Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells.

Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH4Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

GLUT1-mediated glucose import in B cells is critical for anaplerotic balance and humoral immunity.

Glucose uptake increases during B cell activation and antibody-secreting cell (ASC) differentiation, but conflicting findings prevent a clear metabolic profile at different stages of B cell activation. Deletion of the glucose transporter type 1 (GLUT1) gene in mature B cells (GLUT1-cKO) results in normal B cell development, but it reduces germinal center B cells and ASCs. GLUT1-cKO mice show decreased antigen-specific antibody titers after vaccination. In vitro, GLUT1-deficient B cells show impaired activation, whereas established plasmablasts abolish glycolysis, relying on mitochondrial activity and fatty acids. Transcriptomics and metabolomics reveal an altered anaplerotic balance in GLUT1-deficient ASCs. Despite attempts to compensate for glucose deprivation by increasing mitochondrial mass and gene expression associated with glycolysis, the tricarboxylic acid cycle, and hexosamine synthesis, GLUT1-deficient ASCs lack the metabolites for energy production and mitochondrial respiration, limiting protein synthesis. We identify GLUT1 as a critical metabolic player defining the germinal center response and humoral immunity.

Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling the flux of the TCA cycle.

To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and T cell-dependent as well as T cell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.

Shift-work-related sleep disruption and the risk of decline in cognitive function: The CRUISE Study.

Acute total sleep deprivation and partial sleep deprivation have negative impacts on cognitive performance. Studies in subjects who regularly experience sleep loss, however, are rare and often restricted to examination of internal sleeping disorders. To address this issue, we set up a pilot study to explore the effects of a week characterized by sleep disruption on cognitive functioning, using a case-control setting in a maritime pilot group with chronic exposure to intermittent extrinsic, work-related sleep disruption. Twenty maritime pilots (aged 30-50 years) were compared to sex- and education-matched controls with normal sleep behaviour, from the same age range. We assessed subjective and objective cognitive function, including attention, psychomotor speed, memory and executive function using the Cambridge Neuropsychological Test Automated Battery (CANTAB). Although we were able to confirm poorer sleep in maritime pilots and subjective complaints in some cognitive domains, we did not find objective cognitive deficits in the maritime pilot group compared to controls without sleep disruption. This could suggest that in this group of healthy, young maritime pilots, exposure to sleep disruption resulted in some subjective cognitive complaints, but objective deficits of cognitive function were not detected in comparison with a non-pilot control group. However, given the small sample size, the absence of an effect does not exclude the possibility that sleep disruption could result in cognitive deficits in general. Therefore, our findings have to be confirmed in future prospective studies with a larger sample size and matched controls, regarding age, education and work history.

Home-EEG assessment of possible compensatory mechanisms for sleep disruption in highly irregular shift workers - The ANCHOR study.

STUDY OBJECTIVES: While poor sleep quality has been related to increased risk of Alzheimer's disease, long-time shift workers (maritime pilots) did not manifest evidence of early Alzheimer's disease in a recent study. We explored two hypotheses of possible compensatory mechanisms for sleep disruption: Increased efficiency in generating deep sleep during workweeks (model 1) and rebound sleep during rest weeks (model 2). METHODS: We used data from ten male maritime pilots (mean age: 51.6±2.4 years) with a history of approximately 18 years of irregular shift work. Subjective sleep quality was assessed with the Pittsburgh Sleep Quality Index (PSQI). A single lead EEG-device was used to investigate sleep in the home/work environment, quantifying total sleep time (TST), deep sleep time (DST), and deep sleep time percentage (DST%). Using multilevel models, we studied the sleep architecture of maritime pilots over time, at the transition of a workweek to a rest week. RESULTS: Maritime pilots reported worse sleep quality in workweeks compared to rest weeks (PSQI = 8.2±2.2 vs. 3.9±2.0; p<0.001). Model 1 showed a trend towards an increase in DST% of 0.6% per day during the workweek (p = 0.08). Model 2 did not display an increase in DST% in the rest week (p = 0.87). CONCLUSIONS: Our findings indicated that increased efficiency in generating deep sleep during workweeks is a more likely compensatory mechanism for sleep disruption in the maritime pilot cohort than rebound sleep during rest weeks. Compensatory mechanisms for poor sleep quality might mitigate sleep disruption-related risk of developing Alzheimer's disease. These results should be used as a starting point for future studies including larger, more diverse populations of shift workers.

Effects of long-term sleep disruption on cognitive function and brain amyloid-ß burden: a case-control study.

BACKGROUND: Recent evidence indicates that disrupted sleep could contribute to the development of Alzheimer's disease by influencing the production and/or clearance of the amyloid-ß protein. We set up a case-control study to investigate the association between long-term work-induced sleep disruption, cognitive function, and brain amyloid-ß burden. METHODS: Nineteen male maritime pilots (aged 48-60 years) with chronic work-related sleep disruption and a sex-, age-, and education-matched control sample (n = 16, aged 50-60 years) with normal sleep completed the study. Primary sleep disorders were ruled out with in-lab polysomnography. Additional sleep measurements were obtained at home using actigraphy, sleep-wake logs, and a single-lead EEG device. Cognitive function was assessed with a neuropsychological test battery, sensitive to early symptomatic Alzheimer's disease. Brain amyloid-ß burden was assessed in maritime pilots using 18F-flutemetamol amyloid PET-CT. RESULTS: Maritime pilots reported significantly worse sleep quality (Pittsburgh Sleep Quality Index (PSQI) = 8.8 ± 2.9) during work weeks, compared to controls (PSQI = 3.2 ± 1.4; 95% CI 0.01 to 2.57; p = 0.049). This was confirmed with actigraphy-based sleep efficiency (86% ± 3.8 vs. 89.3% ± 4.3; 95% CI 0.43 to 6.03; p = 0.03). Home-EEG recordings showed less total sleep time (TST) and deep sleep time (DST) during work weeks compared to rest weeks (TST 318.56 (250.21-352.93) vs. TST 406.17 (340-425.98); p = 0.001; DST 36.75 (32.30-58.58) vs. DST 51.34 (48.37-69.30); p = 0.005)). There were no differences in any of the cognitive domains between the groups. For brain amyloid-ß levels, mean global cortical standard uptake value ratios of 18F-flutemetamol were all in the normal range (1.009 ± 0.059; 95% CI 0.980 to 1.037), confirmed by visual reads. CONCLUSIONS: Capitalizing on the particular work-rest schedule of maritime pilots, this study with a small sample size observed that long-term intermittent sleep disruption had no effects on global brain amyloid-ß levels or cognitive function.

B Cell Speed and B-FDC Contacts in Germinal Centers Determine Plasma Cell Output via Swiprosin-1/EFhd2.

Plasma cells secreting affinity-matured antibodies develop in germinal centers (GCs), where B cells migrate persistently and directionally over defined periods of time. How modes of GC B cell migration influence plasma cell development remained unclear. Through genetic deletion of the F-actin bundling protein Swiprosin-1/EF-hand domain family member 2 (EFhd2) and by two-photon microscopy, we show that EFhd2 restrains B cell speed in GCs and hapten-specific plasma cell output. Modeling the GC reaction reveals that increasing GC B cell speed promotes plasma cell generation. Lack of EFhd2 also reduces contacts of GC B cells with follicular dendritic cells in vivo. Computational modeling uncovers that both GC output and antibody affinity depend quantitatively on contacts of GC B cells with follicular dendritic cells when B cells migrate more persistently. Collectively, our data explain how GC B cells integrate speed and persistence of cell migration with B cell receptor affinity.

Long-Term Occupational Sleep Loss and Post-Retirement Cognitive Decline or Dementia.

INTRODUCTION: Recent evidence suggests that poor sleep is a risk factor that contributes to the development of Alzheimer's disease (AD). Most studies have focused on short-term effects of sleep deprivation on cognitive function, whereas longitudinal studies are limited to self-reported sleep and the risk of later-life dementia. Because sleep loss could be an early manifestation of neurodegenerative disease, reverse causality in these studies cannot be excluded. OBJECTIVE: In this explorative, observational study, we investigated the effects of extended periods of extrinsically (work-related) caused sleep loss on later-life cognitive function, early dementia symptoms, and current sleep quality. METHODS: We approached a community of retired male maritime pilots (approx. n = 500) through a newsletter. We investigated 50 respondents (mean age 71.7 years ± 7.7), with a history of >25 years of work on irregular schedules, which resulted in extended periods of sleep loss. Validated questionnaires on cognitive complaints (Cognitive Failure Questionnaire [CFQ]), early dementia symptoms (Early Dementia Questionnaire [EDQ]), current sleep quality (Pittsburgh Sleep Quality Index [PSQI] and sleep-wake diaries), quality of life (QoL, EQ-5D), and mood (Hospital Anxiety and Depression Scale [HADS]) were administered by a single investigator (J.T.), who also completed an observer rating of cognitive function. RESULTS: Scores on the CFQ, EDQ, PSQI, EQ-5D, and HADS were within normal ranges adjusted for age, sex, and education. The observer rating was not indicative of cognitive decline. CONCLUSION: We found no evidence that long-term exposure to work-related sleep loss had resulted in cognitive decline or early dementia symptoms in this sample of retired maritime pilots.

Sleep-Cognition Hypothesis In maritime Pilots, what is the effect of long-term work-related poor sleep on cognition and amyloid accumulation in healthy middle-aged maritime pilots: methodology of a case-control study.

INTRODUCTION: Evidence indicates a bidirectional relationship between poor sleep and Alzheimer's disease (AD). While AD may lead to disruption of normal sleep, poor sleep in itself may play a causal role in the development of AD by influencing the production and/or clearance of the amyloid-beta (Aß) protein. This led to the hypothesis that extended periods (>10 years) of sleep loss could lead to Aß accumulation with subsequent cognitive AD-related decline. This manuscript describes the methodology of the SCHIP study, a cohort study in maritime pilots that aims at investigating the relationship between prolonged work-related sleep loss, cognitive function and amyloid accumulation among healthy middle-aged maritime pilots, to test the hypothesis that prolonged sleep loss increases the risk of AD-related cognitive decline. METHODS: Our study sample consists of a group of healthy middle-aged maritime pilots (n=20), who have been exposed to highly irregular work schedules for more than 15 years. The maritime pilots will be compared to a group of healthy, age and education-matched controls (n=20) with normal sleep. Participants will complete 10 days of actigraphy (Actiwatch 2, Philips Respironics) combined with a sleep-wake diary. They will undergo one night of polysomnography, followed by comprehensive assessment of cognitive function. Additionally, participants will undergo amyloid positron emission tomography-CT to measure brain amyloid accumulation and MRI to investigate atrophy and vascular changes. ANALYSIS: All analyses will be performed using IBM SPSS V.20.0 (SPSS). We will perform independent samples t-tests to compare all outcome parameters. ETHICS AND DISSEMINATION: The study protocol was approved by our institutional ethical review board (NL55712.091.16, file number 2016-2337) and will be performed according to Good Clinical Practice rules. Data and results will be published in 2020.

Pain-Suppressed Behaviors in the Red-tailed Hawk (Buteo jamaicensis).

Our ability to provide analgesia in wild and exotic patients is hampered by a lack of species-specific information on effective drugs and protocols. One contributing factor is the difficulty of applying data from traditional laboratory tests of nociception to clinical conditions frequently involving combinations of inflammatory, mechanical, and neuropathic pain. Pain-suppressed behaviors have become a valuable predictor of clinical utility in other species; in this study we extend this framework to red -tailed hawks in a wildlife hospital, in an attempt to develop a new, humane testing method for birds of prey. We scored six behaviors in hawks hospitalized either for orthopedic trauma or for non-painful conditions. These behaviors included: movement about the cage, grooming, head motions, foot shifts, beak clacks, and rouse. Movement, head motions, and beak clacks were all significantly reduced in hawks with recent orthopedic injury, but not in hawks with healed or minor injuries (P<0.05 for all behaviors). However, it should be noted that due to stringent admission criteria, and the difficulties inherent in studying naturally-occuring injury in wild patients, this study only included -subjects in four experimental groups, and this limited our ability to fully investigate confounds within our data. A follow-up experiment was conducted to determine potential effects of buprenorphine, a mu opioid agonist, on the behaviors listed above. Buprenorphine in the absence of pain caused minor, non-significant decreases in most behaviors, and had no effect on head movement frequency. This suggests that head movements in particular may be sensitive to pain but not to sedative side-effects of buprenorphine. Overall, red -tailed hawks with recent orthopedic trauma show consistent and marked red uctions in several normal maintenance behaviors. Head movements, reported for the first time in this study as a potential marker of pain in birds, in particular seem to be insensitive to sedative side effects of buprenorphine, while being a sensitive measu re of affective state in hawks with painful injuries. These behaviors can be scored humanely and with minimal expense, and should be considered for further research on pain and analgesia in avian species.

Autism training in pediatric residency: evaluation of a case-based curriculum.

Despite recent studies indicating the high prevalence of autism spectrum disorders (ASDs), there has been little focus on improving ASD education during pediatric residency training. The objective of this study was to evaluate a new curriculum developed in partnership with the Centers for Disease Control and Prevention and the Maternal and Child Health Bureau about ASDs. "Autism Case Training (ACT): A Developmental-Behavioral Pediatrics Curriculum" consists of 7 case-based teaching modules. Modules were facilitated by faculty at 26 pediatric residency programs and data were obtained on 114 residents. Pre- and post-test data revealed significant short-term improvements in residents' knowledge and self-assessed competence regarding ASDs. Findings suggest that the ACT curriculum is effective in enhancing training about ASDs in pediatric residency programs.