Dual-task effects between tone counting and mathematical calculations.

We examined the impact of performing a tone counting task of varying cognitive loads and mathematical calculations simultaneously, compared to performance on the same tasks done individually. Participants performed continuous mathematical calculations, performed a high and a low cognitive load tone counting task, and also performed the math and counting tasks simultaneously. Performing the two tasks together resulted in significant dual-task interference. We also compared these results to previous studies employing the tone counting tasks with physically demanding tasks (climbing, kayaking and running). The interference between tone counting and mathematical calculations was worse than the interference between tone counting and running and kayaking. For climbing, the difference in interference was more nuanced with evidence indicating climbing uniquely asserts task prioritization. These findings have implications for operations requiring dual or multi-tasking.

Performance, Hemodynamics, and Stress in a Two-Day Vigilance Task: Practical and Theoretical Implications.

OBJECTIVE: To explore vigilance task performance, cerebral blood flow velocity (CBFV), workload, and stress in a within-subjects, two-session experiment. BACKGROUND: Vigilance, or sustained attention, tasks are often characterized by a decline in operator performance and CBFV with time on task, and high workload and stress. Though performance is known to improve with practice, past research has not included measures of CBFV, stress, and workload in a within-subjects multi-session design, which may also provide insight into ongoing theoretical debate. METHOD: Participants performed a vigilance task on two separate occasions. Performance, CBFV, workload, and self-reported stress were measured. RESULTS: Within each session, results were consistent with the vigilance profile found in prior research. Across sessions, performance improved but the time on task decrement remained. Mean CBFV and workload ratings did not differ between sessions, but participants reported significantly less distress, worry, and engagement after session two compared to one. CONCLUSION: Though practice may not disrupt the standard vigilance profile, it may serve to improve overall performance and reduce stress. However, repeated exposure may have negative implications for engagement and mind-wandering. APPLICATION: It is important to better understand the relationship between experience, performance, physiological response, and self-reported stress and workload in vigilance because real-world environments often require operators to do the same task over many occasions. While performance improvement and reduced distress is an encouraging result, the decline in engagement requires further research. Results across sessions fail to provide support to the mind-wandering theory of vigilance.

The impact of cognitive load on kayaking and kayaking on cognitive performance.

OBJECTIVE: We examined the impact of performing a cognitive task of varying loads and kayaking simultaneously, compared to performance on the same tasks individually. BACKGROUND: When two tasks are performed together, performance often suffers compared to performance on either task alone. Interference not only occurs in competing cognitive tasks, but has also been found with certain physically demanding activities, such as climbing. METHOD: Skilled kayakers performed a kayak course on open water, performed a high and a low cognitive load tone counting task, and also performed the kayak and counting tasks simultaneously. RESULTS: Despite some past research finding dual task facilitation with laboratory aerobic activities, simultaneous kayaking and tone counting led to dual-task interference. CONCLUSION: Concurrent counting and kayaking led to performance impairments in both tasks, relative to single task performance. APPLICATION: The present results are applicable to occupations involving concurrent demanding physical activity and cognitive task performance, such as the work of first responders and military operators. PRéCIS: Kayaking, like climbing, appears to hinder cognitive performance more than the low-risk physical tasks carried out in laboratory conditions.

Genetic screen for suppressors of increased silencing in rpd3 mutants in Saccharomyces cerevisiae identifies a potential role for H3K4 methylation.

Several studies have identified the paradoxical phenotype of increased heterochromatic gene silencing at specific loci that results from deletion or mutation of the histone deacetylase (HDAC) gene RPD3. To further understand this phenomenon, we conducted a genetic screen for suppressors of this extended silencing phenotype at the HMR locus in Saccharomyces cerevisiae. Most of the mutations that suppressed extended HMR silencing in rpd3 mutants without completely abolishing silencing were identified in the histone H3 lysine 4 methylation (H3K4me) pathway, specifically in SET1, BRE1, and BRE2. These second-site mutations retained normal HMR silencing, therefore, appear to be specific for the rpd3Δ extended silencing phenotype. As an initial assessment of the role of H3K4 methylation in extended silencing, we rule out some of the known mechanisms of Set1p/H3K4me mediated gene repression by HST1, HOS2, and HST3 encoded HDACs. Interestingly, we demonstrate that the RNA Polymerase III complex remains bound and active at the HMR-tDNA in rpd3 mutants despite silencing extending beyond the normal barrier. We discuss these results as they relate to the interplay among different chromatin-modifying enzyme functions and the importance of further study of this enigmatic phenomenon.

The impact of cognitive load on climbing and climbing on cognitive performance.

OBJECTIVE: We examined the impact of increasing cognitive load on climbing performance and the impact of climbing on concurrent cognitive task performance. BACKGROUND: Generally when two tasks are performed simultaneously performance of one or both suffers relative to performance of each alone. Such dual task decrement is not confined to competing cognitive tasks, but has also been found when one task involves demanding physical activity. METHOD: Skilled climbers performed a traverse climb alone and in combination with low and high cognitive load counting tasks, which were also performed alone. RESULTS: In more realistic physical settings, physical and cognitive tasks will interfere, unlike what some literature using laboratory physical tasks may indicate. CONCLUSION: Compared to single task (climb only) performance concurrent counting and climbing resulted in impaired performance. However, climbers appeared to prioritize climbing over cognitive task performance. APPLICATION: The results and this program of research have implications for occupations that involve concurrent demanding physical activity and cognitive task performance. PRéCIS: High risk, physical tasks in real world conditions appear to hinder cognitive performance more so than low-risk physical tasks carried out in laboratory conditions.

Combined genetic analysis of juvenile idiopathic arthritis clinical subtypes identifies novel risk loci, target genes and key regulatory mechanisms.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is the most prevalent form of juvenile rheumatic disease. Our understanding of the genetic risk factors for this disease is limited due to low disease prevalence and extensive clinical heterogeneity. The objective of this research is to identify novel JIA susceptibility variants and link these variants to target genes, which is essential to facilitate the translation of genetic discoveries to clinical benefit. METHODS: We performed a genome-wide association study (GWAS) in 3305 patients and 9196 healthy controls, and used a Bayesian model selection approach to systematically investigate specificity and sharing of associated loci across JIA clinical subtypes. Suggestive signals were followed-up for meta-analysis with a previous GWAS (2751 cases/15 886 controls). We tested for enrichment of association signals in a broad range of functional annotations, and integrated statistical fine-mapping and experimental data to identify target genes. RESULTS: Our analysis provides evidence to support joint analysis of all JIA subtypes with the identification of five novel significant loci. Fine-mapping nominated causal single nucleotide polymorphisms with posterior inclusion probabilities ≥50% in five JIA loci. Enrichment analysis identified RELA and EBF1 as key transcription factors contributing to disease risk. Our integrative approach provided compelling evidence to prioritise target genes at six loci, highlighting mechanistic insights for the disease biology and IL6ST as a potential drug target. CONCLUSIONS: In a large JIA GWAS, we identify five novel risk loci and describe potential function of JIA association signals that will be informative for future experimental works and therapeutic strategies.

Characterisation of CD4+ T-cell subtypes using single cell RNA sequencing and the impact of cell number and sequencing depth.

CD4+ T-cells represent a heterogeneous collection of specialised sub-types and are a key cell type in the pathogenesis of many diseases due to their role in the adaptive immune system. By investigating CD4+ T-cells at the single cell level, using RNA sequencing (scRNA-seq), there is the potential to identify specific cell states driving disease or treatment response. However, the impact of sequencing depth and cell numbers, two important factors in scRNA-seq, has not been determined for a complex cell population such as CD4+ T-cells. We therefore generated a high depth, high cell number dataset to determine the effect of reduced sequencing depth and cell number on the ability to accurately identify CD4+ T-cell subtypes. Furthermore, we investigated T-cell signatures under resting and stimulated conditions to assess cluster specific effects of stimulation. We found that firstly, cell number has a much more profound effect than sequencing depth on the ability to classify cells; secondly, this effect is greater when cells are unstimulated and finally, resting and stimulated samples can be combined to leverage additional power whilst still allowing differences between samples to be observed. While based on one individual, these results could inform future scRNA-seq studies to ensure the most efficient experimental design.

Diversity of peripheral blood human NK cells identified by single-cell RNA sequencing.

Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2-activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16-, CD56dimCD16+CD57-, and CD56dimCD16+CD57+. CD56dimCD16+CD57- cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon-responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.

Sperm Head-Tail Linkage Requires Restriction of Pericentriolar Material to the Proximal Centriole End.

The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, we identify a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of pericentrin-like protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage.

Dual-Task Interference Between Swimming and Verbal Memory.

OBJECTIVE: A dual-task study was performed to explore the performance effects for swimming, word recall, and the combination of the two tasks performed simultaneously. BACKGROUND: Dual-task interference studies have been performed for a variety of tasks; however, there has not been much dual-task interference research where one of the tasks is a naturalistic physically strenuous task. Swimming is a unique physical task that requires spatial orientation on three dimensional axes, similar to that of flying, but has no risk of falling. Previous studies have been conducted in other activity combinations with word-free recall, such as running and climbing, but swimming has yet to be explored. METHOD: A verbal memory recall task and swimming task were performed in isolated (single-task) and simultaneous conditions. A comparison of effects across these different activities was also explored. RESULTS: Swimming and the word-recall task resulted in significant dual-task interference: almost as much as when word recall was paired with another verbal task, but more than running and less than climbing. CONCLUSION: Consistent with other dual-task studies, this study observed dual-task interference between the physical swimming task and the cognitive verbal memory task. APPLICATION: Future technologies and training for personnel who engage in water rescue or commercial diving, such as underwater welding and fiber optic cable, may be improved by these findings.

Micro-computed tomography as a platform for exploring Drosophila development.

Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering the mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here, we present an accessible method for non-invasive imaging of Drosophila melanogaster at high resolution using micro-computed tomography (µ-CT). We show how rapid processing of intact animals, at any developmental stage, provides precise quantitative assessment of tissue size and morphology, and permits analysis of inter-organ relationships. We then use µ-CT imaging to study growth defects in the Drosophila brain through the characterization of abnormal spindle (asp) and WD repeat domain 62 (Wdr62), orthologs of the two most commonly mutated genes in human microcephaly patients. Our work demonstrates the power of combining µ-CT with traditional genetic, cellular and developmental biology tools available in model organisms to address novel biological mechanisms that control animal development and disease.

Direct Targeting of the mTOR (Mammalian Target of Rapamycin) Kinase Improves Endothelial Permeability in Drug-Eluting Stents-Brief Report.

Objective- Drug-eluting stents eluting canonical mTOR (mammalian target of rapamycin) inhibitors are widely used to treat coronary artery disease but accelerate the development of atherosclerosis within the stent (neoatherosclerosis)-a leading cause of late stent failure. We recently showed that canonical mTOR inhibitors bind FKBP12.6 (12.6-kDa FK506-binding protein 12), displace it from calcium release channels, resulting in activation of PKCα (protein kinase Cα) and dissociation of p-120-catenin (p120) from VE-CAD (vascular endothelial cadherin; promoting endothelial barrier dysfunction [EBD]). However, the relevance of these findings to drug-eluting stents remains unknown. Newer generation direct mTOR kinase inhibitors do not bind FKBP12.6 and offer the potential of improving endothelial barrier function while maintaining antirestenotic efficacy, but their actual effects are unknown. We examined the effects of 2 different pharmacological targeting strategies-canonical mTOR inhibitor everolimus and mTOR kinase inhibitors Torin-2-on EBD after stenting. Approach and Results- Using the rabbit model of stenting and a combination of Evans blue dye, confocal and scanning electron microscopy studies, everolimus-eluting stents resulted in long-term EBD compared with bare metal stents. EBD was mitigated by using stents that eluted mTOR kinase inhibitors (Torin-2-eluting stent). At 60 days after stent placement, everolimus-eluting stents demonstrated large areas of Evans blue dye staining and evidence of p120 VE-CAD dissociation consistent with EBD. These findings were absent in bare metal stents and significantly attenuated in Torin-2-eluting stent. As proof of concept of the role of EBD in neoatherosclerosis, 100 days after stenting, animals were fed an enriched cholesterol diet for an additional 30 days. Everolimus-eluting stents demonstrated significantly more macrophage infiltration (consistent with neoatherosclerosis) compared with both bare metal stents and Torin-2-eluting stent. Conclusions- Our results pinpoint interactions between FKBP12.6 and canonical mTOR inhibitors as a major cause of vascular permeability and neoatherosclerosis, which can be overcome by using mTOR kinase inhibitors. Our study suggests further refinement of molecular targeting of the mTOR complex may be a promising strategy (Graphic Abstract).

A Pilot Investigation of Individual and Dyad Instructional Arrangements.

An essential goal for individuals with autism spectrum disorder (ASD) is to reach maximal independence on a variety of tasks that facilitate academic and vocational engagement and community integration. One-to-one instructional arrangements do not adequately prepare individuals with autism to function within various group contexts and limit opportunities for positive social interactions with one or more peers. Furthermore, group instructional formats have multiple benefits, including potentially increased instructional time and additional learning opportunities. The purpose of this pilot study was to evaluate the acquisition and maintenance of verbal behavior targets in individual and dyad instruction, as well as to compare levels of engagement across these instructional arrangements. Results suggest that three of the four participants acquired more targets during individual instruction, and three of the four participants maintained more targets within individual instruction. In addition, three of the four participants spent less time in instruction and more time on break during dyad instruction. These findings demonstrate the diversity of outcomes for dyad instruction for people with ASD. Directions for future research and suggestions for clinical implementation are provided.

Ossabaw Pigs With a PCSK9 Gain-of-Function Mutation Develop Accelerated Coronary Atherosclerotic Lesions: A Novel Model for Preclinical Studies.

BACKGROUND: Ossabaw pigs are unique miniature swine with genetic predisposition to develop metabolic syndrome and coronary atherosclerosis after extended periods receiving atherogenic diets. We have hypothesized that transgenic Ossabaw swine expressing chimp PCSK9 (proprotein convertase subtilisin-like/kexin type 9) containing the D374Y gain of function would develop familial hypercholesterolemia and coronary artery plaques more rapidly than Landrace swine with the same transgene. METHODS AND RESULTS: Ossabaw and Landrace PCSK9 gain-of-function founders were generated by Sleeping Beauty transposition and cloning. Histopathologic findings in the Ossabaw founder animal showed more advanced plaques and higher stenosis than in the Landrace founder, underscoring the Ossabaw genetic predisposition to atherosclerosis. We chose to further characterize the Ossabaw PCSK9 gain-of-function animals receiving standard or atherogenic diets in a 6-month longitudinal study using computed tomography, magnetic resonance (MR) imaging, intravascular ultrasound, and optical coherence tomography, followed by pathological analysis of atherosclerosis focused on the coronary arteries. The Ossabaw model was consistently hypercholesterolemic, with or without dietary challenge, and by 6 months had consistent and diffuse fibrofatty or fibroatheromatous plaques with necrosis, overlying fibrous caps, and calcification in up to 10% of coronary plaques. CONCLUSIONS: The Ossabaw PCSK9 gain-of-function model provides consistent and robust disease development in a time frame that is practical for use in preclinical therapeutic evaluation to drive innovation. Although no animal model perfectly mimics the human condition, this genetic large-animal model is a novel tool for testing therapeutic interventions in the context of developing and advanced coronary artery disease.

CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis.

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non-foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1α and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1α via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1α/VEGF-A pathway.

Brief Report: The Genetic Profile of Rheumatoid Factor-Positive Polyarticular Juvenile Idiopathic Arthritis Resembles That of Adult Rheumatoid Arthritis.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) comprises 7 heterogeneous categories of chronic childhood arthritides. Approximately 5% of children with JIA have rheumatoid factor (RF)-positive arthritis, which phenotypically resembles adult rheumatoid arthritis (RA). Our objective was to compare and contrast the genetics of RF-positive polyarticular JIA with those of RA and selected other JIA categories, to more fully understand the pathophysiologic relationships of inflammatory arthropathies. METHODS: Patients with RF-positive polyarticular JIA (n = 340) and controls (n = 14,412) were genotyped using the Immunochip array. Single-nucleotide polymorphisms were tested for association using a logistic regression model adjusting for admixture proportions. We calculated weighted genetic risk scores (wGRS) of reported RA and JIA risk loci, and we compared the ability of these wGRS to predict RF-positive polyarticular JIA. RESULTS: As expected, the HLA region was strongly associated with RF-positive polyarticular JIA (P = 5.51 × 10-31 ). Nineteen of 44 RA risk loci and 6 of 27 oligoarticular/RF-negative polyarticular JIA risk loci were associated with RF-positive polyarticular JIA (P < 0.05). The RA wGRS predicted RF-positive polyarticular JIA (area under the curve [AUC] 0.71) better than did the oligoarticular/RF-negative polyarticular JIA wGRS (AUC 0.59). The genetic profile of patients with RF-positive polyarticular JIA was more similar to that of RA patients with age at onset 16-29 years than to that of RA patients with age at onset ≥70 years. CONCLUSION: RF-positive polyarticular JIA is genetically more similar to adult RA than to the most common JIA categories and thus appears to be a childhood-onset presentation of autoantibody-positive RA. These findings suggest common disease mechanisms, which could lead to novel therapeutic targets and shared treatment strategies.

Accumulation of Ubiquitin and Sequestosome-1 Implicate Protein Damage in Diacetyl-Induced Cytotoxicity.

Inhaled diacetyl vapors are associated with flavorings-related lung disease, a potentially fatal airway disease. The reactive α-dicarbonyl group in diacetyl causes protein damage in vitro. Dicarbonyl/l-xylulose reductase (DCXR) metabolizes diacetyl into acetoin, which lacks this α-dicarbonyl group. To investigate the hypothesis that flavorings-related lung disease is caused by in vivo protein damage, we correlated diacetyl-induced airway damage in mice with immunofluorescence for markers of protein turnover and autophagy. Western immunoblots identified shifts in ubiquitin pools. Diacetyl inhalation caused dose-dependent increases in bronchial epithelial cells with puncta of both total ubiquitin and K63-ubiquitin, central mediators of protein turnover. This response was greater in Dcxr-knockout mice than in wild-type controls inhaling 200 ppm diacetyl, further implicating the α-dicarbonyl group in protein damage. Western immunoblots demonstrated decreased free ubiquitin in airway-enriched fractions. Transmission electron microscopy and colocalization of ubiquitin-positive puncta with lysosomal-associated membrane proteins 1 and 2 and with the multifunctional scaffolding protein sequestosome-1 (SQSTM1/p62) confirmed autophagy. Surprisingly, immunoreactive SQSTM1 also accumulated in the olfactory bulb of the brain. Olfactory bulb SQSTM1 often congregated in activated microglial cells that also contained olfactory marker protein, indicating neuronophagia within the olfactory bulb. This suggests the possibility that SQSTM1 or damaged proteins may be transported from the nose to the brain. Together, these findings strongly implicate widespread protein damage in the etiology of flavorings-related lung disease.

Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of ß-catenin.

Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called two-dimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on ß-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of ß-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development.