Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic ...compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
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•The inhibited Sec translocon adopts a conformation optimal for substrate engagement•The inhibitor mycolactone wedges open the lateral gate of Sec61α•Mycolactone blocks the path taken by the signal peptide during engagement•Resistance mutations are likely to operate by modulating translocon dynamics
Gérard et al. determine the structure of the mammalian Sec translocon in an inhibited state. Mycolactone holds the translocon in a conformation optimal for substrate engagement, wedging open the cytosolic side of the lateral gate, while also blocking the path taken by the signal peptide during engagement.
OBJECTIVETo investigate the hypothesis that patients diagnosed with psychogenic nonepileptic seizures (PNES) on video-EEG monitoring (VEM) have increased mortality by comparison to the general ...population.
METHODSThis retrospective cohort study included patients evaluated in VEM units of 3 tertiary hospitals in Melbourne, Australia, between January 1, 1995, and December 31, 2015. Diagnosis was based on consensus opinion of experienced epileptologists and neuropsychiatrists at each hospital. Mortality was determined in patients diagnosed with PNES, epilepsy, or both conditions by linkage to the Australian National Death Index. Lifetime history of psychiatric disorders in PNES was determined from formal neuropsychiatric reports.
RESULTSA total of 5,508 patients underwent VEM. A total of 674 (12.2%) were diagnosed with PNES, 3064 (55.6%) with epilepsy, 175 (3.2%) with both conditions, and 1,595 (29.0%) received other diagnoses or had no diagnosis made. The standardized mortality ratio (SMR) of patients diagnosed with PNES was 2.5 (95% confidence interval CI 2.0–3.3). Those younger than 30 had an 8-fold higher risk of death (95% CI 3.4–19.8). Direct comparison revealed no significant difference in mortality rate between diagnostic groups. Among deaths in patients diagnosed with PNES (n = 55), external causes contributed 18%, with 20% of deaths in those younger than 50 years attributed to suicide, and “epilepsy” was recorded as the cause of death in 24%.
CONCLUSIONSPatients diagnosed with PNES have a SMR 2.5 times above the general population, dying at a rate comparable to those with drug-resistant epilepsy. This emphasizes the importance of prompt diagnosis, identification of risk factors, and implementation of appropriate strategies to prevent potential avoidable deaths.
•Cep120 contains three C2 domains followed by a coiled-coil dimerization domain.•The N-terminal Cep120 C2 domain (C2A) is a novel tubulin binding C2 domain.•Cep120 C2A promotes microtubule formation.
...Centrioles are microtubule-based structures that play essential roles in cell division and cilia biogenesis. Cep120 is an important protein for correct centriole formation and mutations in the Cep120 gene cause severe human diseases like Joubert syndrome and complex ciliopathies. Here, we show that Cep120 contains three consecutive C2 domains that are followed by a coiled-coil dimerization domain. Surprisingly, unlike the classical C2 domains, all three Cep120 C2 domains lack calcium- and phospholipid-binding activities. However, biophysical and biochemical assays revealed that the N-terminal Cep120 C2 domain (C2A) binds to both tubulin and microtubules, and promotes microtubule formation. Structural analyses coupled with mutagenesis identified a highly conserved, positively charged residue patch on the surface of Cep120 C2A, which mediates the interaction with tubulin and microtubules. Together, our results establish Cep120 C2A as a unique microtubule-binding domain. They further provide insights into the molecular mechanism of Cep120 during centriole biogenesis.
The most widespread form of malaria is caused by Plasmodium vivax. To replicate, this parasite must invade immature red blood cells through a process requiring interaction of the P. vivax Duffy ...binding protein (PvDBP) with its human receptor, the Duffy antigen receptor for chemokines. Naturally acquired antibodies that inhibit this interaction associate with clinical immunity, suggesting PvDBP as a leading candidate for inclusion in a vaccine to prevent malaria due to P. vivax. Here, we isolated a panel of monoclonal antibodies from human volunteers immunized in a clinical vaccine trial of PvDBP. We screened their ability to prevent PvDBP from binding to the Duffy antigen receptor for chemokines, and their capacity to block red blood cell invasion by a transgenic Plasmodium knowlesi parasite genetically modified to express PvDBP and to prevent reticulocyte invasion by multiple clinical isolates of P. vivax. This identified a broadly neutralizing human monoclonal antibody that inhibited invasion of all tested strains of P. vivax. Finally, we determined the structure of a complex of this antibody bound to PvDBP, indicating the molecular basis for inhibition. These findings will guide future vaccine design strategies and open up possibilities for testing the prophylactic use of such an antibody.
Objective
Lennox‐Gastaut syndrome (LGS) is an archetypal developmental and epileptic encephalopathy, for which novel treatments are emerging. Diagnostic criteria for LGS have recently been defined by ...the International League Against Epilepsy (ILAE). We aimed to apply these criteria in a real‐world setting.
Methods
We applied ILAE diagnostic criteria to a cohort of patients diagnosed with LGS by epileptologists following inpatient video‐EEG monitoring (VEM) at tertiary comprehensive epilepsy centers between 1995 and 2015. We also assessed mortality in this cohort.
Results
Sixty patients diagnosed with LGS and had complete records available for review were identified. Among them, 29 (48%) patients met ILAE diagnostic criteria for LGS (ILAE‐DC group). Thirty‐one did not meet criteria (non‐ILAE‐DC) due to the absence of documented tonic seizures (n = 7), EEG features (n = 12), or both tonic seizures and EEG features (n = 10), intellectual disability (n = 1), or drug resistance (n = 1). The ILAE‐DC group had a shorter duration of epilepsy at VEM than the non‐ILAE‐DC group (median = 12.0 years vs. 23.7 years, respectively; p = 0.015). The proportions of patients with multiple seizure types (100% vs. 96.7%), ≤2.5 Hz slow spike‐and‐wave EEG activity (100% vs. 90%), seizure‐related injuries (27.6% vs. 25.8%), and mortality (standardized mortality ratio 4.60 vs. 5.12) were similar between the groups.
Significance
Up to 52% of patients diagnosed with LGS following VEM may not meet recently accepted ILAE criteria for LGS diagnosis. This may reflect both the limitations of retrospective medical record review and a historical tendency of applying the LGS diagnosis to a broad spectrum of severe, early‐onset drug‐resistant epilepsies with drop attacks. The ILAE criteria allow the delineation of LGS based on distinct electroclinical features, potentiating accurate diagnosis, prognostication, and management formulation. Nonetheless, mortality outcomes between those who did and did not meet ILAE diagnostic criteria for LGS were similarly poor, and both groups suffered high rates of seizure‐related injury.
Plain Language Summary
More than half of patients diagnosed with Lennox‐Gastaut Syndrome (LGS) at three Australian epilepsy monitoring units between 1995 and 2015 did not meet the recently devised International League Against Epilepsy (ILAE) diagnostic criteria for LGS. Mortality was equally high in those who did and did not meet the ILAE diagnostic criteria, and seizure‐related injury was common. The ILAE diagnostic criteria will guide accurate diagnosis, management, prognostication, and research in patients with LGS, however may be limited in their practical application to patients with a longer duration of epilepsy, or to those for whom detailed assessment is difficult.
Objective
In vitro data prompted U.S Food and Drug Administration warnings that lamotrigine, a common sodium channel modulating anti‐seizure medication (NaM‐ASM), could increase the risk of sudden ...death in patients with structural or ischaemic cardiac disease, however, its implications for Sudden Unexpected Death in Epilepsy (SUDEP) are unclear.
Methods
This retrospective, nested case–control study identified 101 sudden unexpected death in epilepsy (SUDEP) cases and 199 living epilepsy controls from Epilepsy Monitoring Units (EMUs) in Australia and the USA. Differences in proportions of lamotrigine and NaM‐ASM use were compared between cases and controls at the time of admission, and survival analyses from the time of admission up to 16 years were conducted. Multivariable logistic regression and survival analyses compared each ASM subgroup adjusting for SUDEP risk factors.
Results
Proportions of cases and controls prescribed lamotrigine (P = 0.166), one NaM‐ASM (P = 0.80), or ≥2NaM‐ASMs (P = 0.447) at EMU admission were not significantly different. Patients taking lamotrigine (adjusted hazard ratio aHR = 0.56; P = 0.054), one NaM‐ASM (aHR = 0.8; P = 0.588) or ≥2 NaM‐ASMs (aHR = 0.49; P = 0.139) at EMU admission were not at increased SUDEP risk up to 16 years following admission. Active tonic–clonic seizures at EMU admission associated with >2‐fold SUDEP risk, irrespective of lamotrigine (aHR = 2.24; P = 0.031) or NaM‐ASM use (aHR = 2.25; P = 0.029). Sensitivity analyses accounting for incomplete ASM data at follow‐up suggest undetected changes to ASM use are unlikely to alter our results.
Significance
This study provides additional evidence that lamotrigine and other NaM‐ASMs are unlikely to be associated with an increased long‐term risk of SUDEP, up to 16 years post‐EMU admission.
To explore the impact of psychiatric comorbidities on all-cause mortality in adults with epilepsy from a cohort of patients admitted for video-EEG monitoring (VEM) over 2 decades.
A retrospective ...medical record audit was conducted on 2,709 adults admitted for VEM and diagnosed with epilepsy at 3 Victorian comprehensive epilepsy programs from 1995 to 2015. A total of 1,805 patients were identified in whom the record of a clinical evaluation by a neuropsychiatrist was available, excluding 27 patients who died of a malignant brain tumor known at the time of VEM admission. Epilepsy and lifetime psychiatric diagnoses were determined from consensus opinion of epileptologists and neuropsychiatrists involved in the care of each patient. Mortality and cause of death were determined by linkage to the Australian National Death Index and National Coronial Information System.
Compared with the general population, mortality was higher in people with epilepsy (PWE) with a psychiatric illness (standardized mortality ratio SMR 3.6) and without a psychiatric illness (SMR 2.5). PWE with a psychiatric illness had greater mortality compared with PWE without (hazard ratio 1.41, 95% confidence interval 1.02-1.97) after adjusting for age and sex. No single psychiatric disorder by itself conferred increased mortality in PWE. The distribution of causes of death remained similar between PWE with psychiatric comorbidities and those without.
The presence of comorbid psychiatric disorders in adults with epilepsy is associated with increased mortality, highlighting the importance of identifying and treating psychiatric comorbidities in these patients.
The nuclear factor I (NFI) family of transcription factors play an important role in normal development of multiple organs. Three NFI family members are highly expressed in the brain, and deletions ...or sequence variants in two of these, NFIA and NFIX, have been associated with intellectual disability (ID) and brain malformations. NFIB, however, has not previously been implicated in human disease. Here, we present a cohort of 18 individuals with mild ID and behavioral issues who are haploinsufficient for NFIB. Ten individuals harbored overlapping microdeletions of the chromosomal 9p23-p22.2 region, ranging in size from 225 kb to 4.3 Mb. Five additional subjects had point sequence variations creating a premature termination codon, and three subjects harbored single-nucleotide variations resulting in an inactive protein as determined using an in vitro reporter assay. All individuals presented with additional variable neurodevelopmental phenotypes, including muscular hypotonia, motor and speech delay, attention deficit disorder, autism spectrum disorder, and behavioral abnormalities. While structural brain anomalies, including dysgenesis of corpus callosum, were variable, individuals most frequently presented with macrocephaly. To determine whether macrocephaly could be a functional consequence of NFIB disruption, we analyzed a cortex-specific Nfib conditional knockout mouse model, which is postnatally viable. Utilizing magnetic resonance imaging and histology, we demonstrate that Nfib conditional knockout mice have enlargement of the cerebral cortex but preservation of overall brain structure and interhemispheric connectivity. Based on our findings, we propose that haploinsufficiency of NFIB causes ID with macrocephaly.
Numerous placebo-controlled studies have demonstrated the ability of ketamine, an NMDA receptor antagonist, to induce rapid (within hours), transient antidepressant effects when administered ...intravenously (IV) at subanesthetic doses (0.5 mg/kg over 40 min). However, the optimal antidepressant dose remains unknown. We aimed to compare to active placebo the rapid acting antidepressant properties of a broad range of subanesthetic doses of IV ketamine among outpatients with treatment-resistant depression (TRD). A range of IV ketamine doses were compared to active placebo in the treatment of adult TRD over a 3-day period following a single infusion over 40 min. This was an outpatient study conducted across six US academic sites. Outpatients were 18-70 years old with TRD, defined as failure to achieve a satisfactory response (e.g., less than 50% improvement of depression symptoms) to at least two adequate treatment courses during the current depressive episode. Following a washout period, 99 eligible subjects were randomly assigned to one of the five arms in a 1:1:1:1:1 fashion: a single intravenous dose of ketamine 0.1 mg/kg (n = 18), a single dose of ketamine 0.2 mg/kg (n = 20), a single dose of ketamine 0.5 mg/kg (n = 22), a single dose of ketamine 1.0 mg/kg (n = 20), and a single dose of midazolam 0.045 mg/kg (active placebo) (n = 19). The study assessments (HAM-D-6, MADRS, SDQ, PAS, CGI-S, and CGI-I) were performed at days 0, 1, 3 (endpoint), 5, 7, 14, and 30 to assess the safety and efficacy. The overall group × time interaction effect was significant for the primary outcome measure, the HAM-D-6. In post hoc pairwise comparisons controlling for multiple comparisons, standard dose (0.5 mg/kg) and high dose (1 mg/kg) of intravenous ketamine were superior to active placebo; a low dose (0.1 mg/kg) was significant only prior to adjustment (p = 0.02, p-adj = 0.14, d = -0.82 at day 1). Most of the interaction effect was due to differences at day 1, with no significant adjusted pairwise differences at day 3. This pattern generally held for secondary outcomes. The infusions of ketamine were relatively well tolerated compared to active placebo, except for greater dissociative symptoms and transient blood pressure elevations with the higher doses. Our results suggest that there is evidence for the efficacy of the 0.5 mg/kg and 1.0 mg/kg subanesthetic doses of IV ketamine and no clear or consistent evidence for clinically meaningful efficacy of lower doses of IV ketamine. Trial Registration: NCT01920555.
Coronavirus disease (COVID-19) is a global threat to health. Its inflammatory characteristics are incompletely understood.
To define the cytokine profile of COVID-19 and to identify evidence of ...immunometabolic alterations in those with severe illness.
Levels of IL-1β, IL-6, IL-8, IL-10, and sTNFR1 (soluble tumor necrosis factor receptor 1) were assessed in plasma from healthy volunteers, hospitalized but stable patients with COVID-19 (COVID
patients), patients with COVID-19 requiring ICU admission (COVID
patients), and patients with severe community-acquired pneumonia requiring ICU support (CAP
patients). Immunometabolic markers were measured in circulating neutrophils from patients with severe COVID-19. The acute phase response of AAT (alpha-1 antitrypsin) to COVID-19 was also evaluated.
IL-1β, IL-6, IL-8, and sTNFR1 were all increased in patients with COVID-19. COVID
patients could be clearly differentiated from COVID
patients, and demonstrated higher levels of IL-1β, IL-6, and sTNFR1 but lower IL-10 than CAP
patients. COVID-19 neutrophils displayed altered immunometabolism, with increased cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, HIF-1α (hypoxia-inducible factor-1α), and lactate. The production and sialylation of AAT increased in COVID-19, but this antiinflammatory response was overwhelmed in severe illness, with the IL-6:AAT ratio markedly higher in patients requiring ICU admission (
< 0.0001). In critically unwell patients with COVID-19, increases in IL-6:AAT predicted prolonged ICU stay and mortality, whereas improvement in IL-6:AAT was associated with clinical resolution (
< 0.0001).
The COVID-19 cytokinemia is distinct from that of other types of pneumonia, leading to organ failure and ICU need. Neutrophils undergo immunometabolic reprogramming in severe COVID-19 illness. Cytokine ratios may predict outcomes in this population.
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