Exaggerated Nighttime Sleep and Defective Sleep Homeostasis in a Drosophila Knock-In Model of Human Epilepsy

Despite an established link between epilepsy and sleep behavior, it remains unclear how specific epileptogenic mutations affect sleep and subsequently influence seizure susceptibility. Recently, Sun et al. (2012) created a fly knock-in model of human generalized epilepsy with febrile seizures plus (GEFS+), a wide-spectrum disorder characterized by fever-associated seizing in childhood and lifelong affliction. GEFS+ flies carry a disease-causing mutation in their voltage-gated sodium channel (VGSC) gene and display semidominant heat-induced seizing, likely due to reduced GABAergic inhibitory activity at high temperature. Here, we show that at room temperature the GEFS+ mutation dominantly modifies sleep, with mutants exhibiting rapid sleep onset at dusk and increased nighttime sleep as compared to controls. These characteristics of GEFS+ sleep were observed regardless of sex, mating status, and genetic background. GEFS+ mutant sleep phenotypes were more resistant to pharmacologic reduction of GABA transmission by carbamazepine (CBZ) than controls, and were mitigated by reducing GABA_A receptor expression specifically in wake-promoting pigment dispersing factor (PDF) neurons. These findings are consistent with increased GABAergic transmission to PDF neurons being mainly responsible for the enhanced nighttime sleep of GEFS+ mutants. Additionally, analyses under other light conditions suggested that the GEFS+ mutation led to reduced buffering of behavioral responses to light on and off stimuli, which contributed to characteristic GEFS+ sleep phenotypes. We further found that GEFS+ mutants had normal circadian rhythms in free-running dark conditions. Interestingly, the mutants lacked a homeostatic rebound following mechanical sleep deprivation, and whereas deprivation treatment increased heat-induced seizure susceptibility in control flies, it unexpectedly reduced seizure activity in GEFS+ mutants. Our study has revealed the sleep architecture of a Drosophila VGSC mutant that harbors a human GEFS+ mutation, and provided unique insight into the relationship between sleep and epilepsy.     

Modest Interference with Actin Dynamics in Primary T Cell Activation by Antigen Presenting Cells Preferentially Affects Lamellal Signaling

Dynamic subcellular distributions of signaling system components are critical regulators of cellular signal transduction through their control of molecular interactions. Understanding how signaling activity depends on such distributions and the cellular structures driving them is required for comprehensive insight into signal transduction. In the activation of primary murine T cells by antigen presenting cells (APC) signaling intermediates associate with various subcellular structures, prominently a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell. While actin dynamics are well established as general regulators of cellular organization, their role in controlling signaling organization in primary T cell:APC couples and the specific cellular structures driving it is unresolved. Using modest interference with actin dynamics with a low concentration of Jasplakinolide as corroborated by costimulation blockade we show that T cell actin preferentially controls lamellal signaling localization and activity leading downstream to calcium signaling. Lamellal localization repeatedly related to efficient T cell function. This suggests that the transient lamellal actin matrix regulates T cell signaling associations that facilitate T cell activation.     

TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation

TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8–mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)–dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.     

Identification of Medically Actionable Secondary Findings in the 1000 Genomes

The American College of Medical Genetics and Genomics (ACMG) recommends that clinical sequencing laboratories return secondary findings in 56 genes associated with medically actionable conditions. Our goal was to apply a systematic, stringent approach consistent with clinical standards to estimate the prevalence of pathogenic variants associated with such conditions using a diverse sequencing reference sample. Candidate variants in the 56 ACMG genes were selected from Phase 1 of the 1000 Genomes dataset, which contains sequencing information on 1,092 unrelated individuals from across the world. These variants were filtered using the Human Gene Mutation Database (HGMD) Professional version and defined parameters, appraised through literature review, and examined by a clinical laboratory specialist and expert physician. Over 70,000 genetic variants were extracted from the 56 genes, and filtering identified 237 variants annotated as disease causing by HGMD Professional. Literature review and expert evaluation determined that 7 of these variants were pathogenic or likely pathogenic. Furthermore, 5 additional truncating variants not listed as disease causing in HGMD Professional were identified as likely pathogenic. These 12 secondary findings are associated with diseases that could inform medical follow-up, including cancer predisposition syndromes, cardiac conditions, and familial hypercholesterolemia. The majority of the identified medically actionable findings were in individuals from the European (5/379) and Americas (4/181) ancestry groups, with fewer findings in Asian (2/286) and African (1/246) ancestry groups. Our results suggest that medically relevant secondary findings can be identified in approximately 1% (12/1092) of individuals in a diverse reference sample. As clinical sequencing laboratories continue to implement the ACMG recommendations, our results highlight that at least a small number of potentially important secondary findings can be selected for return. Our results also confirm that understudied populations will not reap proportionate benefits of genomic medicine, highlighting the need for continued research efforts on genetic diseases in these populations.     

LptB-LptF coupling mediates the closure of the substrate-binding cavity in the LptB2FGC transporter through a rigid-body mechanism to extract LPS

Lipopolysaccharides (LPS) are essential envelope components in many Gram-negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP-binding cassette (ABC) transporter LptB₂FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF’s coupling helix being important in coordinating cavity collapse with LptB dimerization.     

Analysis of DNA adducts by accelerator mass spectrometry in human breast tissue after administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and benzo[a]pyrene

Epidemiological evidence has suggested an association between meat consumption and the risk of breast cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine found in cooked meat, has been implicated in the aetiology of breast cancer and has been shown to induce tumour formation in rodent mammary glands. In addition, polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) which has also been shown to induce tumour formation at a number of sites in rodents including the breast, are produced during the cooking of meat through the pyrolysis of fats. The aim of this study was to examine the bioavailability of these compounds to human breast tissue and their ability to bind to DNA to form DNA adducts. Patients undergoing breast surgery at York District Hospital were orally administered prior to surgery a capsule containing 20 μg of ¹⁴C PhIP (182 kBq, specific activity 2.05 GBq/mmol) or 5 μg of ¹⁴C B[a]P (36 kBq, specific activity 1.81 GBq/mmol). At surgery, normal and tumour breast tissue was resected and tissue concentrations of carcinogen measured by liquid scintillation counting and DNA adduct levels by accelerator mass spectrometry (AMS) were subsequently determined. It was found that both ¹⁴C PhIP and ¹⁴C B[a]P were able to reach the target organ where they had the ability to form DNA adducts. The level of adducts ranged from 26.22–477.35 and 6.61–208.38 adducts/10¹² nucleotides following administration of ¹⁴C PhIP and ¹⁴C B[a]P, respectively, with no significant difference observed between levels in normal or tumour tissue. In addition, the data obtained in this study were comparable to adduct levels previously found in colon samples following administration of the same compounds to individuals undergoing colorectal surgery. This is the first report that these two carcinogens bind to human breast DNA after administration of a defined low dose.