Subunit asymmetry and roles of conformational switching in the hexameric AAA+ ring of ClpX

The hexameric AAA+ ring of Escherichia coli ClpX, an ATP-dependent machine for protein unfolding and translocation, functions with the ClpP peptidase to degrade target substrates. For efficient function, ClpX subunits must switch between nucleotide-loadable (L) and nucleotide-unloadable (U) conformations, but the roles of switching are uncertain. Moreover, it is controversial whether working AAA+-ring enzymes assume symmetric or asymmetric conformations. Here, we show that a covalent ClpX ring with one subunit locked in the U conformation catalyzes robust ATP hydrolysis, with each unlocked subunit able to bind and hydrolyze ATP, albeit with highly asymmetric position-specific affinities. Preventing U↔L interconversion in one subunit alters the cooperativity of ATP hydrolysis and reduces the efficiency of substrate binding, unfolding and degradation, showing that conformational switching enhances multiple aspects of wild-type ClpX function. These results support an asymmetric and probabilistic model of AAA+-ring activity.National Institutes of Health (U.S.) (Grant GM-101988)Massachusetts Institute of Technology (Poitras Predoctoral Fellowship

Mechanochemical basis of protein degradation by a double-ring AAA+ machine

Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ​ClpA enzyme from Escherichia coli, in complex with the ​ClpP peptidase, mechanically degrades proteins. We demonstrate that ​ClpA unfolds some protein substrates substantially faster than does the single-ring ​ClpX enzyme, which also degrades substrates in collaboration with ​ClpP. We find that ​ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ​ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Grant AI-16892

Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection

BACKGROUND Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients. Recurrences are common after antibiotic therapy. Actoxumab and bezlotoxumab are human monoclonal antibodies against C. difficile toxins A and B, respectively. METHODS We conducted two double-blind, randomized, placebo-controlled, phase 3 trials, MODIFY I and MODIFY II, involving 2655 adults receiving oral standard-of-care antibiotics for primary or recurrent C. difficile infection. Participants received an infusion of bezlotoxumab (10 mg per kilogram of body weight), actoxumab plus bezlotoxumab (10 mg per kilogram each), or placebo; actoxumab alone (10 mg per kilogram) was given in MODIFY I but discontinued after a planned interim analysis. The primary end point was recurrent infection (new episode after initial clinical cure) within 12 weeks after infusion in the modified intention-to-treat population. RESULTS In both trials, the rate of recurrent C. difficile infection was significantly lower with bezlotoxumab alone than with placebo (MODIFY I: 17% [67 of 386] vs. 28% [109 of 395]; adjusted difference, −10.1 percentage points; 95% confidence interval [CI], −15.9 to −4.3; P<0.001; MODIFY II: 16% [62 of 395] vs. 26% [97 of 378]; adjusted difference, −9.9 percentage points; 95% CI, −15.5 to −4.3; P<0.001) and was significantly lower with actoxumab plus bezlotoxumab than with placebo (MODIFY I: 16% [61 of 383] vs. 28% [109 of 395]; adjusted difference, −11.6 percentage points; 95% CI, −17.4 to −5.9; P<0.001; MODIFY II: 15% [58 of 390] vs. 26% [97 of 378]; adjusted difference, −10.7 percentage points; 95% CI, −16.4 to −5.1; P<0.001). In prespecified subgroup analyses (combined data set), rates of recurrent infection were lower in both groups that received bezlotoxumab than in the placebo group in subpopulations at high risk for recurrent infection or for an adverse outcome. The rates of initial clinical cure were 80% with bezlotoxumab alone, 73% with actoxumab plus bezlotoxumab, and 80% with placebo; the rates of sustained cure (initial clinical cure without recurrent infection in 12 weeks) were 64%, 58%, and 54%, respectively. The rates of adverse events were similar among these groups; the most common events were diarrhea and nausea. CONCLUSIONS Among participants receiving antibiotic treatment for primary or recurrent C. difficile infection, bezlotoxumab was associated with a substantially lower rate of recurrent infection than placebo and had a safety profile similar to that of placebo. The addition of actoxumab did not improve efficacy. (Funded by Merck; MODIFY I and MODIFY II ClinicalTrials.gov numbers, NCT01241552 and NCT01513239.

The use of open data as a material for learning

Open data has potential value as a material for use in learning activities. However, approaches to harnessing this are not well understood or in mainstream use in education. In this research, early adopters from a diverse range of educational projects and teaching settings were interviewed to explore their rationale for using open data in teaching, how suitable activity designs could be achieved, and the practical challenges of using open data. A thematic analysis was conducted to identify patterns and relationships in these open data-based practices that have already emerged. A document analysis of teaching materials and other related artefacts was used to augment and validate the findings. Drawing on this, common approaches and issues are identified, and a conceptual framework to support greater use of open data by educators is described. This paper also highlights where existing concepts in education and educational technology research, including inquiry-based learning, authenticity, motivation, dialogue, and personalisation can help us to understand the value and challenges of using open data in education

Moving forward during major goal blockage: situational goal adjustment in women facing infertility

Individuals confronting chronic medical conditions often face profound challenges to cherished life goals. The primary aim of this study was to examine the associations of goal adjustment with psychological adjustment in the context of infertility. At study entry (T1; n = 97) and 6 months later (T2; n = 47), women in fertility treatment completed measures of goal blockage, goal adjustment ability, and psychological adjustment. At T1, greater perceived and actual goal blockage were related to negative psychological adjustment. Ability to disengage from the goal of biological parenthood was associated with less infertility-specific thought intrusion, whereas engagement with other goals was related to fewer depressive symptoms and greater positive states of mind. Greater general goal engagement was protective against the negative relationships between low goal disengagement and the dependent variables. Promoting letting go of the unattainable and investing in the possible may be a useful intervention to foster well-being among individuals experiencing profound goal blockage

Patient-Perceived Changes in the System of Values After Cancer Diagnosis

A cross-sectional study investigated changes in patients’ value systems following a diagnosis of cancer. Fifty patients at 1 to 6 months following cancer diagnosis, were asked to compare their current values with their recollection of past values. Using the Rokeach Value Survey we obtained statistically significant results showing that twenty-seven out of thirty-six values changed their importance from the patients’ perspective: 16 values significantly increased, while 11 values significantly decreased in importance. Changes with respect to nine values were insignificant. We indentified clusters of values increasing in importance the most: Religious morality (Salvation, Forgiving, Helpful, Clean), Personal orientation (Self-Respect, True Friendship, Happiness), Self-constriction (Self-Controlled, Obedient, Honest), Family security (Family Security, Responsible), and Delayed gratification (Wisdom, Inner Harmony). We also observed that the following value clusters decreased in importance: Immediate gratification (An Exciting Life, Pleasure, A Comfortable Life); Self-expansion (Capable, Ambitious, Broadminded), Competence (A Sense of Accomplishment, Imaginative, Intellectual). The remaining values belonged to clusters that as a group changed slightly or not at all. Practical implications of the study are discussed

Cognitive engagement in the problem-based learning classroom

The objective of the present study was to examine to what extent autonomy in problem-based learning (PBL) results in cognitive engagement with the topic at hand. To that end, a short self-report instrument was devised and validated. Moreover, it was examined how cognitive engagement develops as a function of the learning process and the extent to which cognitive engagement determines subsequent levels of cognitive engagement during a one-day PBL event. Data were analyzed by means of confirmatory factor analysis, repeated measures ANOVA, and path analysis. The results showed that the new measure of situational cognitive engagement is valid and reliable. Furthermore, the results revealed that students’ cognitive engagement significantly increased as a function of the learning event. Implications of these findings for PBL are discussed

Selective Transmission of R5 HIV-1 over X4 HIV-1 at the Dendritic Cell–T Cell Infectious Synapse Is Determined by the T Cell Activation State

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of T cell immunity against HIV. On the other hand, due to the susceptibility of DCs to HIV infection, virus replication is strongly enhanced in DC–T cell interaction via an immunological synapse formed during the antigen presentation process. When HIV-1 is isolated from individuals newly infected with the mixture of R5 and X4 variants, R5 is predominant, irrespective of the route of infection. Because the early massive HIV-1 replication occurs in activated T cells and such T-cell activation is induced by antigen presentation, we postulated that the selective expansion of R5 may largely occur at the level of DC–T cell interaction. Thus, the immunological synapse serves as an infectious synapse through which the virus can be disseminated in vivo. We used fluorescent recombinant X4 and R5 HIV-1 consisting of a common HIV-1 genome structure with distinct envelopes, which allowed us to discriminate the HIV-1 transmitted from DCs infected with the two virus mixtures to antigen-specific CD4+ T cells by flow cytometry. We clearly show that the selective expansion of R5 over X4 HIV-1 did occur, which was determined at an early entry step by the activation status of the CD4+ T cells receiving virus from DCs, but not by virus entry efficiency or productivity in DCs. Our results imply a promising strategy for the efficient control of HIV infection

The CDKL5 disorder is an independent clinical entity associated with early-onset encephalopathy

The clinical understanding of the CDKL5 disorder remains limited, with most information being derived from small patient groups seen at individual centres. This study uses a large international data collection to describe the clinical profile of the CDKL5 disorder and compare with Rett syndrome (RTT). Information on individuals with cyclin-dependent kinase-like 5 (CDKL5) mutations (n=86) and females with MECP2 mutations (n=920) was sourced from the InterRett database. Available photographs of CDKL5 patients were examined for dysmorphic features. The proportion of CDKL5 patients meeting the recent Neul criteria for atypical RTT was determined. Logistic regression and time-to-event analyses were used to compare the occurrence of Rett-like features in those with MECP2 and CDKL5 mutations. Most individuals with CDKL5 mutations had severe developmental delay from birth, seizure onset before the age of 3 months and similar non-dysmorphic features. Less than one-quarter met the criteria for early-onset seizure variant RTT. Seizures and sleep disturbances were more common than in those with MECP2 mutations whereas features of regression and spinal curvature were less common. The CDKL5 disorder presents with a distinct clinical profile and a subtle facial, limb and hand phenotype that may assist in differentiation from other early-onset encephalopathies. Although mutations in the CDKL5 gene have been described in association with the early-onset variant of RTT, in our study the majority did not meet these criteria. Therefore, the CDKL5 disorder should be considered separate to RTT, rather than another variant

Motivated learning with digital learning tasks: what about autonomy and structure?

Article about Motivated learning with digital learning tasks: what about autonomy and structure