Autothreshold algorithm feasibility and safety in left bundle branch pacing.

AIMS: Autothreshold algorithms enable remote monitoring of patients with conventional pacing, but there is limited information on their performance in left bundle branch pacing (LBBP). Our objective was to analyse the behaviour of the autothreshold algorithm in LBBP and compare it with conventional pacing and manual thresholds during initial device programming (acute phase), after 1-7 days (subacute), and 1-3 months later (chronic). METHODS AND RESULTS: A prospective, non-randomized, single-centre comparative study was conducted. Consecutive patients with indication for cardiac pacing were enrolled. Implants were performed in the left bundle branch area or the right ventricle endocardium at the discretion of the operator. Left bundle branch pacing was determined according to published criteria. Autothreshold algorithm was activated in both groups whenever allowed by the device. Seventy-five patients were included, with 50 undergoing LBBP and 25 receiving conventional pacing. Activation of the autothreshold algorithm was more feasible in later phases, showing a favourable trend towards bipolar pacing. Failures in algorithm activation were primarily due to insufficient safety margins (82.8% in LBBP and 90% in conventional pacing). The remainder was attributed to atrial tachyarrhythmias (10.3% and 10%, respectively) and electrical noise (the remaining 6.9% in the LBBP group). In the LBBP group, there were not statistically significant differences between manual and automatic thresholds, and both remained stable during follow-up (mean increase of 0.50 V). CONCLUSION: The autothreshold algorithm is feasible in LBBP, with a favourable trend towards bipolar pacing. Automatic thresholds are similar to manual in patients with LBBP, and they remain stable during follow-up.

Comparison of automated subcutaneous defibrillator screening between different pacing sites in cardiac pacing device carriers.

AIMS: The compatibility of cardiac pacing with the presence of a subcutaneous implantable cardioverter-defibrillator (S-ICD) has been investigated, but S-ICD screening test results have not been compared among different pacing sites. The objective was to compare S-ICD screening results among different cardiac pacing sites and to assess the electrocardiographic predictors of success. METHODS AND RESULTS: This prospective single-centre study conducted automated S-ICD screening in 102 carriers of cardiac pacing devices in conduction system (CSP), biventricular (BVP), right ventricular outflow tract (RVOT), or right ventricular apex (RVA) pacing sites. The study included 102 patients: 40 with CSP (20 left bundle pacing and 20 His bundle pacing), 21 with BVP, and 20 and 21 with RVOT and RVA pacing, respectively. The percentage of positive screenings was significantly higher for CSP (97.5%) than for the other patient groups (BVP 71.4%, RVOT 70%, and RVA 19%). In multivariate analysis, positive screening was associated with a narrower QRS (OR 0.95 [0.92-0.98] P = 0.001) and higher R/T ratio in precordial leads (1.76 [1.18-2.61]). CONCLUSION: A higher S-ICD eligibility rate of cardiac pacing device carriers was obtained in CSP than in conventional pacing (RVA or RVOT) or BVP. The presence of narrower paced QRS width and paced corrected QT interval and of higher R/T ratio in precordial and limb leads are electrocardiographic predictors of a positive response to screening.

Septal fascicle involvement during a Purkinje-related ventricular tachycardia: Electroanatomic correlation using omnipolar technology.

Idiopathic verapamil-sensitive fascicular ventricular tachycardia (VT) is the most common form of Purkinje-related ventricular tachycardia (PRVT). Left septal fascicle (LSF) involvement and its connections with the other fascicles, have been recently reported as a pathophysiologic mechanism for this form of PRVT. We describe a case of idiopathic PRVT with LSF involvement using omnipolar technology (OT) mapping in relation to a false tendon. Ablation in the area with concealed fusion entrainment did not terminate the tachycardia. However, radiofrequency application in the area of LSF with manifest fusion entrainment, resulted in immediate tachycardia termination. Six months follow-up showed no tachycardia recurrence.

DNA specificities modulate the binding of human transcription factor A to mitochondrial DNA control region.

Human mitochondrial DNA (h-mtDNA) codes for 13 subunits of the oxidative phosphorylation pathway, the essential route that produces ATP. H-mtDNA transcription and replication depends on the transcription factor TFAM, which also maintains and compacts this genome. It is well-established that TFAM activates the mtDNA promoters LSP and HSP1 at the mtDNA control region where DNA regulatory elements cluster. Previous studies identified still uncharacterized, additional binding sites at the control region downstream from and slightly similar to LSP, namely sequences X and Y (Site-X and Site-Y) (Fisher et al., Cell 50, pp 247-258, 1987). Here, we explore TFAM binding at these two sites and compare them to LSP by multiple experimental and in silico methods. Our results show that TFAM binding is strongly modulated by the sequence-dependent properties of Site-X, Site-Y and LSP. The high binding versatility of Site-Y or the considerable stiffness of Site-X tune TFAM interactions. In addition, we show that increase in TFAM/DNA complex concentration induces multimerization, which at a very high concentration triggers disruption of preformed complexes. Therefore, our results suggest that mtDNA sequences induce non-uniform TFAM binding and, consequently, direct an uneven distribution of TFAM aggregation sites during the essential process of mtDNA compaction.

Structure-Based Design of an RNase Chimera for Antimicrobial Therapy.

Bacterial resistance to antibiotics urges the development of alternative therapies. Based on the structure-function of antimicrobial members of the RNase A superfamily, we have developed a hybrid enzyme. Within this family, RNase 1 exhibits the highest catalytic activity and the lowest cytotoxicity; in contrast, RNase 3 shows the highest bactericidal action, alas with a reduced catalytic activity. Starting from both parental proteins, we designed a first RNase 3/1-v1 chimera. The construct had a catalytic activity much higher than RNase 3, unfortunately without reaching an equivalent antimicrobial activity. Thus, two new versions were created with improved antimicrobial properties. Both of these versions (RNase 3/1-v2 and -v3) incorporated an antimicrobial loop characteristic of RNase 3, while a flexible RNase 1-specific loop was removed in the latest construct. RNase 3/1-v3 acquired both higher antimicrobial and catalytic activities than previous versions, while retaining the structural determinants for interaction with the RNase inhibitor and displaying non-significant cytotoxicity. Following, we tested the constructs' ability to eradicate macrophage intracellular infection and observed an enhanced ability in both RNase 3/1-v2 and v3. Interestingly, the inhibition of intracellular infection correlates with the variants' capacity to induce autophagy. We propose RNase 3/1-v3 chimera as a promising lead for applied therapeutics.

Utility and feasibility of the CartoUnivu™ system for atrial flutter ablation in patients with mechanical prosthetic valves.

Ablation of macroreentrant atrial tachycardia in patients with mechanical prosthetic valves represents a challenge for electrophysiologists, because of the complexity of the procedure and the potential complications. Moreover, the need for fluoroscopy in this type of procedure is greater, due to the risk of interference between the prosthetic valve and the ablation or mapping catheter. We present two cases of patients with mechanical prosthetic valves and atrial flutter who underwent successful ablation with no complications using the CartoUnivu™ tool, which integrates the electroanatomical map and the fluoroscopy image.

Influence of baseline QRS on the left ventricular ejection fraction recovery after frequent premature ventricular complex ablation.

AIMS: Frequent premature ventricular complexes (PVCs) can induce or worsen left ventricular systolic dysfunction. We aimed to investigate the influence of the baseline QRS in the response after PVC ablation in patients with depressed left ventricular ejection fraction (LVEF). METHODS AND RESULTS: Two hundred and fifteen [59 ± 13 years old, 152 (71%) men] consecutive patients with left ventricular (LV) systolic dysfunction and frequent PVCs referred for ablation were included and followed-up for 12 months. Echocardiographic response was defined as an improvement of at least five absolute points in LVEF. Clinical, electrocardiogram, and electrophysiological characteristics were analysed. Mean baseline QRS duration was 110 ms [97-140]. Premature ventricular complex burden significantly decreased after ablation from 23% [16-33] at baseline to 1% [0-8] at 12 months, P < 0.001. Mean PVC burden reduction was 18 [8-30] points. There was a significant improvement of LVEF from 35% [29-40] at baseline to 44% [35-55] at 12 months, P < 0.001. One hundred and thirty (61%) patients were considered as echocardiographic responders. Baseline QRS duration (ms) [odds ratio (OR) 0.98 (0.97-0.99), P = 0.01] was an independent predictor of echocardiographic response. Mean LVEF improvement was 16 [10-21] points when the baseline QRS duration was <90 ms; 12 [4-20] when it was 90-110 ms; 5 [0-15] when it was 110 ± 130 ms; and 0 [0-6] points when it was >130 ms. CONCLUSIONS: In patients with LV systolic dysfunction, intrinsic QRS duration is inversely related to the probability and the degree of echocardiographic response after frequent PVC ablation. Patients with a QRS duration >130 ms at baseline have the poorer response after ablation.

Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI.

Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach, comprising five mutations for which the exact role has not yet been deciphered. In this work, we cocrystallized a reengineered version of iSpinach in complex with the DFHBI and solved the X-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach sheds some light on the possible function of the different beneficial mutations carried by iSpinach.

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding.

RNase P is a universal enzyme that removes 5' leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP-tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP-tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.

Data Fusion Based on Subspace Decomposition for Distributed State Estimation in Multi-Hop Networks.

This paper deals with the problem of estimating the distributed states of a plant using a set of interconnected agents. Each of these agents must perform a real-time monitoring of the plant state, counting on the measurements of local plant outputs and on the exchange of information with the rest of the network. These inter-agent communications take place within a multi-hop network. Therefore, the transmitted information suffers a delay that depends on the position of the sender and receiver in a communication graph. Without loss of generality, it is considered that the transmission rate and the plant sampling rate are both identical. The paper presents a novel data-fusion-based observer structure based on subspace decomposition, and addresses two main subproblems: the observer design to stabilize the estimation error, and an optimal observer design to minimize the estimation uncertainties when plant disturbances and measurements noises come into play. The performance of the proposed design is tested in simulation.

The hexameric structure of the human mitochondrial replicative helicase Twinkle.

The mitochondrial replicative helicase Twinkle is involved in strand separation at the replication fork of mitochondrial DNA (mtDNA). Twinkle malfunction is associated with rare diseases that include late onset mitochondrial myopathies, neuromuscular disorders and fatal infantile mtDNA depletion syndrome. We examined its 3D structure by electron microscopy (EM) and small angle X-ray scattering (SAXS) and built the corresponding atomic models, which gave insight into the first molecular architecture of a full-length SF4 helicase that includes an N-terminal zinc-binding domain (ZBD), an intermediate RNA polymerase domain (RPD) and a RecA-like hexamerization C-terminal domain (CTD). The EM model of Twinkle reveals a hexameric two-layered ring comprising the ZBDs and RPDs in one layer and the CTDs in another. In the hexamer, contacts in trans with adjacent subunits occur between ZBDs and RPDs, and between RPDs and CTDs. The ZBDs show important structural heterogeneity. In solution, the scattering data are compatible with a mixture of extended hexa- and heptameric models in variable conformations. Overall, our structural data show a complex network of dynamic interactions that reconciles with the structural flexibility required for helicase activity.

Transfer RNA: From pioneering crystallographic studies to contemporary tRNA biology.

Transfer RNAs (tRNAs) play a key role in protein synthesis as adaptor molecules between messenger RNA and protein sequences on the ribosome. Their discovery in the early sixties provoked a worldwide infatuation with the study of their architecture and their function in the decoding of genetic information. tRNAs are also emblematic molecules in crystallography: the determination of the first tRNA crystal structures represented a milestone in structural biology and tRNAs were for a long period the sole source of information on RNA folding, architecture, and post-transcriptional modifications. Crystallographic data on tRNAs in complex with aminoacyl-tRNA synthetases (aaRSs) also provided the first insight into protein:RNA interactions. Beyond the translation process and the history of structural investigations on tRNA, this review also illustrates the renewal of tRNA biology with the discovery of a growing number of tRNA partners in the cell, the involvement of tRNAs in a variety of regulatory and metabolic pathways, and emerging applications in biotechnology and synthetic biology.

Structural determinants for tRNA selective cleavage by RNase 2/EDN.

tRNA-derived fragments (tRFs) have emerged as key players of immunoregulation. Some RNase A superfamily members participate in the shaping of the tRFs population. By comparing wild-type and knockout macrophage cell lines, our previous work revealed that RNase 2 can selectively cleave tRNAs. Here, we confirm the in vitro protein cleavage pattern by screening of synthetic tRNAs, single-mutant variants, and anticodon-loop DNA/RNA hairpins. By sequencing of tRF products, we identified the cleavage selectivity of recombinant RNase 2 with base specificity at B1 (U/C) and B2 (A) sites, consistent with a previous cellular study. Lastly, protein-hairpin complexes were predicted by MD simulations. Results reveal the contribution of the α1, loop 3 and loop 4, and ß6 RNase 2 regions, where residues Arg36/Asn39/Gln40/Asn65/Arg68/Arg132 provide interactions, spanning from P-1 to P2 sites that are essential for anticodon loop recognition. Knowledge of RNase 2-specific tRFs generation might guide new therapeutic approaches for infectious and immune-related diseases.

Bipolar ablation involving coronary venous system for refractory left ventricular summit arrhythmias.

Background: Catheter ablation of premature ventricular complexes (PVCs) and ventricular tachycardia (VT) from the left ventricular summit (LVS) may require advanced ablation techniques. Bipolar ablation from the coronary veins and adjacent endocardial structures can be effective for refractory LVS arrhythmias. Objective: The aim of this study was to investigate the outcomes of bipolar ablation performed between the coronary venous system and adjacent endocardial left ventricular outflow tract (LVOT) or right ventricular outflow tract (RVOT). Methods: This multicenter study included consecutive patients with LVS PVC/VT who underwent bipolar ablation between the anterior interventricular vein (AIV) or great cardiac vein (GCV) and the endocardial LVOT/RVOT after failed unipolar ablation. Ablation was started with powers of 10-20 W and uptitrated to achieve an impedance drop of at least 10%. Angiography was performed in all cases to confirm a safe distance (>5 mm) of the catheter from the major coronary arteries. Results: Between 2013 and 2023, bipolar radiofrequency ablation between the AIV/GCV and the adjacent LVOT/RVOT was attempted in 20 patients (4 female; age 57 ± 16 years). Unipolar ablation from sites of early activation (AIV/GCV, LVOT, aortic cusps, RVOT) failed to effectively suppress the PVC/VT in all subjects. Bipolar ablation was delivered with a maximum power of 30 ± 8 W and total duration of 238 ± 217 s and led to acute PVC/VT elimination in all patients. No procedural-related complications occurred. Over a follow-up period of 30 ± 24 months, the freedom from arrhythmia recurrence was 85% (1 recurrence in the VT group and 2 in the PVC group). PVC burden was reduced from 22% ± 10% to 4% ± 8% (P <.001). Conclusion: In cases of LVS PVC/VT refractory to unipolar ablation, bipolar ablation between the coronary venous system and adjacent endocardial LVOT/RVOT is safe and effective if careful titration of power and intraprocedural angiography are performed to ensure a safe distance from the coronary arteries.

Comparative outcomes of extracorporeal membrane oxygenation for COVID-19 delivered in experienced European centres during successive SARS-CoV-2 variant outbreaks (ECMO-SURGES): an international, multicentre, retrospective cohort study.

BACKGROUND: To inform future research and practice, we aimed to investigate the outcomes of patients who received extracorporeal membrane oxygenation (ECMO) for acute respiratory distress syndrome (ARDS) due to different variants of SARS-CoV-2. METHODS: This retrospective study included consecutive adult patients with laboratory-confirmed SARS-CoV-2 infection who received ECMO for ARDS in 21 experienced ECMO centres in eight European countries (Austria, Belgium, England, France, Germany, Italy, Portugal, and Spain) between Jan 1, 2020, and Sept 30, 2021. We collected data on patient characteristics, clinical status, and management before and after the initiation of ECMO. Participants were grouped according to SARS-CoV-2 variant (wild type, alpha, delta, or other) and period of the pandemic (first [Jan 1-June 30] and second [July 1-Dec 31] semesters of 2020, and first [Jan 1-June 30] and second [July 1-Sept 30] semesters of 2021). Descriptive statistics and Kaplan-Meier survival curves were used to analyse evolving characteristics, management, and patient outcomes over the first 2 years of the pandemic, and independent risk factors of mortality were determined using multivariable Cox regression models. The primary outcome was mortality 90 days after the initiation of ECMO, with follow-up to Dec 30, 2021. FINDINGS: ECMO was initiated in 1345 patients. Patient characteristics and management were similar for the groups of patients infected with different variants, except that those with the delta variant had a younger median age and less hypertension and diabetes. 90-day mortality was 42% (569 of 1345 patients died) overall, and 43% (297/686) in patients infected with wild-type SARS-CoV-2, 39% (152/391) in those with the alpha variant, 40% (78/195) in those with the delta variant, and 58% (42/73) in patients infected with other variants (mainly beta and gamma). Mortality was 10% higher (50%) in the second semester of 2020, when the wild-type variant was still prevailing, than in other semesters (40%). Independent predictors of mortality were age, immunocompromised status, a longer time from intensive care unit admission to intubation, need for renal replacement therapy, and higher Sequential Organ Failure Assessment haemodynamic component score, partial pressure of arterial carbon dioxide, and lactate concentration before ECMO. After adjusting for these variables, mortality was significantly higher with the delta variant than with the other variants, the wild-type strain being the reference. INTERPRETATION: Although crude mortality did not differ between variants, adjusted risk of death was highest for patients treated with ECMO infected with the delta variant of SARS-CoV-2. The higher virulence and poorer outcomes associated with the delta strain might relate to higher viral load and increased inflammatory response syndrome in infected patients, reinforcing the need for a higher rate of vaccination in the population and updated selection criteria for ECMO, should a new and highly virulent strain of SARS-CoV-2 emerge in the future. Mortality was noticeably lower than in other large, multicentre series of patients who received ECMO for COVID-19, highlighting the need to concentrate resources at experienced centres. FUNDING: None.