The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational ...modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s). Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database) with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate sequence using machine learning techniques. It is freely available at http://lightning.med.monash.edu.au/PROSPER/.
Educators often use stories, humor, surprise, images, or other ploys to catch students' interest. This form of interest, often called "situational interest" because of how it is triggered by ...contextual cues, can facilitate learning to some degree. Yet it also may carry risks. This article proposes one such potential risk: flawed metacognition, in the form of inflated judgments of learning (JOLs) and overconfident estimates of future performance (calibration bias). Two experiments tested this hypothesis. In each, college students (Ns = 201, 196) read passages on a novel topic (the physics of lighEDU-2020-0310 tning in Study 1; the Hare Krishna organization in Study 2), crafted to be relatively dull or interesting. They then reported their interest, JOLs, and performance estimates before completing a test of their topic knowledge. Otherwise, their methods differed in several ways, such as how situational interest was induced and how performance was measured. Despite those differences, the findings from each study confirmed that situational interest inflated participants' JOLs and promoted overconfident performance estimates. Furthermore, Study 2 showed that those inaccurate metacognitive judgments affect studying decisions; due to their inflated JOLs, participants allocated less of their limited time for restudy to the fun passages. Potential moderators are considered, as are implications for interest theory, for metacognition theory, and for educators more generally.
Educational Impact and Implications StatementEngaging students has long been a major priority for educators and students alike, compelling teachers to find ways to trigger and maintain students' "situational interest" through humor, animation, and other ploys. We explored a possible metacognitive risk to those ploys. In two experiments, we found that raising students' interest (relative to a control group) in novel topics made them overestimate how well they learned the topics and how well they could later answer questions on these topics. This overconfidence, in turn, convinced them to avoid studying the fun topics when considering which topics to revisit for additional study. The findings raise intriguing implications for educators about when and how best to trigger students' interest when learning.
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QRS duration is an established risk factor among patients with heart failure. How change in QRS duration relates to heart failure outcomes has had limited study. In this post-hoc analysis of the ...Beta-Blocker Evaluation of Survival Trial, we demonstrated that QRS duration change from baseline to 3 months is independently associated with long-term survival and left ventricle ejection fraction.
Mitochondria are found in all eukaryotic cells and derive from a bacterial endosymbiont
1, 2. The evolution of a protein import system was a prerequisite for the conversion of the endosymbiont into ...a true organelle. Tom40, the essential component of the protein translocase of the outer membrane, is conserved in mitochondria of almost all eukaryotes but lacks bacterial orthologs
3–6. It serves as the gateway through which all mitochondrial proteins are imported. The parasitic protozoa
Trypanosoma brucei and its relatives do not have a Tom40-like protein, which raises the question of how proteins are imported by their mitochondria
7, 8. Using a combination of bioinformatics and in vivo and in vitro studies, we have discovered that
T. brucei likely employs a different import channel, termed ATOM (
archaic
translocase of the
outer
mitochondrial membrane). ATOM mediates the import of nuclear-encoded proteins into mitochondria and is essential for viability of trypanosomes. It is not related to Tom40 but is instead an ortholog of a subgroup of the Omp85 protein superfamily that is involved in membrane translocation and insertion of bacterial outer membrane proteins
9. This suggests that the protein import channel in trypanosomes is a relic of an archaic protein transport system that was operational in the ancestor of all eukaryotes.
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► ATOM, the outer membrane translocase of trypanosomatids, is of bacterial type ► ATOM provides a missing link for the evolution of mitochondrial protein import ► ATOM suggests that trypanosomatids are early-diverging eukaryotes
In response to the health threats posed by toxic lead to humans, scavenging wildlife and the environment, there is currently a focus on transitioning from lead-based to lead-free bullets for shooting ...of wild animals. We compared efficiency metrics and terminal ballistic performance for lead-based and lead-free (non-lead) bullets for aerial shooting of wild pigs (Sus scrofa) in eastern Australia. Ballistic testing revealed that lead-based and lead-free bullets achieved similar performance in precision and muzzle kinetic energy (E0) levels (3337.2 J and 3345.7 J, respectively). An aerial shooting trial was conducted with wild pigs shot with one type of lead-based and one type of lead-free bullets under identical conditions. Observations were made from 859 shooting events (n = 430 and 429 respectively), with a sub-set of pigs examined via gross post-mortem (n = 100 and 108 respectively), and a further sub-set examined via radiography (n = 94 and 101 respectively). The mean number of bullets fired per pig killed did not differ greatly between lead-based and lead-free bullets respectively (4.09 vs 3.91), nor did the mean number of bullet wound tracts in each animal via post-mortem inspection (3.29 vs 2.98). However, radiography revealed a higher average number of fragments per animal (median >300 vs median = 55) and a broader distribution of fragments with lead-based bullets. Our results suggest that lead-based and lead-free bullets are similarly effective for aerial shooting of wild pigs, but that the bullet types behave differently, with lead-based bullets displaying a higher degree of fragmentation. These results suggest that aerial shooting may be a particularly important contributor to scavenging wildlife being exposed to lead and that investigation of lead-free bullets for this use should continue.
The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to ...increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis.
Recognition of DNA by the innate immune system is central to antiviral and antibacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in ...melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.
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► Electrostatic attraction underlies innate dsDNA recognition by the HIN domains ► Both OB folds and the linker between them engage the dsDNA backbone ► An autoinhibited state of AIM2 is activated by DNA that liberates the PYD domain ► DNA serves as an oligomerization platform for the inflammasome assembly
ObjectiveThe frequency and predictors of improvement in left ventricular ejection fraction (LVEF) in ischaemic cardiomyopathy and its association with mortality is poorly understood. We sought to ...assess the predictors of LVEF improvement ≥10% and its effect on mortality.MethodsWe compared characteristics of patients enrolled in The Surgical Treatment for Ischaemic Heart Failure (STICH) trial with and without improvement of LVEF ≥10% at 24 months. A logistic regression model was constructed to determine the independent predictors of LVEF improvement. A Cox proportional hazards model was created to assess the independent association of improvement in LVEF ≥10% with mortality.ResultsOf the 1212 patients enrolled in STICH, 618 underwent echocardiographic assessment of LVEF at baseline and 24 months. Of the patients randomised to medical therapy plus coronary artery bypass graft surgery (CABG), 58 (19%) had an improvement in LVEF >10% compared with 51 (16%) patients assigned to medical therapy alone (p=0.30). Independent predictors of LVEF improvement >10% included prior myocardial infarction (OR 0.44, 95% CI: 0.28 to 0.71, p=0.001) and lower baseline LVEF (OR 0.94, 95% CI: 0.91 to 0.97, p<0.001). Improvement in LVEF >10% (HR 0.61, 95% CI: 0.44 to 0.84, p=0.004) and randomisation to CABG (HR 0.72, 95% CI: 0.57 to 0.90, p=0.004) were independently associated with a reduced hazard of mortality.ConclusionsImprovement of LVEF ≥10% at 24 months was uncommon in patients with ischaemic cardiomyopathy, did not differ between patients assigned to CABG and medical therapy or medical therapy alone and was independently associated with reduced mortality.Trial registration number NCT00023595.
Background The optimal threshold of left ventricular ejection fraction (LVEF) that should prompt aortic valve replacement (AVR) in asymptomatic patients with high-gradient severe aortic stenosis (AS) ...is controversial. The aim of this study was to assess the relationship between LVEF and mortality benefit in comparing early AVR versus watchful waiting in asymptomatic patients with severe AS. Methods and Results MEDLINE, Embase, Web of Science, and Google Scholar were searched for observational studies and randomized controlled trials on adults with asymptomatic severe AS. Severe AS was defined by a peak aortic velocity ≥4 m/s and/or mean aortic valve gradient ≥40 mm Hg and/or calculated aortic valve area <1.0 cm
or an indexed valve area <0.6 cm
. Studies comparing AVR with conservative management were included and meta-analysis on all-cause mortality was performed. Ten eligible studies were identified with a total of 3332 patients. In 5 observational studies comparing early AVR versus watchful waiting, our meta-analysis showed early AVR was associated with lower mortality with a hazard ratio (HR) of 0.41 (CI, 0.23-0.71;
<0.01). In 4 observational studies comparing AVR versus no AVR, our meta-analysis showed AVR was associated with lower mortality with a HR of 0.31 (CI, 0.17-0.58;
<0.001). In a meta-regression analysis pooling all 10 studies, there was no statistically significant correlation between study mean LVEF and the size of mortality benefit of AVR (
=0.83). Conclusions Among asymptomatic patients with high-gradient severe AS, AVR was associated with a mortality benefit across the spectrum of LVEF. Our study calls into question the need of an LVEF threshold for recommending AVR in this patient population.
The β‐barrel assembly machinery (BAM) complex drives the assembly of β-barrel proteins into the outer membrane of gram-negative bacteria. It is composed of five subunits: BamA, BamB, BamC, BamD, and ...BamE. We find that the BAM complex isolated from the outer membrane of Escherichia coli consists of a core complex of BamA:B:C:D:E and, in addition, a BamA:B module and a BamC:D module. In the absence of BamC, these modules are destabilized, resulting in increased protease susceptibility of BamD and BamB. While the N-terminus of BamC carries a highly conserved region crucial for stable interaction with BamD, immunofluorescence, immunoprecipitation, and protease-sensitivity assays show that the C-terminal domain of BamC, composed of two helix-grip motifs, is exposed on the surface of E. coli. This unexpected topology of a bacterial lipoprotein is reminiscent of the analogous protein subunits from the mitochondrial β-barrel insertion machinery, the SAM complex. The modular arrangement and topological features provide new insight into the architecture of the BAM complex, towards a better understanding of the mechanism driving β-barrel membrane protein assembly.
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► The bacterial BAM can dissociate into smaller stable modules. ► The BamC:D module is stabilized via a highly conserved region at the N-terminus of BamC. ► The large folded C-terminus of BamC is present on the bacterial cell surface. ► BamC provides a novel topology for surface‐exposed proteins, changing the current view of BAM assembly.
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