Search

Membrane-embedded β-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. β-barrels are highly structured domains formed by a series of antiparallel β-strands. Each β-strand is locked in position by hydrogen bonds between its polypeptide backbone and those of the two neighbouring strands in the barrel structure. Some transmembrane β-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembrane β-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as a rapid method to investigate the folding of transmembrane β-barrels., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In vivo site-directed photocrosslinking provides a means to probe the vicinity of proteins in their native cellular environment. Because this method relies on the incorporation of unnatural amino acid analogs that are similar in size to natural amino acids, crosslink products are indicative of direct protein-protein interactions. Here, we present the use of this approach to monitor both transient and stable interactions of two proteins of the envelope of Escherichia coli. First, we describe a protocol to characterize the interactions of a secretory protein as it transverses the bacterial envelope with temporal and spatial resolution. We combine site-directed photocrosslinking with radiolabeling of proteins and lipids. Second, we describe a method to purify a photocrosslinked partner protein and to analyze it by mass spectrometry. We use in-gel protein digestion and peptide fragmentation by MALDI-TOF/TOF tandem mass spectrometry to determine the site of interaction on the photocrosslinked partner., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We report the case of a 73-year-old male admitted for epigastric pain and syncope with increased troponin level and a rare electrocardiogram (a single‑lead ST-elevation). Coronary angiography showed multi-vessel coronary artery disease. The patient underwent coronary angioplasty with drug-eluting stenting on left anterior descending coronary artery and drug eluting ballooning on first diagonal ostium. Coronary revascularization was completed with a staged stenting on left circumflex artery and right coronary artery. In rare cases of acute coronary syndrome, even isolated ST single lead anomalies may underlie multivessel coronary disease., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Insertion of lipopolysaccharide (LPS) into the bacterial outer membrane (OM) is mediated by a druggable OM translocon consisting of a β-barrel membrane protein, LptD, and a lipoprotein, LptE. The β-barrel assembly machinery (BAM) assembles LptD together with LptE at the OM. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls translocon activation. Here we report the discovery of LptM (formerly YifL), a lipoprotein conserved in Enterobacteriaceae, that assembles together with LptD and LptE at the BAM complex. LptM stabilizes a conformation of LptD that can efficiently acquire native disulfide bonds, whereas its inactivation makes disulfide bond isomerization by DsbC become essential for viability. Our structural prediction and biochemical analyses indicate that LptM binds to sites in both LptD and LptE that are proposed to coordinate LPS insertion into the OM. These results suggest that, by mimicking LPS binding, LptM facilitates oxidative maturation of LptD, thereby activating the LPS translocon., (© 2023. Springer Nature Limited.)

Background and Aim of the Study: The response to the increase in heart rate (HR) could be characterized by failure in both left ventricular (LV) and left atrial (LA) functions. This study aimed to evaluate the relationship between the increase in paced HR and the changes in LV and LA functions, assessed by two-dimensional speckle tracking analysis., Methods: In a group of patients with an implantable cardioverter defibrillator (ICD) or pacemaker, the atrial paced rhythm was progressively increased from 60 to 70, from 70 to 80, and from 80 to 90 beats per minute (bpm). For each paced HR, using two-dimensional speckle tracking analysis, LA reservoir (LAr), LA conduit (LAc), LA contraction (LAct), and LV global longitudinal strain (LV-GLS) were evaluated every 10 bpm., Results: Of the 45 patients enrolled, a significant reduction in LAr was observed at higher HR. However, when the patients were dichotomized according to the HR-related response of LV-GLS, the worsening of LAr was observed in those with LV-GLS worsening and not in those without (maximum LAR absolute changes -2.7 ± 7.2% vs. +2.7 ± 7.2%, respectively, p .028). Moreover, the worsening of LA and LV strain measures was associated with an increase in the estimated filling pressures., Conclusions: In patients with atrial paced rhythm, the increase in HR could be associated with worsening of LA and LV functions, as assessed by two-dimensional speckle tracking analyses. These results offer new data on HR-related atrioventricular function and could be useful for guiding the optimal HR responsiveness of the implanted devices., (© 2023 The Authors. Pacing and Clinical Electrophysiology published by Wiley Periodicals LLC.)

Background: In patients with recent ACS, the latest ESC/EAS guidelines for management of dyslipidaemia recommend intensification of LDL- C -lowering therapy ., Objective: Report a real-world picture of lipid-lowering therapy prescribed and cholesterol targets achieved in post-ACS patients before and after a specific educational program., Methods: Retrospective data collection prior to the educational course and prospective data collection after the course of consecutive very high-risk patients with ACS admitted in 2020 in 13 Italian cardiology departments, and with a non-target LDL-C level at discharge., Results: Data from 336 patients were included, 229 in the retrospective phase and 107 in the post-course prospective phase. At discharge, statins were prescribed in 98.1% of patients, alone in 62.3% of patients (65% of which at high doses) and in combination with ezetimibe in 35.8% of cases (52% at high doses). A significant reduction was obtained in total and LDL cholesterol (LDL-C) from discharge to the first control visit. Thirty-five percent of patients achieved a target LDL-C <55 mg/dL according to ESC 2019 guidelines. Fifty percent of patients achieved the <55 mg/dL target for LDL-C after a mean of 120 days from the ACS event., Conclusions: Our analysis, though numerically and methodologically limited, suggests that management of cholesterolaemia and achievement of LDL-C targets are largely suboptimal and need significant improvement to comply with the lipid-lowering guidelines for very high CV risk patients. Earlier high intensity statin combination therapy should be encouraged in patients with high residual risk., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V.)

Background: Catheter ablation (CA) is routinely used for the treatment of arrhythmias. Vascular complications are the most common complications during these procedures. Previous data reported that ultrasound (US)-guided puncture is a useful method to avoid vascular complications. We reported our experience using US-guided puncture in patients undergoing CA for arrhythmias., Methods: A total of 273 patients (mean age 57 ± 17 years; 58% male) were referred to our center for CA of arrhythmias from January 2016 to December 2019. All procedures were performed by expert operators, and US-guided vascular access was performed on all patients. Doppler sonography was performed the day after the procedure on all patients., Results: Eighty-four patients (31%) underwent atrioventricular nodal reentrant tachycardia ablation, 49 patients (18%) atrioventricular reentrant tachycardia ablation, 14 patients (5%) atrial tachycardia ablation, 25 patients (9%) atrial flutter ablation, 63 patients (23%) atrial fibrillation ablation, and 38 patients (14%) ventricular tachycardia ablation. Vascular pseudo-aneurysms and arteriovenous fistula were defined as major complications; furthermore, venous thrombosis and inguinal hematomas were as defined minor complications. The percentage of major vascular complications was 0.3% (1 arteriovenous fistula) and the percentage of minor vascular complications was 0.3% (1 venous thrombosis)., Discussion: Ultrasound-guided vascular puncture in patients undergoing CA is useful to improve procedural success and reduce complications., Competing Interests: Authors declare no conflict of interests for this article., (© 2022 The Authors. Journal of Arrhythmia published by John Wiley & Sons Australia, Ltd on behalf of the Japanese Heart Rhythm Society.)

Although children with Covid-19 generally present with mild symptoms or are often asymptomatic, there is increasing recognition of a delayed multi-organ inflammatory syndrome (MIS-C) following SARS-CoV-2 infection. We report the case of MIS-C associated arrhythmic myocarditis which recovered after anti-inflammatory therapy and immunoglobulin infusion., Competing Interests: Declaration of Competing Interest None to disclose., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The first approach for Type-2 myocardial infarction (T2MI) consists of the elimination of the condition determining the oxygen supply/demand mismatch. However, the long-term impact of medical therapy with beta blockers, statins, aspirin, and P2Y12 inhibitors, used in the case of Type-1 myocardial infarction has been poorly investigated and remains unclear. We, therefore, sought to assess the impact of medical therapy on 1-year mortality in patients with T2MI using a meta-regression analysis. A meta-regression analysis was performed with studies involving in patients with T2MI: 1-year all-cause mortality, rates of beta blockers, statins, aspirin, and P2Y12 inhibitors use were recorded and analyzed. After careful study selection, 8 observational studies were pooled in the analysis, including 3,756 in patients. During meta-regression analysis, a borderline correlation between rates of aspirin, P2Y12 inhibitors, and statins use and 1-year mortality (p = 0.087, p = 0.05, and p = 0.067, respectively) was found; no significant correlation was found at multivariable analysis. In conclusion, in a meta-regression analysis, no significant correlation was found between rates of use of usual drug therapy indicated for Type-1 myocardial infarction (statins, aspirin, P2Y12 inhibitors, β-blockers) and 1-year mortality in T2MI patients., Competing Interests: Disclosures The authors have no conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Percutaneous coronary intervention (PCI) represents the best therapeutic option for type-1 myocardial infarction (T1MI) in the majority of clinical settings; its role in the treatment of type-2 myocardial infarction (T2MI), however, remains unclear. We therefore sought to assess in a meta-regression analysis the impact of PCI rates on mortality in patients with T2MI according to available observational studies., Methods: We performed a meta-regression analysis including all the studies involving in-patients affected by T2MI. We excluded studies not reporting the rate of T2MI patients undergoing PCI and not specifying absolute in-hospital or 1-year all-cause mortality. In the meta-regression analysis we used the in-hospital mortality and 1-year mortality as dependent variables and the rate of PCI as independent; regression was weighted for studies' size., Results: After careful examination, 8 studies were selected for the assessment of in-hospital mortality and 8 for 1-year-mortality. We included 3155 and 3756 in-patients for in-hospital and 1-year mortality respectively. At meta-regression analysis, a borderline correlation between PCI rate and in-hospital mortality (p 0.05) and a statistically significant correlation with 1-year mortality (p < 0.01) in T2MI patients were found., Conclusions: In a meta-regression analysis higher rates of PCI on T2MI in-patients were associated with lower mortality rates both in-hospital and at 1 year. Whether this association is related to the direct effect of PCI or better general conditions of T2MI patients undergoing a PCI still remains unclear., Competing Interests: Declaration of Competing Interest None to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In Proteobacteria, integral outer membrane proteins (OMPs) are crucial for the maintenance of the envelope permeability barrier to some antibiotics and detergents. In Enterobacteria, envelope stress caused by unfolded OMPs activates the sigmaE (σ E ) transcriptional response. σ E upregulates OMP biogenesis factors, including the β-barrel assembly machinery (BAM) that catalyses OMP folding. Here we report that DolP (formerly YraP), a σ E -upregulated and poorly understood outer membrane lipoprotein, is crucial for fitness in cells that undergo envelope stress. We demonstrate that DolP interacts with the BAM complex by associating with outer membrane-assembled BamA. We provide evidence that DolP is important for proper folding of BamA that overaccumulates in the outer membrane, thus supporting OMP biogenesis and envelope integrity. Notably, mid-cell recruitment of DolP had been linked to regulation of septal peptidoglycan remodelling by an unknown mechanism. We now reveal that, during envelope stress, DolP loses its association with the mid-cell, thereby suggesting a mechanistic link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division., Competing Interests: DR, YY, LO, FR, CT, VM, LC, CM, CF, GM, JR, JM, AC, CA, DB, RI No competing interests declared, (© 2021, Ranava et al.)

In the recent past, low-molecular-weight heparin (LMWH) was the first choice in the treatment of cancer related venous thrombo-embolism (VTE). Evidence supporting the preferential use of direct anticoagulants (DOACs) in patients with cancer, instead, is less robust so far. We therefore aimed to assess in an updated meta-analysis of randomized controlled trials whether the use of DOACs may be associated with a more favorable profile when compared to LMWH. We performed a meta-analysis of RCTs enrolling patients with VTE and cancer. We assessed Mantel-Haenszel pooled estimates of risk ratios (RRs) and 95% CIs for recurrence of VTE, major bleeding, and mortality comparing subjects treated with DOACs with those with LMWH. After study selection, three RCTs (HOKUSAI-Cancer, SELECT-D and ADAM-VTE) were included for the analysis with an overall population of 1739 patients. DOACs patients had a lower incidence of 6-month recurrent VTE when compared to LMWHs (RR 0.56, 95% CI 0.40-0.79; p < 0.001). Incidence of major bleeding was not significantly different between DOACs and LMWH treated patients (RR 1.56, 95% CI 0.95-2.47, p = n.s.), and mortality rates were comparable (RR 1.03, 95% CI 0.91-2.47, p = n.s.). In a meta-analysis of RCTs therapy with DOACs was superior to LMWH in terms of efficacy and lower recurrence of VTE with a comparable safety profile in terms of bleeding events and complications.

Background: Dual antiplatelet therapy and anticoagulants may be required in the case of coexistence of coronary artery disease and atrial fibrillation (AF) undergoing (PCI), with associated increased bleeding rates. The introduction of direct oral anticoagulants (DOACs), however, significantly reduced the incidence of bleeding complications in this clinical setting of patients. We therefore sought to assess whether the recent publication of the AUGUSTUS and ENTRUST-AF PCI studies significantly impacted current evidence on the use of DOACs in AF patients treated with PCI., Methods: We performed a meta-analysis of randomized controlled studies enrolling patients with nonvalvular AF undergoing PCI. We assessed pooled estimates of risk ratios (RRs) and 95%CIs for any bleeding (AB), cardiovascular events (CVE), and death at follow-up: 12,542 patients have been included in the analysis. We particularly analyzed data comparing dual anti-thrombotic therapy (DOAC plus single anti-platelet therapy) with triple (DOAC plus dual anti-platelet therapy)., Results: When compared with patients receiving standard triple therapy with warfarin, patients receiving DOACs had a significantly lower risk of AB (RR 0.65; 95% CI, 0.61-0.70, p < 0.00001) and of MB (RR 0.63; 95% CI, 0.53-0.73, p < 0.00001). The risk of cardiovascular events and mortality were comparable between DOAC and VKA groups (RR 1.05, 95% CI 0.93-1.18, RR 1.14, 95% CI 0.94-1.37, respectively, p n.s.). Similar results were observed comparing triple therapy vs dual therapy., Conclusions: DOACs are safer than and as effective as warfarin when used in patients with AF undergoing PCI; dual therapy with DOACs is comparable to triple therapy in terms of safety and efficacy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Introduction: Catheter ablation (CA) of atrial fibrillation (AF) is an important rhythm control strategy for patients with drug-refractory AF. We aimed to perform an updated meta-analysis of direct oral anticoagulants (DOACs) vs vitamin K antagonists (VKAs) as uninterrupted anticoagulation in patients undergoing AF ablation to assess safety and efficacy of DOAC, after the publication of recent data on edoxaban in CA of AF., Methods: We performed a meta-analysis of RCTs enrolling patients undergoing AF ablation. We assessed Mantel-Haenszel pooled estimates of risk ratios (RRs) and 95% CIs for thromboembolic events, major bleeding (MB), and non-major bleeding (NMB)., Results: A total of 2118 patients have been included in the analysis. Compared with patients receiving VKA, patients receiving DOACs had a lower, although non-significant, risk of thromboembolic events (RR, 0.40; 95% CI, 0.09-1.76; P = 0.23). MB rates in patients treated with DOACs were statistically significantly lower than VKA (RR, 0.61; 95% CI, 0.39-0.93, P = 0.02). The incidence of NMB was not significantly different (RR, 0.98; 95% CI, 0.83-1.57, p n.s.)., Conclusions: In a meta-analysis of RCTs, an uninterrupted DOACs strategy for CA of AF appears to be superior to uninterrupted VKA in terms of safety; a non-significant trend favoring DOACs in terms of efficacy is also evident.

Mitochondrial preproteins contain amino-terminal presequences directing them to the presequence translocase of the mitochondrial inner membrane (TIM23 complex). Depending on additional downstream import signals, TIM23 either inserts preproteins into the inner membrane or translocates them into the matrix. Matrix import requires the coupling of the presequence translocase-associated motor (PAM) to TIM23. The molecular mechanisms coordinating preprotein recognition by TIM23 in the intermembrane space (IMS) with PAM activation in the matrix are unknown. Here we show that subsequent to presequence recognition in the IMS, the Tim50 matrix domain facilitates the recruitment of the coupling factor Pam17. Next, the IMS domain of Tim50 promotes PAM recruitment to TIM23. Finally, the Tim50 transmembrane segment stimulates the matrix-directed import-driving force exerted by PAM. We propose that recognition of preprotein segments in the IMS and transfer of signal information across the inner membrane by Tim50 determine import motor activation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The TIM23 complex mediates membrane insertion of presequence-containing mitochondrial proteins via a stop-transfer mechanism. Stop-transfer signals consist of hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex. However, it remains elusive which features of stop-transfer signals are discriminated by Mgr2. To determine the effects of Mgr2 on the TIM23-mediated stop-transfer pathway, we measured membrane insertion of model transmembrane segments of varied hydrophobicity and flanking charges in Mgr2-deletion or -overexpression yeast strains. We found that upon deletion of Mgr2, the threshold hydrophobicity for membrane insertion, as well as the requirement for matrix-facing positive charges, is reduced. These results imply that the Mgr2-mediated gatekeeper function is important for controlling membrane sorting of marginal stop-transfer signals., (© 2019 Federation of European Biochemical Societies.)

Mitochondria are pivotal organelles for cellular signaling and metabolism, and their dysfunction leads to severe cellular stress. About 60-70% of the mitochondrial proteome consists of preproteins synthesized in the cytosol with an amino-terminal cleavable presequence targeting signal. The TIM23 complex transports presequence signals towards the mitochondrial matrix. Ultimately, the mature protein segments are either transported into the matrix or sorted to the inner membrane. To ensure accurate preprotein import into distinct mitochondrial sub-compartments, the TIM23 machinery adopts specific functional conformations and interacts with different partner complexes. Regulatory subunits modulate the translocase dynamics, tailoring the import reaction to the incoming preprotein. The mitochondrial membrane potential and the ATP generated via oxidative phosphorylation are key energy sources in driving the presequence import pathway. Thus, mitochondrial dysfunctions have rapid repercussions on biogenesis. Cellular mechanisms exploit the presequence import pathway to monitor mitochondrial dysfunctions and mount transcriptional and proteostatic responses to restore functionality., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Persistent ST-segment elevation in acute coronary syndrome is associated with both short and long-term complications. By contrast, there is limited information about ST-elevation and its evolution during takotsubo (stress) cardiomyopathy (TTC)., Aim: To evaluate whether persistent downsloping ST-elevation in the early stages of TTC might correlate with short and long-term clinical events., Methods: One-hundred fifty-eight consecutive subjects with TTC were prospectively enrolled and assessed by electrocardiogram. Patients were classified in two groups according to the presence of downsloping ST-elevation ≥5 mm lasting at least 24 hr ("lambda-wave" ST-elevation group vs. without downsloping ST-elevation) in at least one/two contiguous leads., Results: Five (3.2%) patients, all female with a mean left ventricular ejection fraction 32 ± 5%, were included in the lambda-wave ST-elevation group. These patients were characterized by a higher prevalence of physical stressor (100% vs. 49%, p = 0.04) and higher admission and peak levels of troponin-I levels during hospitalization. Peak of ST-elevation in the lambda-wave ST-elevation group was reached 6 hr after admission and gradually decreased after 24 hr. In-hospital complications were observed in all the patients presenting lambda ST-elevation (100% vs. 23%, p = 0.03, OR: 29.1, p = 0.04); one patient presented endoventricular thrombosis and two died of cardiogenic shock. At long-term follow-up (mean 443 days), adverse events were observed in 80% of patients with lambda-wave ST-elevation (RR of adverse events at follow-up 32, p < 0.01)., Conclusion: Persistent downsloping lambda-wave ST-elevation during the acute phase of stress cardiomyopathy may be associated with a higher risk of adverse events at short and long-term follow-up., (© 2018 Wiley Periodicals, Inc.)

We report the case of an incomplete diagnosis of chronic thromboembolic pulmonary hypertension, with relevant prognostic implications, missing the presence of a primary pulmonary artery angiosarcoma. After the late neoplasm diagnosis, the patient, treated for months with riociguat, was considered inoperable and died soon after. This case highlights the need to manage patients with suspected pulmonary arterial hypertension by expert referral centers with specific and multi-professional expertise (heart and thoracic imaging) in order to avoid incomplete or delayed diagnoses.

The outer membrane (OM) of Gram-negative bacteria is an essential organelle that protects cells from external aggressions and mediates the secretion of virulence factors. Efficient assembly of integral OM β-barrel proteins (OMPs) is crucial for the correct functioning of the OM. Biogenesis of OMPs occurs in a stepwise manner that is finalized by the β-barrel assembly machinery (BAM complex). Some OMPs further require the translocation and assembly module (TAM) for efficient and correct integration into the OM. Both the BAM complex and the TAM contain a protein of the Omp85 superfamily and distinct interacting factors. Their mechanism of action, however, remains largely elusive. We summarize and discuss recent structural and biochemical analyses that are helping to elucidate the molecular pathways of OMP assembly.

Assessing the efficacy of revascularization therapy in patients with ST-segment elevation myocardial infarction (STEMI) is extremely important in order to guide subsequent management and assess prognosis. We aimed to determine the relationship between corrected QT-interval (QTc) changes on standard sequential ECG and myocardial salvage index in anterior STEMI patients after successful primary percutaneous coronary intervention. Fifty anterior STEMI patients treated by primary percutaneous coronary intervention underwent quantitative ECG analysis and cardiac magnetic resonance. For each patient the difference (ΔQTc) between the QTc of ischemic myocardium (maximum QTc in anterior leads) versus remote myocardium (minimum QTc in inferior leads) during the first six days after STEMI was measured. The QTc in anterior leads was significantly longer than QTc in inferior leads (p<0.0001). At multivariate analysis, ΔQTC and peak troponin I were the only independent predictors for late gadolium enhancement while ΔQTc and left ventricular ejection fraction were independent predictors of myocardial salvage index <60%. The receiver operative curve of ΔQTc showed an area under the curve of 0.77 to predict a myocardial salvage index <0.6. In conclusion, in a subset of patients with a first occurrence of early revascularized anterior STEMI, ΔQTc is inversely correlated with CMR-derived myocardial salvage index and may represent a useful parameter for assessing efficacy of reperfusion therapy.

Background: Prolonged QT corrected (QTc) intervals are associated with adverse cardiovascular outcomes both in healthy and high-risk populations. Our objective was to evaluate the QTc intervals during a takotsubo cardiomyopathy (TTC) episodes and their potential prognostic role., Hypothesis: Dynamic changes of QTc interval during hospitalization for TTC could be associated with outcome at follow-up., Methods: Fifty-two consecutive patients hospitalized for TTC were enrolled. Twelve-lead electrocardiogram (ECG) was performed within 3 h after admission and repeated after 3, 5, and 7 days. Patients were classified in 2 groups: group 1 presented the maximal QTc interval length at admission and group 2 developed maximal QTc interval length after admission., Results: Mean admission QTc interval was 493 ± 71 ms and mean QTc peak interval was 550 ± 76 ms (P < 0.001). Seventeen (33%) patients were included in group 1 and 35 (67%) patients in group 2. There were no differences for cardiovascular risk factors and in terms of ECG findings such as ST elevation, ST depression, and inverted T waves. Rates of adverse events during hospitalization among patients of group 1 and 2 were different although not significantly (20% vs 6%, P = 0.22). After 647 days follow-up, patients of group 1 presented higher risk of cardiovascular rehospitalization (31% vs 6%, P = 0.013; log-rank, P < 0.01). At multivariate analysis, including age and gender, a prolonged QTc interval at admission was significantly associated with higher risk of rehospitalization at follow-up (hazard ratio: 1.07 every 10 ms, 95% confidence interval: 1.003-1.14, P = 0.04)., Conclusions: Prolonged QTc intervals at admission during a TTC episode could be associated with a higher risk of cardiovascular rehospitalization at follow-up. Dynamic increase of QTc intervals after admission are characterized by a trend toward a better prognosis., (© 2017 Wiley Periodicals, Inc.)

In Gram-negative bacteria, autotransporters secrete effector protein domains that are linked to virulence. Although they were once thought to be simple and autonomous secretion machines, mounting evidence reveals that multiple factors of the bacterial envelope are necessary for autotransporter assembly. Secretion across the outer membrane of their soluble effector "passenger domain" is promoted by the assembly of an outer membrane-spanning "β-barrel domain". Both reactions require BamA, an essential component of the β-barrel assembly machinery (BAM complex) that catalyzes the final reaction step by which outer membrane proteins are integrated into the lipid bilayer. A large amount of data generated in the last decade has shed key insights onto the mechanistic coordination of autotransporter β-barrel domain assembly and passenger domain secretion. These results, together with the recently solved structures of the BAM complex, offer an unprecedented opportunity to discuss a detailed model of autotransporter assembly. Importantly, some autotransporters benefit from the presence of an additional machinery, the translocation and assembly module (TAM), a two-membrane spanning complex, which contains a BamA-homologous subunit. Although it remains unclear how the BAM complex and the TAM cooperate, it is evident that multiple preparatory steps are necessary for efficient autotransporter biogenesis., (© 2017 John Wiley & Sons Ltd.)

Background: To assess levels of kinesiophobia (fear of movement) in patients hospitalized for acute cardiovascular disease., Hypothesis: Increased levels of kinesiophobia can be found in subjects hospitalized for acute cardiovascular disease., Methods: Seventy-four consecutive patients admitted for acute coronary syndrome and 58 for acute heart failure were enrolled in the study and assessed by the Tampa Scale for the evaluation of kinesiophobia. Subjects were compared with a reference population with stable coronary artery disease and healthy controls., Results: No significant differences were found between acute coronary syndrome and acute heart failure in terms of kinesiophobia, even considering the rates of high kinesiophobia (Tampa score >37) and the 4 groups of questionnaire items (danger, fear, avoidance, dysfunction). Differences, however, were significant comparing our population with an historical population of subjects with stable coronary artery disease and controls (43 ± 5 vs 35 ± 7 vs 33 ± 6, P < 0.0001 in both cases). A significant correlation was found between the grade of kinesiophobia in the Tampa Scale and the age of subjects (r = 0.27, P = 0.001) and inversely with level of education (r = -0.33, P < 0.0001)., Conclusions: Increased levels of kinesiophobia can be found in subjects hospitalized for acute cardiovascular disease. Kinesiophobia is related to age and education. Kinesiophobia should be carefully considered in subjects hospitalized in acute cardiac care units., (© 2017 Wiley Periodicals, Inc.)

Two driving forces energize precursor translocation across the inner mitochondrial membrane. Although the membrane potential (Δψ) is considered to drive translocation of positively charged presequences through the TIM23 complex (presequence translocase), the activity of the Hsp70-powered import motor is crucial for the translocation of the mature protein portion into the matrix. In this study, we show that mitochondrial matrix proteins display surprisingly different dependencies on the Δψ. However, a precursor's hypersensitivity to a reduction of the Δψ is not linked to the respective presequence, but rather to the mature portion of the polypeptide chain. The presequence translocase constituent Pam17 is specifically recruited by the receptor Tim50 to promote the transport of hypersensitive precursors into the matrix. Our analyses show that two distinct Δψ-driven translocation steps energize precursor passage across the inner mitochondrial membrane. The Δψ- and Pam17-dependent import step identified in this study is positioned between the two known energy-dependent steps: Δψ-driven presequence translocation and adenosine triphosphate-driven import motor activity., (© 2017 Schendzielorz et al.)

To efficiently transport proteins into and across cellular membranes, specialized transport machineries engage in recognition events with different domains of their client proteins, forming sequential intermediate complexes. The short-lived nature of these interactions poses a big challenge in the identification of the key factors involved in transport reactions and their mechanism of action. Site-directed photocrosslinking is a powerful method for the detection and accurate mapping of interacting protein domains. This chapter describes a protocol that combines site-directed photocrosslinking to metabolic labeling of proteins and lipids as a method to characterize, with temporal and spatial resolution, the interactions of a secretory protein as it transverses the bacterial envelope.

The visualization of membrane protein complexes in their natural membrane environment is a major goal in an emerging area of research termed structural cell biology. Such approaches provide important information on the spatial distribution of protein complexes in their resident cellular membrane systems and on the structural organization of multi-subunit membrane protein assemblies. We have developed a method to specifically label active membrane protein complexes in their native membrane environment with electron-dense nanoparticles coupled to an activating ligand, in order to visualize them by electron cryo-tomography. As an example, we describe here the depiction of preprotein import sites of mitochondria, formed by the translocase of the outer membrane (TOM complex) and the presequence translocase of the inner membrane (TIM23 complex). Active import sites are selectively labeled via a biotinylated, quantum dot-coupled preprotein that is arrested in translocation across the outer and inner mitochondrial membranes. Additionally, a related method is described for direct labeling of mitochondrial outer membrane proteins that does not depend on binding of a ligand.