The ribosome lowers the entropic penalty of protein folding.

Most proteins fold during biosynthesis on the ribosome1, and co-translational folding energetics, pathways and outcomes of many proteins have been found to differ considerably from those in refolding studies2-10. The origin of this folding modulation by the ribosome has remained unknown. Here we have determined atomistic structures of the unfolded state of a model protein on and off the ribosome, which reveal that the ribosome structurally expands the unfolded nascent chain and increases its solvation, resulting in its entropic destabilization relative to the peptide chain in isolation. Quantitative 19F NMR experiments confirm that this destabilization reduces the entropic penalty of folding by up to 30 kcal mol-1 and promotes formation of partially folded intermediates on the ribosome, an observation that extends to other protein domains and is obligate for some proteins to acquire their active conformation. The thermodynamic effects also contribute to the ribosome protecting the nascent chain from mutation-induced unfolding, which suggests a crucial role of the ribosome in supporting protein evolution. By correlating nascent chain structure and dynamics to their folding energetics and post-translational outcomes, our findings establish the physical basis of the distinct thermodynamics of co-translational protein folding.

Effects of Oxide Fragments on Microstructure and Mechanical Properties of AA6061 Aluminum Alloy Tube Fabricated by Thermomechanical Consolidation of Machining Chips.

An AA6061 aluminum alloy tube was fabricated by compacting machining chips via thermomechanical consolidation, including hot pressing and hot extrusion. The microstructure evolution and formation of oxide particles were investigated in correlation to tensile mechanical properties. It was found that fine Al/Mg oxide particles were formed due to the fracture of oxide layers on the chips and the reaction between Mg and Al2O3 during hot extrusion. The oxide particles inhibited the growth of recrystallized α-Al grains, leading to the formation of a microstructure consisting of coarse elongated grains with sizes of 420 µm and fine equiaxed grains with sizes of 10 µm. After T6 heat treatment, a microstructure with finer grains (grain sizes: 34 µm) formed due to further recrystallization induced by residual strain. The tensile mechanical properties testing results indicated that a good combination of strength (296 MPa) and ductility (7.6%) was achieved in the T6 heat treated samples, which were likely attributed to the high-quality inter-chip bonding, as well as the fine oxide particles which were small enough that further crack nucleation and growth around them were inhibited during tensile deformation. For the purpose of comparison, the microstructure and mechanical properties of the as-extruded and T6 heat treated samples produced by hot extrusion of the cast ingot of AA6061 aluminum alloy were also investigated. The lower tensile strength of solid-state recycled tube sample might be associated with the consumption of Mg atoms by the oxidation reaction, leading to the lower density of ß″ precipitates in precipitation strengthening.

The ribosome stabilizes partially folded intermediates of a nascent multi-domain protein.

Co-translational folding is crucial to ensure the production of biologically active proteins. The ribosome can alter the folding pathways of nascent polypeptide chains, yet a structural understanding remains largely inaccessible experimentally. We have developed site-specific labelling of nascent chains to detect and measure, using 19F nuclear magnetic resonance (NMR) spectroscopy, multiple states accessed by an immunoglobulin-like domain within a tandem repeat protein during biosynthesis. By examining ribosomes arrested at different stages during translation of this common structural motif, we observe highly broadened NMR resonances attributable to two previously unidentified intermediates, which are stably populated across a wide folding transition. Using molecular dynamics simulations and corroborated by cryo-electron microscopy, we obtain models of these partially folded states, enabling experimental verification of a ribosome-binding site that contributes to their high stabilities. We thus demonstrate a mechanism by which the ribosome could thermodynamically regulate folding and other co-translational processes.

Modulating co-translational protein folding by rational design and ribosome engineering.

Co-translational folding is a fundamental process for the efficient biosynthesis of nascent polypeptides that emerge through the ribosome exit tunnel. To understand how this process is modulated by the shape and surface of the narrow tunnel, we have rationally engineered three exit tunnel protein loops (uL22, uL23 and uL24) of the 70S ribosome by CRISPR/Cas9 gene editing, and studied the co-translational folding of an immunoglobulin-like filamin domain (FLN5). Our thermodynamics measurements employing 19F/15N/methyl-TROSY NMR spectroscopy together with cryo-EM and molecular dynamics simulations reveal how the variations in the lengths of the loops present across species exert their distinct effects on the free energy of FLN5 folding. A concerted interplay of the uL23 and uL24 loops is sufficient to alter co-translational folding energetics, which we highlight by the opposite folding outcomes resulting from their extensions. These subtle modulations occur through a combination of the steric effects relating to the shape of the tunnel, the dynamic interactions between the ribosome surface and the unfolded nascent chain, and its altered exit pathway within the vestibule. These results illustrate the role of the exit tunnel structure in co-translational folding, and provide principles for how to remodel it to elicit a desired folding outcome.

Cardiac rehabilitation program: An exploration of patient experiences and perspectives on program dropout.

BACKGROUND: Cardiac rehabilitation programs (CRP) are effective evidence-based secondary prevention programs that reduce morbidity and mortality in patients with cardiovascular disease (CVD). However, participation remains suboptimal, resulting in under-treatment and greater risk for recurrent cardiac events. Understanding the reasons behind CRP dropout is urgently needed to inform the development of programs that best meet patient needs and support sustained engagement. AIMS: The aim of this study was to identify and understand factors impacting CRP dropout from the patient perspective. METHODS: A qualitative study using semi-structured interviews was undertaken to examine the experience of 23 patients who dropped out of a CRP within a large urban hospital in British Columbia, Canada. Data were coded, analyzed using the constant comparison technique, and organized thematically. RESULTS: Participants described multiple challenges when attempting to complete CRP. Analysis of the data led to the identification of three main categories: (1) challenges living with CVD, (2) perceived advantages and disadvantages of CRP, and (3) unmet needs during CRP. LINKING EVIDENCE TO ACTION: In the practice setting, assessment of readiness to engage in CRP, alongside patient preferences and engagement needs, should be undertaken for maximum CRP uptake and completion. Providing diverse modes of CRP delivery, along with exploring the impact of virtual options as compared to traditional in-person programs, will further advance the CRP evidence and may help address pervasive access barriers.

Common sequence motifs of nascent chains engage the ribosome surface and trigger factor.

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and in vitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol.

Nascent chains can form co-translational folding intermediates that promote post-translational folding outcomes in a disease-causing protein.

During biosynthesis, proteins can begin folding co-translationally to acquire their biologically-active structures. Folding, however, is an imperfect process and in many cases misfolding results in disease. Less is understood of how misfolding begins during biosynthesis. The human protein, alpha-1-antitrypsin (AAT) folds under kinetic control via a folding intermediate; its pathological variants readily form self-associated polymers at the site of synthesis, leading to alpha-1-antitrypsin deficiency. We observe that AAT nascent polypeptides stall during their biosynthesis, resulting in full-length nascent chains that remain bound to ribosome, forming a persistent ribosome-nascent chain complex (RNC) prior to release. We analyse the structure of these RNCs, which reveals compacted, partially-folded co-translational folding intermediates possessing molten-globule characteristics. We find that the highly-polymerogenic mutant, Z AAT, forms a distinct co-translational folding intermediate relative to wild-type. Its very modest structural differences suggests that the ribosome uniquely tempers the impact of deleterious mutations during nascent chain emergence. Following nascent chain release however, these co-translational folding intermediates guide post-translational folding outcomes thus suggesting that Z's misfolding is initiated from co-translational structure. Our findings demonstrate that co-translational folding intermediates drive how some proteins fold under kinetic control, and may thus also serve as tractable therapeutic targets for human disease.

Interactions between nascent proteins and the ribosome surface inhibit co-translational folding.

Most proteins begin to fold during biosynthesis on the ribosome. It has been suggested that interactions between the emerging polypeptide and the ribosome surface might allow the ribosome itself to modulate co-translational folding. Here we combine protein engineering and NMR spectroscopy to characterize a series of interactions between the ribosome surface and unfolded nascent chains of the immunoglobulin-like FLN5 filamin domain. The strongest interactions are found for a C-terminal segment that is essential for folding, and we demonstrate quantitative agreement between the strength of this interaction and the energetics of the co-translational folding process itself. Mutations in this region that reduce the extent of binding result in a shift in the co-translational folding equilibrium towards the native state. Our results therefore demonstrate that a competition between folding and binding provides a simple, dynamic mechanism for the modulation of co-translational folding by the ribosome.

Fraudulent News Headline Detection with Attention Mechanism.

E-mail systems and online social media platforms are ideal places for news dissemination, but a serious problem is the spread of fraudulent news headlines. The previous method of detecting fraudulent news headlines was mainly laborious manual review. While the total number of news headlines goes as high as 1.48 million, manual review becomes practically infeasible. For news headline text data, attention mechanism has powerful processing capability. In this paper, we propose the models based on LSTM and attention layer, which fit the context of news headlines efficiently and can detect fraudulent news headlines quickly and accurately. Based on multi-head attention mechanism eschewing recurrent unit and reducing sequential computation, we build Mini-Transformer Deep Learning model to further improve the classification performance.

Automated E-Counseling for Chronic Heart Failure: CHF-CePPORT Trial.

BACKGROUND: International task force statements advocate telehealth programs to promote health-related quality of life for patients with chronic heart failure (CHF). To that end, we evaluated the efficacy and usability of an automated e-counseling program. METHODS: This Canadian multi-site double-blind randomized trial assessed whether usual care plus either internet-based e-counseling (motivational and cognitive-behavioral tools for CHF self-care) or e-based conventional CHF self-care education (e-UC) improved 12-month Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS). Secondary outcomes included program engagement (total logon weeks, logons, and logon hours), total CHF self-care behaviors, diet (fruit and vegetable servings), 6-minute walk test, and 4-day step count. The association between program engagement and health-related quality of life was assessed using KCCQ-OS tertiles. RESULTS: We enrolled 231 patients, median age =59.5 years, 22% female, and elevated median KCCQ-OS=83.0 (interquartile range, 68-93). KCCQ-OS increase ≥5 points was not more prevalent for e-counseling, n=29 (29.6%) versus e-UC, n=32 (34.0%), P=0.51. E-Counseling versus e-UC increased total logon weeks (P=0.02), logon hours (P=0.001), and logons (P<0.001). Only e-counseling showed a positive association between 12-month KCCQ-OS tertile and logon weeks (P=0.04) and logon hours (P=0.004). E-Counseling increased CHF self-care behavior and diet but not 6-minute walk test or 4-day step count. CONCLUSIONS: The primary KCCQ-OS end point was negative for this trial. Only e-counseling showed a positive association between program engagement and 12-month KCCQ-OS tertile, and it improved CHF self-care behavior and diet. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01864369.

Lipoprotein(a) the Insurgent: A New Insight into the Structure, Function, Metabolism, Pathogenicity, and Medications Affecting Lipoprotein(a) Molecule.

Lipoprotein(a) [Lp(a)], aka "Lp little a", was discovered in the 1960s in the lab of the Norwegian physician Kåre Berg. Since then, we have greatly improved our knowledge of lipids and cardiovascular disease (CVD). Lp(a) is an enigmatic class of lipoprotein that is exclusively formed in the liver and comprises two main components, a single copy of apolipoprotein (apo) B-100 (apo-B100) tethered to a single copy of a protein denoted as apolipoprotein(a) apo(a). Plasma levels of Lp(a) increase soon after birth to a steady concentration within a few months of life. In adults, Lp(a) levels range widely from <2 to 2500 mg/L. Evidence that elevated Lp(a) levels >300 mg/L contribute to CVD is significant. The improvement of isoform-independent assays, together with the insight from epidemiologic studies, meta-analyses, genome-wide association studies, and Mendelian randomization studies, has established Lp(a) as the single most common independent genetically inherited causal risk factor for CVD. This breakthrough elevated Lp(a) from a biomarker of atherosclerotic risk to a target of therapy. With the emergence of promising second-generation antisense therapy, we hope that we can answer the question of whether Lp(a) is ready for prime-time clinic use. In this review, we present an update on the metabolism, pathophysiology, and current/future medical interventions for high levels of Lp(a).

Systematic mapping of free energy landscapes of a growing filamin domain during biosynthesis.

Cotranslational folding (CTF) is a fundamental molecular process that ensures efficient protein biosynthesis and minimizes the formation of misfolded states. However, the complexity of this process makes it extremely challenging to obtain structural characterizations of CTF pathways. Here, we correlate observations of translationally arrested nascent chains with those of a systematic C-terminal truncation strategy. We create a detailed description of chain length-dependent free energy landscapes associated with folding of the FLN5 filamin domain, in isolation and on the ribosome, and thus, quantify a substantial destabilization of the native structure on the ribosome. We identify and characterize two folding intermediates formed in isolation, including a partially folded intermediate associated with the isomerization of a conserved cis proline residue. The slow folding associated with this process raises the prospect that neighboring unfolded domains might accumulate and misfold during biosynthesis. We develop a simple model to quantify the risk of misfolding in this situation and show that catalysis of folding by peptidyl-prolyl isomerases is sufficient to eliminate this hazard.

Menstrual Blood Human Papillomavirus DNA and TAP1 Gene Polymorphisms as Potential Biomarkers for Screening and Monitoring of Cervical Squamous Intraepithelial Lesion.

Background: Human papillomavirus (HPV) is a known causative factor in the etiology of cervical cancer. Methods: HPV DNA genotyping was performed in menstrual blood (MB) collected in napkins from patients with cervical intraepithelial neoplasia (CIN), HPV infection and sexually active apparently normal subjects. In the same patient cohort, MB TAP1 I333V and TAP1 D637G gene polymorphisms were examined. Results: The sensitivity, specificity, and positive and negative predictive values of HPV DNA in the detection of CIN or HPV infection were 83% (223 of 268), 98% (131 of 134), 99% (223 of 226), and 74% (131 of 176), respectively. Moreover, HPV DNA was found in 24% (28/118) patients who had loop electrosurgical excision procedure treatment and 0% (0/76) HPV infected or CIN1 patient with proven recovery. On the other hand, the risk of developing high-grade CIN was significantly reduced for AG and GG genotypes compared with AA genotype and for carriers with a G allele compared with those with an A allele for both polymorphisms. Conclusions: MB HPV DNA is a potential noninvasive marker for screening and monitoring of squamous intraepithelial lesion. Together with TAP1 I333V and TAP1 D637G gene polymorphisms, the combined test may be useful for stratifying high-risk patients for better follow-up strategies.

Effect of Lowering Diastolic Pressure in Patients With and Without Cardiovascular Disease: Analysis of the SPRINT (Systolic Blood Pressure Intervention Trial).

Systolic and diastolic blood pressure thresholds, below which cardiovascular events increase, are widely debated. Using data from the SPRINT (Systolic Blood Pressure Intervention Trial), we evaluated the relation between systolic and diastolic pressure and cardiovascular events among 1519 participants with or 7574 without prior cardiovascular disease. Using Cox regression, we examined the composite risk of myocardial infarction, other acute coronary syndrome, stroke, heart failure, or cardiovascular death, and follow-up systolic and diastolic pressure were analyzed as time-dependent covariates for a median of 3.1 years. Models were adjusted for age, sex, baseline systolic pressure, body mass index, 10-year Framingham risk score, and estimated glomerular filtration rate. A J-shaped relationship with diastolic pressure was observed in both treatment arms in patients with or without cardiovascular disease (P nonlinearity≤0.002). When diastolic pressure fell <55 mm Hg, the hazards were at least 25% higher relative to 70 mm Hg (P=0.29). The hazard ratios (95% CI) of diastolic pressure <55 mm Hg versus 55 to 90 mm Hg were 1.68 (1.16-2.43), P value 0.006 and 1.52 (0.99-2.34), P value 0.06 in patients without and with prior cardiovascular disease, respectively. After adjusting for follow-up diastolic pressure, follow-up systolic pressure was not associated with the outcome in those without prior cardiovascular disease (P=0.64). In those with cardiovascular disease, adjusting for diastolic pressure, follow-up systolic pressure was associated with the risk in the intensive arm (hazard ratio per 10 mm Hg decrease, 0.86; 95% CI, 0.75-0.99; P interaction=0.02). Although the observed J-shaped relationship may be because of reverse causality in the SPRINT population, we advise caution in aggressively lowering diastolic pressure.

The Impact of Reduced Cardiac Rehabilitation on Maximal Treadmill Exercise Time: A RANDOMIZED CONTROLLED TRIAL.

PURPOSE: Cardiac rehabilitation programs (CRPs) remain underutilized partly because of access barriers. We therefore evaluated a CRP with fewer center-based sessions (rCRP) compared with standard CRP (sCRP) with respect to changes in exercise capacity and cardiac risk factors. METHODS: In this randomized controlled noninferiority trial, primary and secondary prevention patients at low and moderate risk were randomized to an sCRP (n = 60) or an rCRP (n = 61). Over 4 months, sCRP and rCRP participants attended 32 and 10 on-site cardiac rehabilitation sessions, respectively. The primary outcome was the difference in the change in exercise capacity from baseline at 4 and 16 months between the groups measured in seconds from a maximal treadmill exercise test. Noninferiority of the rCRP was tested with mixed-effects model analysis with a cut point of 60 seconds for the upper value of the group estimate. RESULTS: Attendance was higher for the rCRP group (97% ± 63% vs 71% ± 22%, P = .002). Over 16 months, exercise test time increased for the sCRP (524 ± 168 to 604 ± 172 seconds, P < .01) and the rCRP (565 ± 183 to 640 ± 192 seconds, P < .01). The rCRP was not inferior to the sCRP regarding changes in treadmill time (48.47 seconds, P = .454). The rCRP was not inferior to the sCRP regarding metabolic and anthropometric risk factors. CONCLUSION: Our findings suggest that, for a selected group of low-/moderate-risk patients, the number of center-based CRP exercise sessions can be decreased while maintaining reduced cardiovascular risk factors.

Prevalence and Associated Clinical Characteristics of Exercise-Induced ST-Segment Elevation in Lead aVR.

BACKGROUND: Exercise-induced ST-segment elevation (STE) in lead aVR may be an important indicator of prognostically important coronary artery disease (CAD). However, the prevalence and associated clinical features of exercise-induced STE in lead aVR among consecutive patients referred for exercise stress electrocardiography (ExECG) is unknown. METHODS: All consecutive patients receiving a Bruce protocol ExECG for the diagnosis of CAD at a tertiary care academic center were included over a two-year period. Clinical characteristics, including results of coronary angiography, were compared between patients with and without exercise-induced STE in lead aVR. RESULTS: Among 2227 patients undergoing ExECG, exercise-induced STE ≥1.0mm in lead aVR occurred in 3.4% of patients. Patients with STE in lead aVR had significantly lower Duke Treadmill Scores (DTS) (-0.5 vs. 7.0, p<0.01) and a higher frequency of positive test results (60.2% vs. 7.3%, p<0.01). Furthermore, patients with STE in lead aVR were more likely to undergo subsequent cardiac catheterization than those without STE in lead aVR (p<0.01, odds ratio = 4.2). CONCLUSIONS: Among patients referred for ExECG for suspected CAD, exercise-induced STE in lead aVR was associated with a higher risk DTS, an increased likelihood of a positive ExECG, and referral for subsequent coronary angiography. These results suggest that exercise-induced STE in lead aVR may represent a useful ECG feature among patients undergoing ExECG in the risk stratification of patients.

The First Dedicated Cardiac Rehabilitation Program for Patients With Spontaneous Coronary Artery Dissection: Description and Initial Results.

BACKGROUND: Spontaneous coronary artery dissection (SCAD) is an important cause of myocardial infarction in women, but the role of rehabilitation after SCAD is unclear. METHODS: We designed a dedicated SCAD cardiac rehabilitation (SCAD-CR) program for our SCAD survivors at Vancouver General Hospital. This program encompasses a multidisciplinary approach including exercise rehabilitation, psychosocial counselling, dietary and cardiovascular disease education, and peer group support. Exercise and educational classes were scheduled weekly with a targeted participation of 6 months. Psychosocial counselling, mindful living sessions, social worker and psychiatry evaluations, and peer-group support were offered. RESULTS: We report our first consecutive cohort of 70 SCAD women who joined SCAD-CR from November 2011 to April 2015. The average age was 52.3 ± 8.4 years. Mean participation duration was 12.4 ± 10.5 weeks; 28 completed 6 months, 48 completed ≥ 1 month. At entry, 44 (62.9%) had recurrent chest pains and average metabolic equivalents on exercise treadmill test was 10.1 ± 3.3. At program exit, the proportion with recurrent chest pains was lower (37.1%) and average metabolic equivalents was higher 11.5 ± 3.5 (both P < 0.001). There was a significant improvement in the STOP-D depression questionnaire, with mean scores of 13.0 ± 1.4 before and 8.0 ± 1.7 after the SCAD-CR (P = 0.046). Twenty (28.6%) social worker referrals and 19 (27.1%) psychiatry referrals were made. Mean follow-up was 3.8 ± 2.9 years from the presenting SCAD event, and the major cardiac adverse event rate was 4.3%, lower than our non-SCAD-CR cohort (n = 145; 26.2%; P < 0.001). CONCLUSIONS: This is the first dedicated SCAD-CR program to address the unique exercise and psychosocial needs of SCAD survivors. Our program appears safe and beneficial in improving chest pain, exercise capacity, psychosocial well-being and cardiovascular events.

Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome-nascent chain complexes.

The translational diffusion of macromolecules can be examined non-invasively by stimulated echo (STE) NMR experiments to accurately determine their molecular sizes. These measurements can be important probes of intermolecular interactions and protein folding and unfolding, and are crucial in monitoring the integrity of large macromolecular assemblies such as ribosome-nascent chain complexes (RNCs). However, NMR studies of these complexes can be severely constrained by their slow tumbling, low solubility (with maximum concentrations of up to 10 µM), and short lifetimes resulting in weak signal, and therefore continuing improvements in experimental sensitivity are essential. Here we explore the use of the paramagnetic longitudinal relaxation enhancement (PLRE) agent NiDO2A on the sensitivity of (15)N XSTE and SORDID heteronuclear STE experiments, which can be used to monitor the integrity of these unstable complexes. We exploit the dependence of the PLRE effect on the gyromagnetic ratio and electronic relaxation time to accelerate recovery of (1)H magnetization without adversely affecting storage on N z during diffusion delays or introducing significant transverse relaxation line broadening. By applying the longitudinal relaxation-optimized SORDID pulse sequence together with NiDO2A to 70S Escherichia coli ribosomes and RNCs, NMR diffusion sensitivity enhancements of up to 4.5-fold relative to XSTE are achieved, alongside ~1.9-fold improvements in two-dimensional NMR sensitivity, without compromising the sample integrity. We anticipate these results will significantly advance the use of NMR to probe dynamic regions of ribosomes and other large, unstable macromolecular assemblies.