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In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains. [ABSTRACT FROM AUTHOR]

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequences in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions. [ABSTRACT FROM AUTHOR]

The enzymatic hydrolysis of DNA phosphodiester bonds has been widely studied, but the chemical reaction has not yet been observed. Here we follow the generation of a DNA double-strand break (DSB) by the Desulfurococcus mobilis homing endonuclease I-DmoI, trapping sequential stages of a two-metal-ion cleavage mechanism. We captured intermediates of the different catalytic steps, and this allowed us to watch the reaction by 'freezing' multiple states. We observed the successive entry of two metals involved in the reaction and the arrival of a third cation in a central position of the active site. This third metal ion has a crucial role, triggering the consecutive hydrolysis of the targeted phosphodiester bonds in the DNA strands and leaving its position once the DSB is generated. The multiple structures show the orchestrated conformational changes in the protein residues, nucleotides and metals during catalysis. [ABSTRACT FROM AUTHOR]

DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein-DNA interactions in protein scaffolds is key to providing `toolkits' for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix-loop-helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing. [ABSTRACT FROM AUTHOR]

Transcription activator-like effectors contain a DNA-binding domain organized in tandem repeats. The repeats include two adjacent residues known as the repeat variable di-residue, which recognize a single base pair, establishing a direct code between the dipeptides and the target DNA. This feature suggests this scaffold as an excellent candidate to generate new protein-DNA specificities for biotechnological applications. Here, the crystal structure of AvrBs3 (residues 152-895, molecular mass 82 kDa) in complex with its target DNA sequence is presented, revealing a new mode of interaction with the initial thymine of the target sequence, together with an analysis of both the binding specificity and the thermodynamic properties of AvrBs3. This study quantifies the affinity and the specificity between AvrBs3 and its target DNA. Moreover, in vitro and in vivo analyses reveal that AvrBs3 does not show a strict nucleotide-binding preference for the nucleotide at the zero position of the DNA, widening the number of possible sequences that could be targeted by this scaffold. [ABSTRACT FROM AUTHOR]

Aims The prognostic role of tricuspid regurgitation (TR) associated with organic left-sided valvular heart disease is well known. However, no data are available regarding the prognostic value of functional TR (FTR) in patients with functional mitral regurgitation (FMR) and left ventricular (LV) dysfunction. The purpose of this study was to evaluate the prognostic role of FTR for occurrence of heart failure (HF) and mortality in patients with FMR. Methods and results We enrolled 373 consecutive patients (mean age 68 ±11 years) with LV dysfunction and at least mild FMR and with or without FTR, both quantitated by echocardiography. The median follow-up was 32 months (range 1–120 months); 132 (35.4%) and 97 patients developed HF or died, respectively. The incidence of HF at 3 and 6 years was 36 ±2% and 55 ±4%, respectively. Moderate to severe FTR [hazard ratio (HR) 1.4, 95% confidence interval (CI) 1.1–2.1, P = 0.01) was an independent determinant of HF. The incidence of HF was 41 ±5, 46 ±7, 57 ±7, and 65 ±8% for patients without, and with mild, moderate, and severe FTR respectively (P = 0.03). At 3 and 6 years the survival free of all-cause mortality was 77.5 ±2% and 60 ±3%, respectively. Moderate to severe FTR (HR 1.6, 95% CI 1.2–2.1, P = 0.01) was an independent determinant of overall mortality. At 6 years, survival free of all-cause mortality was 69 ±2.5, 67 ±2.1, 51 ±2.5, and 40 ±4.8% for patients without, and with mild, moderate, and severe FTR, respectively (P = 0.004). Conclusions Moderate or more FTR is independently associated with worse survival and a high incidence of HF episodes in patients with FMR. [ABSTRACT FROM AUTHOR]

The P2 promoter of proP, encoding a transporter for proline and glycine betaine in Escherichia coli, is a unique paradigm, where master regulators of different growth stages, Fis and σS (RpoS), collaborate to achieve promoter activation. It is also the only case described where Fis functions as class II transcriptional activator (centred at −41). Here we show that the degenerate −35 sequence, and the location of the Fis binding site, which forces a suboptimal 16 bp spacing between the −35 and −10 elements, allow only σS but not σ70 to function at proP (P2). Moreover, the interface between Fis and σS seems better suited to σS, due to a single residue difference between σS and σ70. Nevertheless, Fis can activate RNA polymerase containing σ70 at a proP (P2) promoter variant, in which a typical σ70−35 recognition sequence has been introduced at a 17 bp distance from the −10 hexamer. In summary, we elucidate the rules that govern sigma factor selectivity in the presence of a class II activator, provide new insight into transcriptional activation by Fis from this position, and clarify, why the proP (P2) promoter is precisely activated during a short time window of the growth cycle, when Fis and σS are both present. [ABSTRACT FROM AUTHOR]

The nucleoid-associated transcriptional repressor H-NS forms both dimers and tetramers in vivo. Two types of two-hybrid systems, one capable of detecting protein dimerization and the other protein tetramerization, have been used to determine whether environmental changes could affect the oligomerization capacity of this protein in the cell. Increasing the temperature from 37°C to 48°C and changing the pH between 4.0 and 9.0 did not influence either dimerization or tetramerization, whereas lowering the temperature below 25°C and increasing osmolarity were found to reduce the formation of H-NS tetramers, which are the active form of this protein, without affecting dimerization. These findings provide a rationale to explain the induction of H-NS expression during cold-shock, suggest a mechanism contributing to derepressing osmotic-shock genes transcriptionally regulated by H-NS and indicate that changes of the oligomerization properties of H-NS do not play a role in the H-NS and temperature-dependent control of virulence gene expression. [Copyright &y& Elsevier]

Two types of two-hybrid systems demonstrate that the transcriptional repressor, nucleoid-associated protein H-NS (histone-like, nucleoid structuring protein) forms dimers and tetramers in vivo, the latter being the active form of the protein. The H-NS ‘protein oligomerization’ domain (N-domain) is unable to oligomerize in the absence of the intradomain linker while the ‘DNA-binding’ C-domain clearly displays a protein–protein interaction capacity, which contributes to H-NS tetramerization and which is lost following Pro115 mutation. Linker deletion or substitution with KorB linker abolishes H-NS oligomerization. A model describing H-NS dimerization and tetramerization based on all available data and suggesting the existence in the tetramer of a bundle of four α-helices, each contributed by an H-NS monomer, is presented. [ABSTRACT FROM AUTHOR]

Summary Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs. Graphical Abstract Highlights • Structural landscape of Cas12a in the intermediate state by cryo-EM • smFRET shows the thermodynamic and kinetic characterization of Cas12a • The crRNA-DNA hybrid formation triggers the opening of the catalytic cleft • The displacement of the R-loop after target DNA cleavage shuts down the nuclease The cryo-EM structures of Cas12a and smFRET reveal the activation mechanism for target DNA cleavage and indiscriminate ssDNA degradation. The displacement of the R-loop by crRNA suggests a mechanism to stop unspecific ssDNA degradation, resetting the endonuclease to target a new DNA sequence. [ABSTRACT FROM AUTHOR]

In terms of pathophysiology, tricuspid regurgitation (TR), right ventricular function and pulmonary artery pressure are linked to each other. Our aim was to analyze whether the echocardiography-derived right ventricular free wall longitudinal strain/pulmonary artery systolic pressures (RVFWLS/PASP) ratio can improve risk stratification in patients with severe tricuspid regurgitation (TR). In this single-center retrospective study, 250 consecutive patients with severe TR were enrolled from December 2015 to December 2018. Baseline clinical and echocardiographic parameters were collected. Echocardiography-derived TAPSE/PASP and RVFWLS/PASP were evaluated. The primary endpoint was all-cause mortality. Out of 250 consecutive patients, 171 meet inclusion criteria. Patients were predominantly female, with several cardiovascular risk factors and comorbidities. RVFWLS/PASP ≤0.34%/mmHg (AUC 0.68, p < 0.001, sensitivity 70%, specificity 67%) was associated with baseline clinical RV heart failure (p = 0.03). After univariate and multivariate analyses, RVFWLS/PASP, but not TAPSE/PASP, independently correlated with all-cause mortality (HR 0.004, p = 0.02). Patients with RVFWLS/PASP >0.26%/mmHg (AUC 0.74, p < 0.001, sensitivity 77%, specificity 52%) showed higher survival rates (p = 0.02). In addition at 24 months follow-up, the Kaplan-Meyer curves showed patients with RVFWLS >14% & RVFWLS/PASP >0.26%/mmHg had the best survival rate compared to patients without. RVFWLS/PASP is independently associated with baseline RV heart failure and poor long-term prognosis in patients with severe TR. The prognostic role of RVFWLS /PASP ratio in severe TR. [Display omitted] [ABSTRACT FROM AUTHOR]

Bacteria and archaea use the CRISPR-Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR-Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5′-TTN-3′ protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1-crRNA-DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C2221, with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments. [ABSTRACT FROM AUTHOR]

Aims: Infection by SARS‐CoV‐2 may result in a systemic disease and a proportion of patients ranging 15%–44% experienced cardiac injury (CI) diagnosed by abnormal troponin levels. The aim of the present study was to analyse the clinical characteristics of a large series of hospitalized patients for COVID‐19 in order to identify predisposing and/or protective factors of CI and the outcome. Methods and results: This is an observational, retrospective study on patients hospitalized in two Italian centres (San Raffaele Hospital and Cremona Hospital) for COVID‐19 and at least one high‐sensitivity cardiac troponin (hs‐cTnt) measurement during hospitalization. CI was defined if at least one hs‐cTnt value was above the 99th percentile. The primary end‐point was the occurrence of CI during hospitalization. We included 750 patients (median age 67, IQR 56–77 years; 69% males), of whom 46.9% had history of hypertension, 14.7% of chronic coronary disease and 22.3% of chronic kidney disease (CKD). Abnormal troponin levels (median troponin 74, IQR 34–147 ng/l) were detected in 390 patients (52%) during the hospitalization. At multivariable analysis age, CKD, cancer, C‐reactive protein (CRP) levels were independently associated with CI. Independent predictors of very high troponin levels were chronic kidney disease and CRP levels. Patients with CI showed higher rate of all‐cause mortality (40.0% vs. 9.1%, p = 0.001) compared to those without CI. Conclusion: This large, multicentre Italian study confirmed the high prevalence of CI and its prognostic role in hospitalized patients with COVID‐19, highlighting the leading role of systemic inflammation for the occurrence of CI. [ABSTRACT FROM AUTHOR]

Background: Left atrial appendage (LAA) thrombosis increases the risk of stroke and its management has to be assessed. The aim of the present study is to evaluate short and long‐term safety and efficacy of a standardized approach of percutaneous LAA closure (LAAC) routinely using a cerebral protection device (CPD) in patients with LAA thrombosis or sludge (LAAT). Methods: We prospectively enrolled 14 consecutive patients with atrial fibrillation complicated by LAAT presenting in a high‐volume tertiary center. In seven patients (50%) LAAT was found after anticoagulant withdrawal for severe bleedings and in the remaining half LAAT was found despite appropriate anticoagulant therapy. All patients were treated with a standardized interventional approach of LAAC routinely using a CPD and guided by transoesophageal echocardiography. Results: Mean age was 68 ± 14 years and nine patients (64%) were male. Mean CHA2DS2‐VASc and HAS‐BLED scores were 3.3 ± 1.6 and 2.3 ± 1.1, respectively. Six patients (42.8%) presented organized thrombi while eight LAA sludge (57.1%). In 13 patients (92.8%) CPD was positioned through a right radial arterial access. Procedural success was achieved in all patients. In one patient we assisted to embolization of the thrombus during deployment of the device in the absence of neurological consequences. During a mean follow up of 426 ± 307 days, one patient died for non‐cardiac cause while no embolic event or major bleeding were reported. Conclusion: In an unselected cohort, LAAC with the systematic use of CPD was a feasible, safe and effective therapeutic option for LAAT both acutely and after long‐term follow‐up. [ABSTRACT FROM AUTHOR]

Background: Myocardial injury (MI) can be detected during the acute phase of Coronavirus disease 19 (COVID‐19) and is associated with a dismal prognosis. Recent imaging studies described the persistence of cardiac abnormalities after the recovery. The aim of the study was to investigate the spectrum of cardiac abnormalities at mid‐term follow‐up in patients recovered from COVID‐19 using clinical assessment, laboratory tests, and imaging evaluation with comprehensive echocardiography. Methods: This is an observational, cross‐sectional study assessing an unselected cohort of consecutive patients recovered from COVID‐19. MI was defined by elevated plasma levels of high sensitive troponin T (hsTnT). At the follow‐up, a complete examination including echocardiography was performed. Results: The 123 patients included were divided into two groups according to the presence of MI during hospitalization: group A (without MI) and group B (with MI). After a median of 85 days, group B patients were more frequently symptomatic for dyspnea and had significantly higher values of hsTnT and N‐Terminal prohormone of Brain Natriuretic Peptide (NT‐proBNP), compared to Group A. No differences between the two groups in left nor right ventricle dimension and ejection fraction were found. However, in group B a significant reduction of mean left ventricle global longitudinal strain was observed (‐15.7±.7 vs ‐18.1±.3 in group A, p < 0.001), together with higher frequency of impaired diastolic function and higher values of pulmonary pressure. Conclusions: In patients recovered from COVID‐19, echocardiography with speckle‐tracking analysis may be an useful imaging tool to identify subclinical myocardial dysfunction and potentially guide management strategies. [ABSTRACT FROM AUTHOR]

Aims: Aortic stenosis (AS) grading by 2D‐transthoracic echocardiography (2D‐TTE) aortic valve area (AVA) calculation is limited by left ventricular outflow tract (LVOT) area underestimation. The combination of Doppler parameters with 3D LVOT area obtained by multidetector computed tomography (MDCT) can improve AS grading, reconciling discordant 2D‐TTE findings. This study aimed to systematically evaluate the role of 3D‐transesophageal echocardiography (3D‐TEE) in AS grading using MDCT as reference standard. Methods and results: 288 patients (81 ± 6.3 years, 52.4% female) with symptomatic AS underwent 2D‐TTE, 3D‐TEE, and MDCT for transcatheter aortic valve implantation. Doppler parameters were combined with 3D LVOT areas measured by manual and semi‐automated software 3D‐TEE and by MDCT to calculate AVA, reassessing AS severity. Both 3D‐TEE modalities demonstrated good correlation with MDCT, with excellent intra‐observer and inter‐observer variability. Compared to MDCT, 3D‐TEE measurements significantly underestimated AVA (PANOVA <.0001), although the difference was clinically acceptable. Compared to 2D‐TTE, 3D‐TEE manual and semi‐automated software reclassified severe AS in 21.9% and 25.2% of cases, respectively (P <.0001), overcame grading parameters discordance in more than 40% of cases in patients with low‐gradient AS (P <.0001) and reduced the proportion of low‐flow states in nearly 75% of cases when combined to stroke volume index assessment (P <.0001). 3D‐TEE imaging modalities showed a reduction in the proportion of patients with low‐gradient and pathological AVA as defined by 2D‐TTE, and improved AVA and mean pressure gradient agreement with current guidelines cutoff values. Conclusion: 3D‐TEE AVA calculation is a reliable tool for AS grading with excellent reproducibility and good correlation with MDCT measurements. [ABSTRACT FROM AUTHOR]

Single-molecule Fo¨ rster Resonance energy transfer (smFRET) is an adaptable method for studying the structure and dynamics of biomolecules. The development of high throughput methodologies and the growth of commercial instrumentation have outpaced the development of rapid, standardized, and automated methodologies to objectively analyze the wealth of produced data. Here we present DeepFRET, an automated, open-source standalone solution based on deep learning, where the only crucial human intervention in transiting from raw microscope images to histograms of biomolecule behavior, is a user-adjustable quality threshold. Integrating standard features of smFRET analysis, DeepFRET consequently outputs the common kinetic information metrics. Its classification accuracy on ground truth data reached >95% outperforming human operators and commonly used threshold, only requiring ~1% of the time. Its precise and rapid operation on real data demonstrates DeepFRET's capacity to objectively quantify biomolecular dynamics and the potential to contribute to benchmarking smFRET for dynamic structural biology. [ABSTRACT FROM AUTHOR]

Cmr-β is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-β, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-β and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5′ tag in the CRISPR RNAs (crRNA) and the 3′ antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes. • The stalk loop is an allosteric sensor signaling the self- versus non-self-recognition • Dynamics of the stalk loop key in triggering Cmr2 (Cas10)-dependent activities • The HD domain of SisCmr2 cleaves non-complementary UA ssRNA • The Cmr7 subunit modulates the cleavage activities and cOA generation Sofos et al. report the mechanisms of the immune response of S. islandicus Cmr-β complex. Cryo-EM structures of the apo, self, and non-self target-RNA-bound complexes combined with biochemical assays show that target RNA binding allows alternate conformations of the "stalk loop" in Cmr3 due to the lack of pairing between the 5′ tag and 3′ antitag. This induces conformational changes in Cmr2, triggering cleavage of ssDNA and non-complementary ssRNA, and cyclic oligoadenylate synthesis. Autoimmunity is avoided by stabilizing the stalk loop in a conformation that inhibits Cmr2 activities. [ABSTRACT FROM AUTHOR]

LAAO is an emerging option for thromboembolic event prevention in patients with NVAF. We previously reported data on comparison between LAAO and DOAC at two-year follow-up in NVAF patients at HBR (HAS-BLED ≥3). Limited data are available on long term follow-up. We aimed to evaluate the efficacy and safety of DOACs versus LAAO indication after 5 years. We enrolled 193 HBR treated with LAAO and 189 HBR patients with DOACs. At baseline, LAAO group had higher HAS-BLED (4.2 vs 3.3, p < 0.001) and lower CHADS-VASc (4.3 vs. 4.7, p = 0.005). After 1:1 PSM, 192 patients were included (LAAO n = 96; DOACs n = 96). At 5-year follow-up the rate of the combined safety and effectiveness endpoint (ISTH major bleeding and thromboembolic events) was significantly higher in LAAO group (p = 0.042), driven by a higher number of thromboembolic events (p = 0.047). The rate of ISTH-major bleeding events was similar (p = 0.221). After PSM no significant difference in the primary effectiveness (LAAO 13.3% vs DOACs 9.5%, p = 0.357) and safety endpoint (LAAO 7.5% vs DOACs 7.5%; p = 0.918) were evident. Overall bleeding rate was significantly higher in DOACs group (25.0% vs 13.7%, p = 0.048), while a non-significant higher number of TIA was reported in LAAO group (5.4% vs 1.1%, p = 0.098). All-cause and cardiovascular mortality were higher in LAAO group at both unmatched and matched analysis. We confirmed safety and effectiveness of both DOAC and LAAO in NVAF patients at HBR, with no significant differences in thromboembolic events or major bleeding were at 5-year follow-up. The observed increased mortality after LAAO warrants further investigations in RCTs. • This study is currently presenting the longest follow-up available comparing LAAO and DOACs for thromboembolic events prevention in 382 patients at high bleeding risk (HAS-BLED score ≥3) with non-valvular atrial fibrillation. • At 5-year follow-up, after propensity score matching both DOACs and LAAO groups showed similar results for primary safety and effectiveness endpoints, despite a higher rate of cardiovascular mortality in the LAAO group. • At unmatched analysis, the rate of thromboembolic events was higher in LAAO group compared to DOACs and that of ISTH-major bleeding did not differ between the two indications. • At unmatched analysis, all-cause mortality and cardiac mortality were significantly higher in LAAO group reflecting the real-word higher risk profile of patients undergoing LAAO. [ABSTRACT FROM AUTHOR]

To evaluate the long-term clinical outcome of a cohort of patients suffering from moderate tricuspid regurgitation (TR), regardless of its etiology. Clinical and echocardiographic follow-up were assessed in 250 patients diagnosed with moderate TR between January 2016 and July 2020. TR progression at follow-up was defined as TR grade increase to at least severe. The primary endpoint was all-cause death; secondary endpoints were cardiovascular (CV) death and the composite of heart failure (HF) hospitalization plus tricuspid valve (TV) intervention. After a median follow-up of 3.6 years, TR progression occurred in 84 patients (34%). At multivariate analyses, atrial fibrillation (AF, OR 1.81, CI 1.01–3.29, p = 0.045) and right ventricular end-diastolic diameter (RVEDD, OR 2.19, CI 1.26–3.78, p = 0.005) were independent predictors of TR progression. The primary endpoint occurred in 59 patients (24%) and was significantly more frequent in the group with TR progression (p = 0.009). At multivariate analyses, chronic kideney disease (OR 2.80, CI 1.30–6.03, p = 0.009), left ventricular ejection fraction (OR 0.97, CI 0.94–0.99, p = 0.041) and TR progression (OR 2.32, CI 1.31–4.12, p = 0.004) were independent predictors of the primary outcome. Moreover, both the secondary endpoints of CV death and HF hospitalization plus TV intervention were more frequent in the TR progression group (p = 0.001 and p < 0.001, respectively). Moderate TR progresses in a significant proportion of patients over a long-term follow-up, leading to a worse prognosis. TR progression is an independent determinant of hard clinical events and AF and RVEDD are associated with TR progression. • We investigated the clinical outcome of 250 patients suffering from moderate all-cause tricuspid regurgitation (TR) • Over a long-term clinical follow-up, moderate TR progressed to at least severe grade in one third of our population • TR progression conveys a significant risk of hard clinical events, independently of its etiologies and of comorbidities • Moderate TR patients should be closely followed-up to avoid the negative prognostic effects of this valvulopathy. [ABSTRACT FROM AUTHOR]

Aims: To explore whether left ventricular reverse remodelling (LVRR) is a predictor of outcomes in patients with functional mitral regurgitation (FMR) undergoing MitraClip procedure.Methods and Results: We analysed 184 consecutive patients with FMR who underwent successful MitraClip procedure. LVRR was defined as a reduction in left ventricular end-systolic volume ≥ 10% from baseline to 6 months. LVRR was observed in 79 (42.9%) patients. Compared with non-LVRR, LVRR patients were more likely to be females, less likely to have an ischaemic aetiology of mitral regurgitation or a prior (<6 months) heart failure (HF) hospitalization, and had smaller left ventricular dimensions. New York Heart Association class improved from baseline up to 1-year follow-up in both groups. Higher rates of overall survival (87.3% vs. 75.2%, P = 0.039), freedom from HF hospitalization (77.2% vs. 60%, P = 0.020), and freedom from the composite endpoint (cardiovascular mortality or HF hospitalization) (74.7% vs. 55.2%; P = 0.012) were observed in LVRR vs. non-LVRR patients at 2-year follow-up. LVRR was associated with a significant reduction of the adjusted relative risk of mortality, HF hospitalization and composite endpoint [hazard ratio (HR) 0.44; 95% confidence interval (CI) 0.20-0.96, P = 0.040; HR 0.55; 95% CI 0.32-0.97, P = 0.038; and HR 0.54; 95% CI 0.32-0.92, P = 0.023, respectively]. Female gender, absence of diabetes, freedom from prior HF hospitalization, non-ischaemic aetiology of mitral regurgitation, and left ventricular end-diastolic diameter < 75 mm were found to be independent predictors of LVRR.Conclusions: Left ventricular reverse remodelling is associated with better long-term outcomes in patients with FMR successfully treated with MitraClip. A careful patient selection may be useful as specific baseline features predict favourable left ventricular remodelling. [Correction added on 17 January 2019, after online publication: the preceding sentence has been changed.]. [ABSTRACT FROM AUTHOR]

Abstract Background MitraClip implantation has evolved as a new tool for treatment of inoperable or high-risk patients with severe functional mitral regurgitation (FMR) due to dilated cardiomyopathy (DCM). Limited data are available regarding MitraClip outcomes comparing patients with ischemic and non-ischemic DCM. Methods From 2008 to 2016, 314 patients received MitraClip for FMR at four institutions: Brescia, Zurich and Milan. Patients were stratified according to MR aetiology in non-ischemic FMR (n = 99) and ischemic FMR (n = 215). Preoperative risk factors, operative variables and outcomes up to 2-year were evaluated. A multivariable Cox Proportional Hazards survival model with covariate adjustments was used to assess the relationship between FMR aetiology and 2-year cardiac mortality. Results As expected, patients with ischemic FMR had significantly more risk factors and comorbidities. Overall procedural success rate was 80% and in-hospital cardiac mortality was 3% without significant differences between aetiology. Two-year overall (25% vs. 19%, p = 0.574) and cardiac (18% vs. 16%, p = 0.990) mortality rates were comparable. No differences were detected in terms of re-hospitalization rates (32%), LVAD implantation (4.5%) and mitral valve surgery (1%). LVEF ≤ 25%, LVEDV > 216 ml, NT-proBNP ≥ 10.000 pg/ml and AF were the strongest baseline predictors of 2-year cardiac mortality. Greater improvements of 6MWT and NYHA functional class were observed in patients with non-ischemic FMR. Conclusions The ischemic or non-ischemic aetiology of DCM did not affect in-hospital and 2-year cardiac mortality after MitraClip in patients with FMR. In case of unfavorable baseline clinical condition, the indication for MitraClip should be carefully weighed in favour of conservative medical therapy alone or left ventricular assist device. Highlights • Greater improvements of 6MWT and NYHA functional class were observed in patients with non-ischemic FMR. • Non-/ischemic FMR aetiology did not affect 2-year cardiac mortality after MitraClip. • LVEF ≤25%, LVEDV >216 ml, NT-proBNP ≥10.000 pg/ml and AF were the strongest baseline predictors of 2-year cardiac mortality. [ABSTRACT FROM AUTHOR]

Background: Transcatheter heart valve thrombosis (THV‐t) and endocarditis (THV‐e) are relevant complications after transcatheter aortic valve implantation (TAVI). Transcatheter heart valve (THV) dysfunction definition is mostly based on Doppler (stenosis/regurgitation) without considering leaflets characteristics. Purpose: To evaluate the additional diagnostic value of leaflets echocardiographic features over Doppler when prosthetic valve complication is suspected. Methods: Among 621 post‐TAVI patients, 128 cases with probable valve complication were identified. THV‐t was finally diagnosed in 13 patients (10%) and THV‐e in 8 (6%), while the remaining 107 (84%) had no definitive diagnosis of thrombosis/endocarditis (THV‐no). We analyzed at 2 time points (baseline and follow‐up) both traditional Doppler parameters and leaflets morpho‐functional features. Results: Both Doppler and leaflets parameters showed high sensitivity (sensitivity 92%) and low specificity (ranging from specificity 32% to 74%) in detecting THV‐t. Interestingly, the combination of mean aortic pressure gradient ≥20 mm Hg and leaflet thickening significantly improved the specificity of echocardiography for diagnosis of THV thrombosis (specificity 94%). On the other hand, echocardiographic diagnosis of THV endocarditis remained limited by very low sensitivity despite showing high specificity. Conclusions: The combination of Doppler and leaflets parameters can improve the echocardiographic diagnosis of THV thrombosis in post‐TAVI patients with suspicious symptoms, via a significant increase in the overall test specificity. This would potentially allow more rational gatekeeping to more expensive/invasive diagnostic examinations (eg, CT scan) or therapeutic trials (eg, unnecessary anticoagulation). [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to assess the impact of the ratio of volume of contrast medium to the glomerular filtration rate (V/GFR) on acute kidney injury (AKI) after transcatheter aortic valve implantation (TAVI) and its impact on long-term mortality.Methods: We retrospectively calculated V/GFR in 397 patients undergoing TAVI. AKI was defined as VARC-modified Risk, Injury, Failure, Loss and End-stage (RIFLE) kidney disease score≥2.Results: The incidence of AKI was 17.9%. The mean V/GFR ratio was 3.0±2.7 in patients without AKI and 7.8±8.8 in patients with AKI (p<0.001). The receiver-operator characteristic curve analysis showed fair discrimination between patients with and without AKI (C-statistic 0.85) at a V/GFR ratio of 3.2. Multivariable regression analysis indicated that V/GFR>3.2 was an independent predictor of both AKI (OR 3.4, 95% CI 1.0-6.1, p<0.001) and long-term mortality (OR 3.3, 95% CI 2.0-5.2, p<0.001).Conclusions: A V/GFR > 3.2 was found to be correlated with a higher incidence of AKI and mortality after TAVI. Therefore, this ratio could potentially be used to calculate the maximum volume of contrast medium that can be administered without significantly increasing the risk of AKI and mortality. Further larger studies are needed to validate these findings. [ABSTRACT FROM AUTHOR]

VT Ablation in MitraClip Patients Introduction Patients with mitral regurgitation are increasingly treated by percutaneous implantation of a MitraClip device (Abbott Park, IL, USA). We investigate the feasibility and safety of the transmitral catheter route for catheter ablation of ventricular tachycardia (VT) in these patients. Methods The mitral valve with the MitraClip in situ was crossed under transesophageal 3-dimensional echocardiographic and fluoroscopic guidance using a steerable sheath for ablation of the left ventricle. Results Five patients (all males, median age 74.0 ± 16.0 years) who had previously a MitraClip implanted were referred for catheter ablation of VT. The left ventricular ejection fraction was 29.0% ± 24.0%. One patient had both an atrial septal defect and a left atrial appendage occluder device in addition to a MitraClip. The duration between MitraClip implantation and ablation was 1019.0 ± 783.0 days. After transseptal puncture, ablation catheter was successfully steered through the mitral valve with the use of fluoroscopy. A complete high-density map of the substrate in sinus rhythm could be obtained in all patients using multipolar mapping catheters. In 1 patient, mapping was carried out using a mini-basket catheter. Procedural endpoints, noninducibility of all VTs, and abolition of all late potentials were achieved in all patients. Procedure time was 255.0 ± 52.5 minute, fluoroscopy time was 23.0 ± 7.3, and the radiation dose was 61.0 ± 37.5 Gycm2. No mitral insufficiency or worsening of regurgitation was documented after the procedure. Conclusions This is the first report demonstrating the feasibility and safety of VT ablation in patients with a MitraClip device using the anterograde transmitral catheter route. [ABSTRACT FROM AUTHOR]

Transcatheter aortic valve implantation (TAVI) is now the treatment of choice for patients with symptomatic aortic stenosis who are inoperable or with high surgical risk. Data with regards to contemporary clinical practice and long-term outcomes are sparse. To evaluate temporal changes in TAVI practice and explore procedural and long-term clinical outcomes of patients in a contemporary "real-world" population, outcomes of 829 patients treated from November 2007 to May 2015, at the San Raffaele Scientific Institute, Milan, Italy, were retrospectively analyzed. Median follow-up was 568 days, with the longest follow-up of 2,677 days. Overall inhospital mortality was 3.5%. During the study period, there was a trend toward treating younger, lower risk patients. Overall mortality rates were 3.5% (30 days), 14% (1 year), 22% (2 years), 29% (3 years), 37% (4 years), 47% (5 years), 53% (6 years), and 72% (7 years). The survival probability at 5 years was significantly higher in patients treated through the transfemoral (TF) route compared to other vascular access sites (log rank p <0.001). Non-TF vascular access and residual paravalvular leak ≥2 (after TAVI) were identified as independent predictors for both all-cause and cardiovascular mortality. No patient required further aortic valve intervention for TAVI prosthesis degeneration. In conclusion, there is a trend toward treating younger, lower-risk patients. Non-TF vascular access approach and ≥2 PVL after TAVI were identified as independent predictors for both overall and cardiovascular mortality with no cases of prosthesis degeneration suggesting acceptable durability. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To determine the functions of in vitro primed Natural Killer (NK) cells in Human Immunodeficiency Virus (HIV-1) infection and the role of IL-2, IL-12 and IL-15 in enhancing the NK survival and activity in terms of viral suppression and of purging of HIV provirus. Methods: Peripheral Blood Mononuclear Cells (PBMCs) and CD4+ T lymphocytes cells obtained from eight healthy donors were infected in vitro with HIV-1 and p24 was measured with and without IL-2, IL-12 and IL-15. We studied the effect of NK pulsed in vitro with IL-2, IL-12 and IL-15 on HIV replication by measurement of p-24 and DNA-provirus load when added into the culture of PBMCs and CD4+ T lymphocytes cells infected in vitro. We evaluated the effect of NK cells pulsed with IL-2, IL-12 and IL-15 on HIV replication and DNA-load into the culture of CD4+ T lymphocytes cells and PBMCs by trans-well chamber. Results: We found high levels of p24 in the supernatants of PBMCs and CD4+ T lymphocytes cells cultured with IL-2, IL-12, and IL-15. We observed a significant reduction of p24 in the culture both of infected PBMCs and CD4+ T lymphocytes cells in which was added NK pulsed with IL-15. We did not obtain the some results with NK pulsed with IL-2 and IL-12. We observed a power effect of NK pulsed with IL-15 on HIV-DNA. The trans-well chamber experiments showed that the effect of NK is both direct and both mediated by realizing of soluble factors. Conclusions: This study highlights some important effects of IL 15 on NK in HIV patients anyway our results are preliminary and descriptive and others studies will be needed to provide rationale for immune therapies. [Copyright &y& Elsevier]

A 67-year-old man with a dilated cardiomyopathy and severe aortic regurgitation (AR) secondary to a traumatic cusp lesion was referred to our institution because of progressive worsening of dyspnea. After formal discussion in the heart team, the patient was scheduled for TAVI (transcatheter aortic valve implantation). The pre procedural computed tomography scan revealed a minimum amount of calcium on the aortic valve and low position of coronary ostia. The TAVI procedure was performed with the implantation of a fully retrievable and repositionable aortic valve prosthesis (Direct Flow 29 mm, Direct Flow Medical, Santa Rosa, California) with an excellent result and no paravalvular leak. The TAVI devices designed for the treatment of calcific aortic stenosis have numerous limitations for the treatment of pure AR such as the risk of residual AR, the lack of repositionability and retrievability, and the need for valve- in-valve implantation. We believe that treatment of selected cases of pure AR with the Direct Flow valve is feasible and takes advantage of the retrievability of the prosthesis. [ABSTRACT FROM AUTHOR]