RNA-only targets yielded predictions topologically similar to experimentally determined structures.

Wide-field fluorescence lifetime imaging of neuron spiking and subthreshold activity in vivo.

The development of voltage-sensitive fluorescent probes suggests fluorescence lifetime as a promising readout for electrical activity in biological systems. Existing approaches fail to achieve the speed and sensitivity required for voltage imaging in neuroscience applications. We demonstrated that wide-field electro-optic fluorescence lifetime imaging microscopy (EO-FLIM) allows lifetime imaging at kilohertz frame-acquisition rates, spatially resolving action potential propagation and subthreshold neural activity in live adult .

Utility of balanced steady-state field precession sequence in the evaluation of retinal and subdural hemorrhages in patients with abusive head trauma.

Abusive head trauma is the leading cause of physical child abuse deaths in children under 5 years of age in the United States. To evaluate suspected child abuse, radiologic studies are typically the first to identify hallmark findings of abusive head trauma including intracranial hemorrhage, cerebral edema, and ischemic injury. Prompt evaluation and diagnosis are necessary as findings may change rapidly.

Tumor protease-activated theranostic nanoparticles for MRI-guided glioblastoma therapy.

As a cancer, Glioblastoma (GBM) is a highly lethal and difficult-to-treat. With the aim of improving therapies to GBM, we developed novel and target-specific theranostic nanoparticles (TNPs) that can be selectively cleaved by cathepsin B (Cat B) to release the potent toxin monomethyl auristatin E (MMAE). We synthesized TNPs composed of a ferumoxytol-based nanoparticle carrier and a peptide prodrug with a Cat-B-responsive linker and the tubulin inhibitor MMAE.

CryoET reveals organelle phenotypes in huntington disease patient iPSC-derived and mouse primary neurons.

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. While experiments with patient-derived induced pluripotent stem cells (iPSCs) can help understand disease, defining pathological biomarkers remains challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice.

Dual-polarity voltage imaging of the concurrent dynamics of multiple neuron types.

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between targeted cell populations. However, current indicators lack the requisite sensitivity for in vivo multipopulation imaging. We describe next-generation green and red voltage sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants pAce and pAceR.

Near-Atomic Resolution Cryo-EM Image Reconstruction of RNA.

The rapid development of cryogenic electron microscopy (cryo-EM) enables the structure determination of macromolecules without the need for crystallization. Protein, protein-lipid, and protein-nucleic acid complexes can now be routinely resolved by cryo-EM single-particle analysis (SPA) to near-atomic or atomic resolution. Here we describe the structure determination of pure RNAs by SPA, from cryo-specimen preparation to data collection and 3D reconstruction.

Efficient manual annotation of cryogenic electron tomograms using IMOD.

Annotation highlights and segmentation isolates features in cryogenic electron tomograms to improve visualization and quantification of features (for example, their size and abundance, and spatial relationships with other features), facilitating phenotypic structural analyses of cellular tomograms. Here, we present a manual annotation protocol using the open-source software IMOD and describe segmentation of three types of common cellular features: membranes, large globules, and filaments. IMOD's interpolation function can improve the speed of manual annotation up to an order of magnitude.

Validation of the Reduced Unified Continuum Formulation Against In Vitro 4D-Flow MRI.

We previously introduced and verified the reduced unified continuum formulation for vascular fluid-structure interaction (FSI) against Womersley's deformable wall theory. Our present work seeks to investigate its performance in a patient-specific aortic setting in which assumptions of idealized geometries and velocity profiles are invalid. Specifically, we leveraged 2D magnetic resonance imaging (MRI) and 4D-flow MRI to extract high-resolution anatomical and hemodynamic information from an in vitro flow circuit embedding a compliant 3D-printed aortic phantom.

Chikungunya virus assembly and budding visualized in situ using cryogenic electron tomography.

Chikungunya virus (CHIKV) is a representative alphavirus causing debilitating arthritogenic disease in humans. Alphavirus particles assemble into two icosahedral layers: the glycoprotein spike shell embedded in a lipid envelope and the inner nucleocapsid (NC) core. In contrast to matrix-driven assembly of some enveloped viruses, the assembly/budding process of two-layered icosahedral particles remains poorly understood.

Planar 2D wireframe DNA origami.

Two-dimensional (2D) DNA origami is widely used for applications ranging from excitonics to single-molecule biophysics. Conventional, single-layer 2D DNA origami exhibits flexibility and curvature in solution; however, that may limit its suitability as a 2D structural template. In contrast, 2D wireframe DNA origami rendered with six-helix bundle edges offers local control over duplex orientations with enhanced in-plane rigidity.

Emergent reliability in sensory cortical coding and inter-area communication.

Reliable sensory discrimination must arise from high-fidelity neural representations and communication between brain areas. However, how neocortical sensory processing overcomes the substantial variability of neuronal sensory responses remains undetermined. Here we imaged neuronal activity in eight neocortical areas concurrently and over five days in mice performing a visual discrimination task, yielding longitudinal recordings of more than 21,000 neurons.

Six-Month Outcomes of Reimplantation of a Coin-Sized Tibial Nerve Stimulator for the Treatment of Overactive Bladder Syndrome With Urgency Urinary Incontinence.

Objective: The eCoin (Valencia Technologies Corporation, Valencia, CA) is a battery-powered, nickel-sized and shaped neuromodulation device for the treatment of overactive bladder, and it is implanted in the lower leg in a short office or outpatient procedure under local anesthesia. A follow-on trial was conducted to evaluate the feasibility, safety, and efficacy of eCoin reimplantation.

Methods: This was a prospective, single-arm, open-label study, including 23 participants with refractory urgency urinary incontinence (UUI) who were previously participants in the eCoin clinical feasibility trial.

Colocalization of Coronary Plaque with Wall Shear Stress in Myocardial Bridge Patients.

Purpose: Patients with myocardial bridges (MBs) have a higher prevalence of atherosclerosis. Wall shear stress (WSS) has previously been correlated with plaque in coronary artery disease patients, but such correlations have not been investigated in symptomatic MB patients. The aim of this paper was to use a multi-scale computational fluid dynamics (CFD) framework to simulate hemodynamics in MB patient, and investigate the co-localization of WSS and plaque.