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The topographic features of the free energy landscapes that govern the thermodynamics and kinetics of conformational transitions in proteins, which in turn are integral for function, are not well understood. This reflects the experimental challenges associated with characterizing these multidimensional surfaces, even for small proteins. Here we focus on a 62-residue protein, gpW, that folds very rapidly into a native structure with an α/β topology in which α-helices are at the N- and C-terminal ends of the molecule with a central β-hairpin positioned orthogonally to the helices. Using relaxation dispersion NMR spectroscopy to probe the conformational fluctuations in gpW at 1 °C, we found that the native state interconverts with a transiently formed, sparsely populated second state with a lifetime of 250 μs, consistent with the global folding-unfolding rate under these conditions. In this low-populated state, the β-hairpin is unfolded whereas the α-helices remain predominantly formed. Our results argue for a hierarchical stability of secondary structural elements and demonstrate the existence of a complex free energy landscape even in this small, fast-folding single-domain protein.

Item does not contain fulltext Fatigue is prevalent among patients with systemic sclerosis (SSc). To date, studies investigating fatigue in SSc have been hampered by the instruments used to measure fatigue in SSc and have included patient-reported rather than objectively-rated measures of disease. The Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) scale is a validated measure for assessing fatigue in SSc that, compared to other instruments, provides good coverage of the full range of the fatigue spectrum. The objective of this study was to assess sociodemographic and objectively-rated disease-related associates of fatigue, as measured by the FACIT-F, in a large sample of patients with SSc. Fatigue was assessed using the FACIT-F scale. Disease severity was assessed using Medsger’s severity scale. Multivariable linear regression was performed to assess the independent associations between sociodemographic and medical variables and fatigue. Among 785 patients, the mean FACIT-F score was 32.2 (SD = 12.1). Being age 40-49 (reference = 60+; standardized regression coefficient (beta) = -0.11), less than post-secondary education (beta = 0.07), having more medical comorbidities (beta = -0.11) and more severe muscle (beta = -0.10), gastrointestinal (beta= -0.15), lung (beta = -0.13), and general system disease severity (beta = -0.13) were independently associated with more fatigue (p < 0.05). Fatigue in SSc was independently associated with more severe disease. These data contribute to a better understanding of fatigue in SSc and help inform patient-centered research in SSc. 7 p.

Alzheimer's disease (AD) is linked to the abnormal accumulation of amyloid ? peptide (A?) aggregates in the brain. Silybin B, a natural compound extracted from milk thistle (Silybum marianum), has been shown to significantly inhibit A? aggregation in vitro and to exert neuroprotective properties in vivo. However, further explorations of silybin B's clinical potential are currently limited by three main factors: (a) poor solubility, (b) instability in blood serum, and (c) only partial knowledge of silybin's mechanism of action. Here, we address these three limitations. We demonstrate that conjugation of a trehalose moiety to silybin significantly increases both water solubility and stability in blood serum without significantly compromising its antiaggregation properties. Furthermore, using a combination of biophysical techniques with different spatial resolution, that is, TEM, ThT fluorescence, CD, and NMR spectroscopy, we profile the interactions of the trehalose conjugate with both A? monomers and oligomers and evidence that silybin may shield the "toxic" surfaces formed by the N-terminal and central hydrophobic regions of A?. Finally, comparative analysis with silybin A, a less active diastereoisomer of silybin B, revealed how even subtle differences in chemical structure may entail different effects on amyloid inhibition. The resulting insight on the mechanism of action of silybins as aggregation inhibitors is anticipated to facilitate the future investigation of silybin's therapeutic potential.

The human genome encodes ∼20 mitochondrial proteases, yet we know little of how they sculpt the mitochondrial proteome, particularly during important mitochondrial events such as the initiation of apoptosis. To characterize global mitochondrial proteolysis we refined our technique, terminal amine isotopic labeling of substrates, for mitochondrial SILAC (MS-TAILS) to identify proteolysis across mitochondria and parent cells in parallel. Our MS-TAILS analyses identified 45% of the mitochondrial proteome and identified protein amino (N)-termini from 26% of mitochondrial proteins, the highest reported coverage of the human mitochondrial N-terminome. MS-TAILS revealed 97 previously unknown proteolytic sites. MS-TAILS also identified mitochondrial targeting sequence (MTS) removal by proteolysis during protein import, confirming 101 MTS sites and identifying 135 new MTS sites, revealing a wobbly requirement for the MTS cleavage motif. To examine the relatively unknown initial cleavage events occurring before the well-studied activation of caspase-3 in intrinsic apoptosis, we quantitatively compared N-terminomes of mitochondria and their parent cells before and after initiation of apoptosis at very early time points. By identifying altered levels of >400 N-termini, MS-TAILS analyses implicated specific mitochondrial pathways including protein import, fission, and iron homeostasis in apoptosis initiation. Notably, both staurosporine and Bax activator molecule-7 triggered in common 7 mitochondrial and 85 cellular cleavage events that are potentially part of an essential core of apoptosis-initiating events. All mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD009054.

Anoxic-epileptic seizures (AES) are rare outcomes of common childhood reflex anoxic syncope that trigger a true epileptic seizure. The term AES was coined by Stephenson in 1983, to differentiate these events from convulsive syncopes and the more common reflex anoxic syncopes. A genetic susceptibility for AES has been postulated; but, its molecular basis has up to now been elusive. We report here two illustrative cases and show the association of de novo SCN8A variants and AES. One of them had focal or generalized seizures and autonomic symptoms triggered by orthostatism; the second had breath-holding spells triggered by pain or exercise leading to tonic-clonic seizures; both had repeatedly normal EEGs and a family history of reflex syncope. The data of three additional AES patients further suggest, for the first time, a link between SCN8A pathogenic variants and AES. The neurodevelopment of four patients was abnormal. Four of the five SCN8A mutations observed here were previously described in patients with seizure disorders. Seizures responded particularly well to sodium channel blockers. Our observation enriches the spectrum of seizures linked with SCN8A pathogenic variants.

International audience; BACKGROUND:Placenta-mediated pregnancy complications include pre-eclampsia, late pregnancy loss, placental abruption, and birth of a small-for-gestational-age (SGA) neonate. These complications are leading causes of maternal, fetal, and neonatal morbidity and mortality in high-income countries. Affected women are at high risk of recurrence in subsequent pregnancies; however, effective strategies to prevent recurrence are absent. Findings from our previous study-level meta-analysis suggested that low-molecular-weight heparin reduced the risk of recurrent placenta-mediated pregnancy complications. However, we identified significant heterogeneity in the results, possibly due to trial design or inclusion criteria. To identify which patients benefit from, and which outcomes are prevented by, low-molecular-weight heparin, we did an individual patient data meta-analysis.METHODS:We did a systematic review in May, 2013, which identified eight eligible randomised trials done between 2000 and 2013 of low-molecular-weight heparin to prevent recurrent placenta-mediated pregnancy complications. We excluded studies on the basis of the wrong population, the study being ongoing, inability to confirm eligibility of participants, intervention stopped too early, and no response from the principal investigator. We requested individual patient data from the study authors for eligible women (women pregnant at the time of the study with a history of previous pregnancy that had been complicated by one or more of the following: pre-eclampsia, placental abruption, birth of an SGA neonate [<10th percentile], pregnancy loss after 16 weeks' gestation, or two losses after 12 weeks' gestation) and recoded, combined, and analysed the data for our meta-analysis. The primary outcome was a composite of early-onset (<34 weeks) or severe pre-eclampsia, birth of an SGA neonate (<5th percentile), late pregnancy loss (≥20 weeks' gestation), or placental abruption leading to delivery, assessed on an intention-to-treat basis. We assessed risk of bias with the Cochrane Risk of Bias tool. This study is registered with PROSPERO, number CRD42013006249.FINDINGS:We analysed data from 963 eligible women in eight trials: 480 randomly assigned to low-molecular-weight heparin and 483 randomly assigned to no low-molecular-weight heparin. Overall, the risk of bias was not substantial enough to affect decisions regarding trial inclusion. Participants were mostly white (795/905; 88%) with a mean age of 30·9 years (SD 5·0) and 403/963 (42%) had thrombophilia. In the primary analysis, low-molecular-weight heparin did not significantly reduce the risk of recurrent placenta-mediated pregnancy complications (low-molecular-weight heparin 62/444 [14%] versus no low-molecular-weight heparin 95/443 (22%) absolute difference -8%, 95% CI -17·3 to 1·4, p=0·09; relative risk 0·64, 95% CI 0·36-1·11, p=0·11). We noted significant heterogeneity between single-centre and multicentre trials. In subgroup analyses, low-molecular-weight heparin in multicentre trials reduced the primary outcome in women with previous abruption (p=0·006) but not in any of the other subgroups of previous complications.INTERPRETATION:Low-molecular-weight heparin does not seem to reduce the risk of recurrent placenta-mediated pregnancy complications in at-risk women. However, some decreases in event rates might have been too small for the power of our study to explore.FUNDING:Canadian Institutes of Health Research.

In this natural experiment, we investigated on-leash and off-leash policies as plausible influences on the behavior of dog-walkers in the City of Calgary, Alberta, Canada. Following policy-mandated public consultations, two of the four parks initially proposed by the City as sites for new off-leash areas retained on-leash designations. Within a year of creating off-leash areas in two parks, we observed more dog-walkers and improved compliance with dog-fouling in one case, but not in the other. Compared to the previous year, we also observed more stationary dog-walkers in both of these parks. Paradoxically, activity levels amongst dog-walkers – including while dogs were off-leash – remained highest for a park that retained an on-leash designation. Off-leash policies in urban parks could have positive as well as negative implications for public health. In addition to off-leash policies, factors that merit consideration regarding dog-walking and dog-fouling include implementation strategies, physical features, socio-demographic characteristics and modifications to park environments.

Background: The birth of a child with a major congenital anomaly may create chronic caregiving stress for mothers, yet little is known about their psychiatric outcomes. Aims: To evaluate the association of the birth of a child with a major congenital anomaly with subsequent maternal psychiatric risk. Methods: This Danish nationwide cohort study included mothers who gave birth to an infant with a major congenital anomaly (n = 19 220) between 1997 and 2015. Comparators were randomly selected mothers, matched on maternal age, year of delivery and parity (n = 195 399). The primary outcome was any new-onset psychiatric diagnosis. Secondary outcomes included specific psychiatric diagnoses, psychiatric in-patient admissions and redeemed psychoactive medicines. Cox models were used to estimate hazard ratios (HRs), adjusted for socioeconomic and medical variables. Results: Mothers of affected infants had an elevated risk for a new-onset psychiatric disorder vs. the comparison group (adjusted HR, 1.16, 95% CI 1.11–1.22). The adjusted HR was particularly elevated during the first postpartum year (1.65, 95% CI 1.42–1.90), but remained high for years, especially among mothers of children with multiorgan anomalies (1.37, 95% CI 1.18–1.57). The risk was also elevated for most specific psychiatric diagnoses, admissions and medicines. Conclusions: Mothers who give birth to a child with a major congenital anomaly are at increased risk of new-onset psychiatric disorders, especially shortly after birth and for mothers of children with more severe anomalies. Our study highlights the need to screen for mental illness in this high-risk population, as well as to integrate adult mental health services and paediatric care.