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Background: Posterior cervical fusion (PCF) with lateral mass screws is a favorable treatment option to revise a symptomatic pseudarthrosis due to reliable rates of arthrodesis; however, this technique introduces elevated risk for wound infection and hospital readmission. A tissue-sparing PCF approach involving facet fixation instrumentation reduces the rates of postoperative complications while stabilizing the symptomatic level to achieve arthrodesis; however, these outcomes have been limited to small study cohorts from individual surgeons commonly with mixed indications for treatment. Materials and Methods: One hundred and fifty cases were identified from a retrospective chart review performed by seven surgeons across six sites in the United States. All cases involved PCF revision for a pseudarthrosis at one or more levels from C3 to C7 following anterior cervical discectomy and fusion (ACDF). PCF was performed using a tissue-sparing technique with facet instrumentation. Cases involving additional supplemental fixation such as lateral mass screws, rods, wires, or other hardware were excluded. Demographics, operative notes, postoperative complications, hospital readmission, and subsequent surgical interventions were summarized as an entire cohort and according to the following risk factors: age, sex, number of levels revised, body mass index (BMI), and history of nicotine use. Results: The average age of patients at the time of PCF revision was 55 ± 11 years and 63% were female. The average BMI was 29 ± 6 kg/m2 and 19% reported a history of nicotine use. Postoperative follow-up visits were available with a median of 68 days (interquartile range = 41-209 days) from revision PCF. There were 91 1-level, 49 2-level, 8 3-level, and 2 4±-level PCF revision cases. The mean operative duration was 52 ± 3 min with an estimated blood loss of 14 ± 1.5cc. Participants were discharged an average of 1 ± 0.05 days following surgery. Multilevel treatment resulted in longer procedure times (single = 45 min, multi = 59 min, P = 0.01) but did not impact estimated blood loss (P = 0.94). Total nights in the hospital increased by 0.2 nights with multilevel treatment (P = 0.01). Sex, age, nicotine history, and BMI had no effect on recorded perioperative outcomes. There was one instance of rehospitalization due to deep-vein thrombosis, one instance of persistent pseudarthrosis at the revised level treated with ACDF, and four instances of adjacent segment disease. In patients initially treated with multilevel ACDF, revisions occurred most commonly on the caudal level (48% of revised levels), followed by the cranial (43%), and least often in the middle level (9%). Conclusions: This chart review of perioperative and safety outcomes provides evidence in support of tissue-sparing PCF with facet instrumentation as a treatment for symptomatic pseudarthrosis after ACDF. The most common locations requiring revision were the caudal and cranial levels. Operative duration and estimated blood loss were favorable when compared to open alternatives. There were no instances of postoperative wound infection, and the majority of patients were discharged the day following surgery. [ABSTRACT FROM AUTHOR]

Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and is regulated by subcellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endolysosomal markers and for the inositol lipid PI4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod, driving enhanced inflammasome activation and cytokine secretion. Together, these data suggest that NLRP3 can sense disruptions in the trafficking of endosomal cargoes, which may explain in part the spatial activation of the NLRP3 inflammasome. These data highlight mechanisms that could be exploited in the therapeutic targeting of NLRP3. Inflammation-inducing traffic jams: The NLRP3 inflammasome assembles and is activated by diverse cellular stresses to produce proinflammatory cytokines. Lee et al. investigated whether the NLRP3 inflammasome was activated by disruptions in endosomal trafficking. By analyzing various cell types, including bone marrow-derived macrophages, the authors found that NLRP3-activating stimuli resulted in perturbations in endosomal trafficking and conversely, that chemical interference with endosomal trafficking potentiated NLRP3 activation, although it was insufficient to activate NLRP3 by itself. Activated NLRP3 was recruited to endolysosomal vesicles, where it colocalized with the lipid PI4P, which the authors speculated may be the additional stimulus required for NLRP3 activation. Thus, NLRP3 senses and is activated by cellular stress resulting from disordered endosomal trafficking. —WW [ABSTRACT FROM AUTHOR]

DOI: 10.2514/1.20588 A human-in-the-loop simulation experiment was conducted at the NASA Langley Research Center's Air Traffic Operations Laboratory in an effort to comprehensively validate tools and procedures intended to enable the small aircraft transportation system higher-volume-operations concept of operations. These procedures were developed with the goal of increasing the rate of operations at nontowered, nonradar airports in near all-weather conditions. A key element of the new design is the establishment of a volume of airspace around designated airports at which pilots accept responsibility for self-separation. Flights operating at these airports are given approach-sequencing information computed by a ground-based automated system. The validation experiment was conducted to determine if a pilot could safely and proficiently fly an airplane while performing the newly developed procedures. Comparative measures of flight path error, perceived workload, and situation awareness were obtained for two types of simulated scenarios. Baseline scenarios were representative of today's system using procedure separation, in which air traffic control grants one approach or departure clearance at a time. Test scenarios represented the newly developed approach and departure procedures. Results from the experiment indicate that low-time pilots were able to fly the test procedures in a simulation environment and maintain self-separation as safely and proficiently as flying today's procedures.

DOI: 10.2514/1.20586 A multilayer approach to the prevention of conflicts due to the loss of aircraft-to-aircraft separation that relies on procedures and onboard automation was implemented as part of the Small Aircraft Transportation Systems Higher-Volume-Operations concept. The multilayer system gives pilots support and guidance during the execution of normal operations and advance warning for procedure deviations or off-nominal operations. This paper describes the major concept elements of this multilayer approach to separation assurance and conflict prevention and provides the rationale for its design. All the algorithms and functionality described in this paper were implemented in an aircraft simulation in the Air Traffic Operations Laboratory at NASA Langley Research Center and on the NASA Cirrus SR22 research aircraft.

One objective of the Small Aircraft Transportation System (SATS) Project is to increase the capacity and utilization of small non-towered, non-radar equipped airports by transferring traffic management activities to an automated system and separation responsibilities to general aviation (GA) pilots. This paper describes the development of a research multi-function display (MED) to support the interaction between pilots and an automated Airport Management Module (AMM). Preliminary results of simulation and flight tests indicate that adding the responsibility of monitoring other traffic for self-separation does not increase pilots' subjective workload levels. Pilots preferred using the enhanced MFD to execute flight procedures, reporting improved situation awareness (SA) over conventional instrument flight rules (IFR) procedures. Keywords: Small Aircraft Transportation System; SATS Higher Volume Operations; MFD and SATS; Data link and general aviation; Data link messaging

This paper summarizes findings from the Small Aircraft Transportation System Higher Volume Operations Flight Experiment. The higher volume operations concept improves efficiency at nontowered, nonradar airports in instrument meteorological conditions. The success of the higher volume operations concept is based on pilot acceptability as determined through objective and subjective assessments when compared with the procedural control operations in use today at nontowered, nonradar-controlled airfields in instrument meteorological conditions. Flight experiment data indicate that the concept is viable. The experiment, flown on a general aviation aircraft, used a subset of the Higher Volume Operations Simulation Experiment scenarios and evaluation pilots to validate the simulation experiment results. Results reveal that all 12 low-time instrument-rated pilots preferred Small Aircraft Transportation System Higher Volume Operations when compared with current procedural separation operations. These pilots also flew the higher volume operations procedures safely and proficiently without additional workload in comparison to today's system. Detailed results of pilot flight technical error and their subjective assessments of workload and situation awareness are presented. DOI: 10.2514/1.20403

The complementarity of site-directed mutagenesis and synthetic substrate analogues are used to investigate the dissociative mechanistic model for protein tyrosine kinase-catalyzed reaction and use of substrate neutral phenol in phosphorylation reaction, instead of chemically more reactive phenoxide anion.

Protein kinase chips, in which kinases are tested for their ability to phosphorylate immobilized substrates, have been developed and used to evaluate the protein kinases encoded by the yeast genome. This new technology promises to be a valuable addition to the biochemists' and cell biologists' arsenal for evaluating the substrate selectivity and function of protein kinases in cell signaling.

Paroxysmal hypnogenic dyskinesia is a rare clinical entity characterized by intermittent dystonia and choreoathetoid movements that begin exclusively during sleep, often with consciousness preserved once the patient is awakened during the episodes. They occur almost every night and are often misdiagnosed as sleeping disorders. Paroxysmal hypnogenic dyskinesia is currently known to be a form of frontal lobe epilepsy, but not in all cases. We present a 19-year-old male patient with paroxysmal hypnogenic dyskinesia who responded to antihistamines. This supports an alternative theory from 1977 (before the cases had been adequately described) that the disorder lies in dysregulation in the basal ganglia. This description now appears similar to acute dystonic reactions such as extrapyramidal symptoms from antipsychotic medications, which also respond to antihistamines. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins can signal to reorganize the actin cytoskeleton, but the mechanism is unclear. Here we report that, in tyrosine kinase Csk-deficient mouse embryonic fibroblast cells, G protein (Gβγ, Gα12, Gα13, and Gαq)-induced, and G protein-coupled receptor-induced, actin stress fiber formation was completely blocked. Reintroduction of Csk into Csk-deficent cells restored the G protein-induced actin stress fiber formation. Chemical rescue experiments with catalytic mutants of Csk demonstrated that the catalytic activity of Csk was required for this process. Furthermore, we uncovered that Gβγ can both translocate Csk to the plasma membrane and directly increase Csk kinase activity. Our genetic and biochemical studies demonstrate that Csk plays a critical role in mediating G protein signals to actin cytoskeletal reorganization. [Copyright &y& Elsevier]

Endocytosis allows cells to internalise a wide range of molecules from their environment and to maintain their plasma membrane composition. It is vital during development and for maintenance of tissue homeostasis. The ability to visualise endocytosis in vivo requires suitable assays to monitor the process. Here, we describe imaging-based assays to visualise endocytosis in the neuroepithelium of living zebrafish embryos. Injection of fluorescent tracers into the brain ventricles followed by live imaging was used to study fluid-phase or receptor-mediated endocytosis, for which we used receptor-associated protein (RAP, encoded by Lrpap1) as a ligand for low-density lipoprotein receptor-related protein (LRP) receptors. Using dual-colour imaging combined with expression of endocytic markers, it is possible to track the progression of endocytosed tracers and to monitor trafficking dynamics. Using these assays, we reveal a role for the Lowe syndrome protein Ocrl in endocytic trafficking within the neuroepithelium. We also found that the RAP-binding receptor Lrp2 (encoded by lrp2a) appears to contribute only partially to neuroepithelial RAP endocytosis. Altogether, our results provide a basis to track endocytosis within the neuroepithelium in vivo and support a role for Ocrl in this process. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.

Protein phosphorylation catalyzed by protein kinases plays a critical role in cellular signaling. Here we review several chemical approaches to understanding protein kinases and the consequences of protein phosphorylation. We discuss the design of bisubstrate analogue inhibitors based on a dissociative transition state, the development of reagents for cross-linking protein kinases with their substrates, the chemical rescue of mutant protein tyrosine kinases, and the application of expressed protein ligation to understanding protein phosphorylation.