Misconceptions in IONM Part III: Stimulation Repetition Rate Effects on Intraoperative Somatosensory Evoked Potential Amplitude and Latency.

The rate at which stimulation is applied to peripheral nerves is critical to generating high-quality intraoperative somatosensory evoked potentials (SSEPs) in a timely manner. Guidelines based on a limited study and anecdotal evidence present differing, incorrect, or incomplete stimulation rate recommendations. We examined the effect stimulating the ulnar and tibial nerves at 1.05, 2.79, 5.69, and 8.44 Hz had on cortical, subcortical, and peripheral response amplitude and latency in 10 subjects with neuromuscular blockade (NMB) and 10 without NMB in the operating room under general anesthesia. As the stimulation repetition rate increased, the amplitude of upper and lower extremity cortical responses decreased equally in both groups. The ulnar nerve N20 cortical response amplitude decreased 27.9% at 2.79 Hz, 48.8% at 5.69 Hz, and 53.8% at 8.44 Hz. The tibial nerve P37 cortical response amplitude decreased 30.3% at 2.79 Hz, 53.8% at 5.69 Hz, and 56.8% at 8.44 Hz. Neither upper or lower extremity peripheral or subcortical amplitudes nor upper and lower extremity subcortical or peripheral latencies were affected by increasing repetition rate in either group. Low SSEP stimulation repetition rates ensure the highest quality cortical responses.

SARS-CoV-2 variants of concern: spike protein mutational analysis and epitope for broad neutralization.

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we describe an antibody fragment (VH ab6) that neutralizes all major variants including the recently emerged BA.1 and BA.2 Omicron subvariants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within previously emerged variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.

Three-Dimensional Visualization of Viral Structure, Entry, and Replication Underlying the Spread of SARS-CoV-2.

The global spread of SARS-CoV-2 has proceeded at an unprecedented rate. Remarkably, characterization of the virus using modern tools in structural biology has also progressed at exceptional speed. Advances in electron-based imaging techniques, combined with decades of foundational studies on related viruses, have enabled the research community to rapidly investigate structural aspects of the novel coronavirus from the level of individual viral proteins to imaging the whole virus in a native context. Here, we provide a detailed review of the structural biology and pathobiology of SARS-CoV-2 as it relates to all facets of the viral life cycle, including cell entry, replication, and three-dimensional (3D) packaging based on insights obtained from X-ray crystallography, cryo-electron tomography, and single-particle cryo-electron microscopy. The structural comparison between SARS-CoV-2 and the related earlier viruses SARS-CoV and MERS-CoV is a common thread throughout this review. We conclude by highlighting some of the outstanding unanswered structural questions and underscore areas that are under rapid current development such as the design of effective therapeutics that block viral infection.

Structural and biochemical rationale for enhanced spike protein fitness in delta and kappa SARS-CoV-2 variants.

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a structural head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation and with unknown biological implications. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

SARS-CoV-2 Omicron variant: Antibody evasion and cryo-EM structure of spike protein-ACE2 complex.

The newly reported Omicron variant is poised to replace Delta as the most prevalent SARS-CoV-2 variant across the world. Cryo-EM structural analysis of the Omicron variant spike protein in complex with human ACE2 reveals new salt bridges and hydrogen bonds formed by mutated residues R493, S496 and R498 in the RBD with ACE2. These interactions appear to compensate for other Omicron mutations such as K417N known to reduce ACE2 binding affinity, resulting in similar biochemical ACE2 binding affinities for Delta and Omicron variants. Neutralization assays show that pseudoviruses displaying the Omicron spike protein exhibit increased antibody evasion. The increase in antibody evasion, together with retention of strong interactions at the ACE2 interface, thus represent important molecular features that likely contribute to the rapid spread of the Omicron variant.

Structural analysis of receptor binding domain mutations in SARS-CoV-2 variants of concern that modulate ACE2 and antibody binding.

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Beta (B.1.351) and Gamma (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the Alpha (B.1.1.7) variant that enhances affinity of the spike protein for its receptor, angiotensin-converting enzyme 2 (ACE2). Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Epsilon variants (B.1.427/429) lack the N501Y mutation yet exhibit antibody evasion. We have engineered spike proteins to express these receptor binding domain (RBD) VoC mutations either in isolation or in different combinations and analyze the effects using biochemical assays and cryoelectron microscopy (cryo-EM) structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

SARS-CoV-2 Omicron Variant: ACE2 Binding, Cryo-EM Structure of Spike Protein-ACE2 Complex and Antibody Evasion

The newly reported Omicron variant is poised to replace Delta as the most rapidly spread SARS-CoV-2 variant across the world. Cryo-EM structural analysis of the Omicron variant spike protein in complex with human ACE2 reveals new salt bridges and hydrogen bonds formed by mutated residues R493, S496 and R498 in the RBD with ACE2. These interactions appear to compensate for other Omicron mutations such as K417N known to reduce ACE2 binding affinity, explaining our finding of similar biochemical ACE2 binding affinities for Delta and Omicron variants. Neutralization assays show that pseudoviruses displaying the Omicron spike protein exhibit increased antibody evasion, with greater evasion observed in sera obtained from unvaccinated convalescent patients as compared to doubly vaccinated individuals (8-vs 3-fold). The retention of strong interactions at the ACE2 interface and the increase in antibody evasion are molecular factors that likely contribute to the increased transmissibility of the Omicron variant.

Emerging SARS-CoV-2 Variants of Concern: Spike Protein Mutational Analysis and Epitope for Broad Neutralization

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we report the discovery of a novel antibody fragment (VH ab6) that neutralizes all major variants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.

Type I Interferon signaling controls the accumulation and transcriptomes of monocytes in the aged lung.

Aging is paradoxically associated with a deteriorated immune defense (immunosenescence) and increased basal levels of tissue inflammation (inflammaging). The lung is particularly sensitive to the effects of aging. The immune cell mechanisms underlying physiological lung aging remain poorly understood. Here we reveal that aging leads to increased interferon signaling and elevated concentrations of chemokines in the lung, which is associated with infiltration of monocytes into the lung parenchyma. scRNA-seq identified a novel Type-1 interferon signaling dependent monocyte subset (MO-ifn) that upregulated IFNAR1 expression and exhibited greater transcriptomal changes with aging than the other monocytes. Blockade of type-1 interferon signaling by treatment with anti-IFNAR1 neutralizing antibodies rapidly ablated MO-ifn cells. Treatment with anti-IFNAR1 antibodies also reduced airway chemokine concentrations and repressed the accumulation of the overall monocyte population in the parenchyma of the aged lung. Together, our work suggests that physiological aging is associated with increased basal level of airway monocyte infiltration and inflammation in part due to elevated type-1 interferon signaling.

Structural and Biochemical Rationale for Enhanced Spike Protein Fitness in Delta and Kappa SARS-CoV-2 Variants

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a novel head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding

The recently emerged SARS-CoV-2 South African (B. 1.351) and Brazil/Japan (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the UK variant that enhances affinity of the spike protein for its receptor, ACE2. Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Californian VoCs (B.1.427/429) lack the N501Y mutation, yet exhibit antibody evasion. We engineered spike proteins to express these RBD VoC mutations either in isolation, or in different combinations, and analyzed the effects using biochemical assays and cryo-EM structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer either increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

Group 2 innate lymphoid cells are numerically and functionally deficient in the triple transgenic mouse model of Alzheimer's disease.

BACKGROUND: The immune pathways in Alzheimer's disease (AD) remain incompletely understood. Our recent study indicates that tissue-resident group 2 innate lymphoid cells (ILC2) accumulate in the brain barriers of aged mice and that their activation alleviates aging-associated cognitive decline. The regulation and function of ILC2 in AD, however, remain unknown. METHODS: In this study, we examined the numbers and functional capability of ILC2 from the triple transgenic AD mice (3xTg-AD) and control wild-type mice. We investigated the effects of treatment with IL-5, a cytokine produced by ILC2, on the cognitive function of 3xTg-AD mice. RESULTS: We demonstrate that brain-associated ILC2 are numerically and functionally defective in the triple transgenic AD mouse model (3xTg-AD). The numbers of brain-associated ILC2 were greatly reduced in 7-month-old 3xTg-AD mice of both sexes, compared to those in age- and sex-matched control wild-type mice. The remaining ILC2 in 3xTg-AD mice failed to efficiently produce the type 2 cytokine IL-5 but gained the capability to express a number of proinflammatory genes. Administration of IL-5, a cytokine produced by ILC2, transiently improved spatial recognition and learning in 3xTg-AD mice. CONCLUSION: Our results collectively indicate that numerical and functional deficiency of ILC2 might contribute to the cognitive impairment of 3xTg-AD mice.

Experimental Investigation and Control of a Hot-Air Tunnel with Improved Performance and Energy Saving.

The paper is focused on the identification, control design, and experimental verification of a two-input two-output hot-air laboratory apparatus representing a small-scale version of appliances widely used in the industry. A decentralized multivariable controller design is proposed, satisfying control-loop decoupling and measurable disturbance rejection. The proposed inverted or equivalent noninverted decoupling controllers serve for the rejection of cross-interactions in controlled loops, whereas open-loop antidisturbance members satisfy the absolute invariance to the disturbances. Explicit controller-structure design formulae are derived, and their equivalence to other decoupling schemes is proven. Three tuning rules are used to set primary controller parameters, which are further discretized. All the control responses are simulated in the Matlab/Simulink environment. In the experimental part, two data-acquisition, communication, and control interfaces are set up. Namely, a programmable logic controller and a computer equipped with the peripheral component interconnect card commonly used in industrial practice are implemented. A simple supervisory control and data acquisition human-machine interface via the Control Web environment is developed. The laboratory experiments prove better temperature control performance measured by integral criteria by 35.3%, less energy consumption by up to 6%, and control effort of mechanical actuator parts by up to 17.1% for our method compared to the coupled or disturbance-ignoring design in practice. It was also observed that the use of a programmable logic controller gives better performance measures for both temperature and air-flow control.

Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies.

The recently reported "UK variant" (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.

Emerging Trends in Metalloid-Dependent Signaling in Plants.

Metalloids are semiconducting elements that constitute a small group in the periodic table of elements. Their occurrence in nature either poses an environmental threat or benefit to plants. The precise mechanisms or manner of crosstalk of metalloid interference and sensing remain open questions. Standard plant nutrient solutions contain the metalloid boron (B) as a micronutrient, while silicon (Si) is considered a beneficial element routinely supplied only to some plants such as grasses. By contrast, arsenic (As) is a severe environmental hazard to most organisms, including plants, while the less abundant metalloids germanium (Ge), antimony (Sb), and tellurium (Te) display variable degrees of toxicity. Here we review the molecular events and mechanisms that could explain the contrasting (or overlapping) action of metalloids on the cell and cell signaling.

Remodeling of Root Growth Under Combined Arsenic and Hypoxia Stress Is Linked to Nutrient Deprivation.

Root architecture responds to environmental stress. Stress-induced metabolic and nutritional changes affect the endogenous root development program. Transcriptional and translational changes realize the switch between stem cell proliferation and cell differentiation, lateral root or root hair formation and root functionality for stress acclimation. The current work explores the effects of stress combination of arsenic toxicity (As) and hypoxia (Hpx) on root development in Arabidopsis thaliana. As revealed previously, combined As and Hpx treatment leads to severe nutritional disorder evident from deregulation of root transcriptome and plant mineral contents. Both As and Hpx were identified to pose stress-specific constraints on root development that lead to unique root growth phenotype under their combination. Besides inhibition of root apical meristem (RAM) activity under all stresses, As induced lateral root growth while root hair density and lengths were strongly increased by Hpx and HpxAs-treatments. A dual stimulation of phosphate (Pi)-starvation response was observed for HpxAs-treated plant roots; however, the response under HpxAs aligned more with Hpx than As. Transcriptional evidence along with biochemical data suggests involvement of PHOSPHATE STARVATION RESPONSE 1; PHR1-dependent systemic signaling. Pi metabolism-related transcripts in close association with cellular iron homeostasis modulate root development under HpxAs. Early redox potential changes in meristematic cells, differential ROS accumulation in root hair zone cell layers and strong deregulation of NADPH oxidases, NADPH-dependent oxidoreductases and peroxidases signify a role of redox and ROS signaling in root architecture remodeling under HpxAs. Differential aquaporin expression suggests transmembrane ROS transport to regulate root hair induction and growth. Reorganization of energy metabolism through NO-dependent alternate oxidase, lactate fermentation, and phosphofructokinase seems crucial under HpxAs. TOR and SnRK-signaling network components were potentially involved in control of sustainable utilization of available energy reserves for root hair growth under combined stress as well as recovery on reaeration. Findings are discussed in context of combined stress-induced signaling in regulation of root development in contrast to As and Hpx alone.

High Potency of a Bivalent Human VH Domain in SARS-CoV-2 Animal Models.

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VH-Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.

Depletion of NK Cells Improves Cognitive Function in the Alzheimer Disease Mouse Model.

Despite mounting evidence suggesting the involvement of the immune system in regulating brain function, the specific role of immune and inflammatory cells in neurodegenerative diseases remain poorly understood. In this study, we report that depletion of NK cells, a type of innate lymphocytes, alleviates neuroinflammation, stimulates neurogenesis, and improves cognitive function in a triple-transgenic Alzheimer disease (AD) mouse model. NK cells in the brains of triple-transgenic AD mouse model (3xTg-AD) mice exhibited an enhanced proinflammatory profile. Depletion of NK cells by anti-NK1.1 Abs drastically improved cognitive function of 3xTg-AD mice. NK cell depletion did not affect amyloid ß concentrations but enhanced neurogenesis and reduced neuroinflammation. Notably, in 3xTg-AD mice depleted of NK cells, microglia demonstrated a homeostatic-like morphology, decreased proliferative response and reduced expression of neurodestructive proinflammatory cytokines. Together, our results suggest a proinflammatory role for NK cells in 3xTg-AD mice and indicate that targeting NK cells might unlock novel strategies to combat AD.

Early coagulopathy in children with isolated blunt head injury is associated with mortality and poor neurological outcomes.

OBJECTIVE: Traumatic brain injury (TBI) is the leading cause of long-term disability and death in children and adolescents globally. Long-term adverse outcomes, including physical, cognitive, and behavioral sequelae, have been reported after TBI in a significant number of pediatric patients. In this study the authors sought to investigate the epidemiology of TBI-associated coagulopathy and its association with mortality and poor neurological outcome in a pediatric population with isolated moderate to severe blunt head injury treated at the authors' institution. METHODS: This retrospective study was conducted in the children's emergency department between January 2010 and December 2016. Children < 18 years old who presented with isolated moderate to severe blunt head injury were included in the study. The authors collected data on patient demographics, clinical presentation, and TBI management. Outcomes studied were death and poor neurological outcome defined by a score of < 7 (death, moderate to severe neurological disability) at 6 months postinjury on the pediatric version of the Glasgow Outcome Scale-Extended (GOS-E Peds). RESULTS: In 155 pediatric patients who presented with isolated moderate to severe blunt head injury, early coagulopathy was observed in 33 (21.3%) patients during the initial blood investigations done in the emergency department. The mean (SD) age of the study group was 7.03 (5.08) years and the predominant mechanism of injury was fall from height (65.2%). The median Abbreviated Injury Scale of the head (AIS head) score was 4 and the median GCS score was 13 (IQR 12-15). TBI-associated coagulopathy was independently associated with GOS-E Peds score < 7 (p = 0.02, adjusted OR 6.07, 95% CI 1.32-27.83). The overall mortality rate was 5.8%. After adjusting for confounders, only AIS head score and hypotension at triage remained significantly associated with TBI-associated coagulopathy. CONCLUSIONS: TBI-associated coagulopathy was independently associated with GOS-E Peds score < 7 at 6 months postinjury. Larger prospective studies are needed to investigate the use of TBI-associated coagulopathy to prognosticate these critical clinical outcomes.

Activation of group 2 innate lymphoid cells alleviates aging-associated cognitive decline.

Increasing evidence has challenged the traditional view about the immune privilege of the brain, but the precise roles of immune cells in regulating brain physiology and function remain poorly understood. Here, we report that tissue-resident group 2 innate lymphoid cells (ILC2) accumulate in the choroid plexus of aged brains. ILC2 in the aged brain are long-lived, are relatively resistant to cellular senescence and exhaustion, and are capable of switching between cell cycle dormancy and proliferation. They are functionally quiescent at homeostasis but can be activated by IL-33 to produce large amounts of type 2 cytokines and other effector molecules in vitro and in vivo. Intracerebroventricular transfer of activated ILC2 revitalized the aged brain and enhanced the cognitive function of aged mice. Administration of IL-5, a major ILC2 product, was sufficient to repress aging-associated neuroinflammation and alleviate aging-associated cognitive decline. Targeting ILC2 in the aged brain may provide new avenues to combat aging-associated neurodegenerative disorders.