Physiological responses during walking in men and women with intermittent claudication.

BACKGROUND: Miyasato et al. show that peak oxygen consumption, walking economy, anaerobic threshold, and cardiovascular responses (heart rate, blood pressure, and rate pressure product) during walking were similar between men and women with peripheral artery disease and intermittent claudication. There were no differences in the physiological responses to walking between men and women with intermittent claudication. Sex per se is not a factor that demands changes in walking prescription for patients with intermittent claudication. OBJECTIVE: Peak oxygen consumption (VO2peak), anaerobic threshold, walking economy, and cardiovascular responses during walking are used to guide and monitor walking training in patients with peripheral artery disease and intermittent claudication. Women with peripheral artery disease and intermittent claudication present greater impairments than men, and evaluating training markers according to sex for decisions regarding walking prescription in this population is important. This study aimed to compare VO2peak, walking economy, anaerobic threshold, and cardiovascular responses during walking in men and women with peripheral artery disease and intermittent claudication. METHODS: Forty patients (20 men and 20 women with similar baseline characteristics) underwent a cardiopulmonary treadmill test (3.2km/h and 2% increase in slope every 2 minutes until maximal leg pain). The VO2 and rate-pressure product were assessed. Data from men and women were compared using t-tests. RESULTS: There were no significant differences between men and women (VO2peak: 15.0±4.8 versus 13.9±2.9mL∙kg-1∙min-1, p=0.38; walking economy: 9.6±2.7 versus 8.4±1.6mL∙kg-1∙min-1, p=0.09; anaerobic threshold: 10.5±3.2 versus 10.5±2.2mL∙kg-1∙min-1, p=0.98; rate pressure product at 1st stage: 13,465± 2,910 versus 14,445±4,379bpm∙mmHg, p=0.41; and rate pressure product at anaerobic threshold:13,673±3,100 versus 16,390±5,870bpm∙mmHg, p=0.08 and rate pressure product at peak exercise: 21,253±6,141 versus 21,923±7,414bpm∙mmHg, p=0.76, respectively). CONCLUSION: Men and women with peripheral artery disease and similar baseline characteristics presented similar responses to walking, suggesting that decisions regarding walking prescription and monitoring can be made regardless of sex in this specific population.

Mapping PTBP2 binding in human brain identifies SYNGAP1 as a target for therapeutic splice switching.

Alternative splicing of neuronal genes is controlled partly by the coordinated action of polypyrimidine tract binding proteins (PTBPs). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal. Here, we define the PTBP2 footprint in the human transcriptome using brain tissue and human induced pluripotent stem cell-derived neurons (iPSC-neurons). We map PTBP2 binding sites, characterize PTBP2-dependent alternative splicing events, and identify novel PTBP2 targets including SYNGAP1, a synaptic gene whose loss-of-function leads to a complex neurodevelopmental disorder. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and nonsense-mediated decay, and that antisense oligonucleotides (ASOs) that disrupt PTBP binding redirect splicing and increase SYNGAP1 mRNA and protein expression. In SYNGAP1 haploinsufficient iPSC-neurons generated from two patients, we show that PTBP2-targeting ASOs partially restore SYNGAP1 expression. Our data comprehensively map PTBP2-dependent alternative splicing in human neurons and cerebral cortex, guiding development of novel therapeutic tools to benefit neurodevelopmental disorders.

Medição automática de valva mitral a partir de ecocardiograma utilizando processamento digital de imagens / Automatic measurement of the mitral valve based on echocardiography using digital image processing

Fundamento: A avaliação da área valvar mitral por meio da reconstrução multiplano na ecocardiografia tridimensional é restrita a softwares específicos e à experiência dos ecocardiografistas. Eles precisam selecionar manualmente o frame do vídeo que contenha a área de abertura máxima da valva mitral, dimensão fundamental para a identificação de estenose mitral. Objetivo: Automatizar o processo de determinação da área de abertura máxima da valva mitral, por meio da aplicação de Processamento Digital de Imagens (PDI) em exames de ecocardiograma, desenvolvendo um algoritmo aberto com leitura de vídeo no formato avi. Método: Este estudo piloto observacional transversal foi realizado com vinte e cinco exames diferentes de ecocardiograma, sendo quinze com abertura normal e dez com estenose mitral reumática. Todos os exames foram realizados e disponibilizados por dois especialistas, com autorização do Comitê de Ética em Pesquisa, que utilizaram dois modelos de aparelhos ecocardiográficos: Vivid E95 (GE Healthcare) e Epiq 7 (Philips), com sondas multiplanares transesofágicas. Todos os vídeos em formato avi foram submetidos ao PDI através da técnica de segmentação de imagens. Resultados: As medidas obtidas manualmente por ecocardiografistas experientes e os valores calculados pelo sistema desenvolvido foram comparados utilizando o diagrama de Bland-Altman. Observou-se maior concordância entre valores no intervalo de 0,4 a 2,7 cm². Conclusão: Foi possível determinar automaticamente a área de máxima abertura das valvas mitrais, tanto para os casos advindos da GE quanto da Philips, utilizando apenas um vídeo como dado de entrada. O algoritmo demonstrou economizar tempo nas medições quando comparado com a mensuração habitual. (AU)

Background: The evaluation of mitral valve area through multiplanar reconstruction in 3-dimensional echocardiography is restricted to specific software and to the experience of echocardiographers. They need to manually select the video frame that contains the maximum mitral valve opening area, as this dimension is fundamental to identification of mitral stenosis. Objective: To automate the process of determining the maximum mitral valve opening area, through the application of digital image processing (DIP) in echocardiography tests, developing an open algorithm with video reading in avi format. Method: This cross-sectional observational pilot study was conducted with 25 different echocardiography exams, 15 with normal aperture and 10 with rheumatic mitral stenosis. With the authorization of the Research Ethics Committee, all exams were performed and made available by 2 specialists who used 2 models of echocardiographic devices: Vivid E95 (GE Healthcare) and Epiq 7 (Philips), with multiplanar transesophageal probes. All videos in avi format were submitted to DIP using the image segmentation technique. Results: The measurements obtained manually by experienced echocardiographers and the values calculated by the developed system were compared using a Bland-Altman diagram. There was greater agreement between values in the range from 0.4 to 2.7 cm². Conclusion: It was possible to automatically determine the maximum mitral valve opening area, for cases from both GE and Philips, using only 1 video as input data. The algorithm has been demonstrated to save time on measurements when compared to the usual method. (AU)

Physiological responses during walking in men and women with intermittent claudication

ABSTRACT Objective Peak oxygen consumption (VO2peak), anaerobic threshold, walking economy, and cardiovascular responses during walking are used to guide and monitor walking training in patients with peripheral artery disease and intermittent claudication. Women with peripheral artery disease and intermittent claudication present greater impairments than men, and evaluating training markers according to sex for decisions regarding walking prescription in this population is important. This study aimed to compare VO2peak, walking economy, anaerobic threshold, and cardiovascular responses during walking in men and women with peripheral artery disease and intermittent claudication. Methods Forty patients (20 men and 20 women with similar baseline characteristics) underwent a cardiopulmonary treadmill test (3.2km/h and 2% increase in slope every 2 minutes until maximal leg pain). The VO2 and rate-pressure product were assessed. Data from men and women were compared using t-tests. Results There were no significant differences between men and women (VO2peak: 15.0±4.8 versus 13.9±2.9mL∙kg-1∙min-1, p=0.38; walking economy: 9.6±2.7 versus 8.4±1.6mL∙kg-1∙min-1, p=0.09; anaerobic threshold: 10.5±3.2 versus 10.5±2.2mL∙kg-1∙min-1, p=0.98; rate pressure product at 1st stage: 13,465± 2,910 versus 14,445±4,379bpm∙mmHg, p=0.41; and rate pressure product at anaerobic threshold:13,673±3,100 versus 16,390±5,870bpm∙mmHg, p=0.08 and rate pressure product at peak exercise: 21,253±6,141 versus 21,923±7,414bpm∙mmHg, p=0.76, respectively). Conclusion Men and women with peripheral artery disease and similar baseline characteristics presented similar responses to walking, suggesting that decisions regarding walking prescription and monitoring can be made regardless of sex in this specific population.

Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA).

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence.

A machine learning framework for the evaluation of myocardial rotation in patients with noncompaction cardiomyopathy.

AIMS: Noncompaction cardiomyopathy (NCC) is considered a genetic cardiomyopathy with unknown pathophysiological mechanisms. We propose to evaluate echocardiographic predictors for rigid body rotation (RBR) in NCC using a machine learning (ML) based model. METHODS AND RESULTS: Forty-nine outpatients with NCC diagnosis by echocardiography and magnetic resonance imaging (21 men, 42.8±14.8 years) were included. A comprehensive echocardiogram was performed. The layer-specific strain was analyzed from the apical two-, three, four-chamber views, short axis, and focused right ventricle views using 2D echocardiography (2DE) software. RBR was present in 44.9% of patients, and this group presented increased LV mass indexed (118±43.4 vs. 94.1±27.1g/m2, P = 0.034), LV end-diastolic and end-systolic volumes (P< 0.001), E/e' (12.2±8.68 vs. 7.69±3.13, P = 0.034), and decreased LV ejection fraction (40.7±8.71 vs. 58.9±8.76%, P < 0.001) when compared to patients without RBR. Also, patients with RBR presented a significant decrease of global longitudinal, radial, and circumferential strain. When ML model based on a random forest algorithm and a neural network model was applied, it found that twist, NC/C, torsion, LV ejection fraction, and diastolic dysfunction are the strongest predictors to RBR with accuracy, sensitivity, specificity, area under the curve of 0.93, 0.99, 0.80, and 0.88, respectively. CONCLUSION: In this study, a random forest algorithm was capable of selecting the best echocardiographic predictors to RBR pattern in NCC patients, which was consistent with worse systolic, diastolic, and myocardium deformation indices. Prospective studies are warranted to evaluate the role of this tool for NCC risk stratification.

Rifaximin or Saccharomyces boulardii in heart failure with reduced ejection fraction: Results from the randomized GutHeart trial.

BACKGROUND: The gut microbiota represents a potential treatment target in heart failure (HF) through microbial metabolites such as trimethylamine N-oxide (TMAO) and systemic inflammation. Treatment with the probiotic yeast Saccharomyces boulardii have been suggested to improve left ventricular ejection fraction (LVEF). METHODS: In a multicentre, prospective randomized open label, blinded end-point trial, we randomized patients with LVEF <40% and New York Heart Association functional class II or III, despite optimal medical therapy, to treatment (1:1:1) with the probiotic yeast Saccharomyces boulardii, the antibiotic rifaximin, or standard of care (SoC) only. The primary endpoint, the baseline-adjusted LVEF at three months, was assessed in an intention-to-treat analysis. FINDINGS: We enrolled a total of 151 patients. After three months' treatment, the LVEF did not differ significantly between the SoC arm and the rifaximin arm (mean difference was -1•2 percentage points; 95% CI -3•2 - 0•7; p=0•22) or between the SoC arm and the Saccharomyces boulardii arm (mean difference -0•2 percentage points; 95% CI -2•2 - 1•9; p=0•87). We observed no significant between-group differences in changes in microbiota diversity, TMAO, or C-reactive protein. INTERPRETATION: Three months' treatment with Saccharomyces boulardii or rifaximin on top of SoC had no significant effect on LVEF, microbiota diversity, or the measured biomarkers in our population with HF. FUNDING: The trial was funded by the Norwegian Association for Public Health, the Blix foundation, Stein Erik Hagen's Foundation for Clinical Heart Research, Ada og Hagbart Waages humanitære og veldedige stiftelse, Alfasigma, and Biocodex.

Synthesis and validation of DOPY: A new gemini dioleylbispyridinium based amphiphile for nucleic acid transfection.

Nucleic acids therapeutics provide a selective and promising alternative to traditional treatments for multiple genetic diseases. A major obstacle is the development of safe and efficient delivery systems. Here, we report the synthesis of the new cationic gemini amphiphile 1,3-bis[(4-oleyl-1-pyridinio)methyl]benzene dibromide (DOPY). Its transfection efficiency was evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs), a nucleic acid tool for gene silencing and gene repair developed in our laboratory. The interaction of DOPY with PPRHs was confirmed by gel retardation assays, and it forms complexes of 155 nm. We also demonstrated the prominent internalization of PPRHs using DOPY compared to other chemical vehicles in SH-SY5Y, PC-3 and DF42 cells. Regarding gene silencing, a specific PPRH against the survivin gene delivered with DOPY decreased survivin protein levels and cell viability more effectively than with N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) in both SH-SY5Y and PC-3 cells. We also validated the applicability of DOPY in gene repair approaches by correcting a point mutation in the endogenous locus of the dhfr gene in DF42 cells using repair-PPRHs. All these results indicate both an efficient entry and release of PPRHs at the intracellular level. Therefore, DOPY can be considered as a new lipid-based vehicle for the delivery of therapeutic oligonucleotides.

Nucleic acids therapeutics using PolyPurine Reverse Hoogsteen hairpins.

PolyPurine Reverse Hoogsteen hairpins (PPRHs) are DNA hairpins formed by intramolecular reverse Hoogsteen bonds which can bind to polypyrimidine stretches in dsDNA by Watson:Crick bonds, thus forming a triplex and displacing the fourth strand of the DNA complex. PPRHs were first described as a gene silencing tool in vitro for DHFR, telomerase and survivin genes. Then, the effect of PPRHs directed against the survivin gene was also determined in vivo using a xenograft model of prostate cancer cells (PC3). Since then, the ability of PPRHs to inhibit gene expression has been explored in other genes involved in cancer (BCL-2, mTOR, topoisomerase, C-MYC and MDM2), in immunotherapy (SIRPα/CD47 and PD-1/PD-L1 tandem) or in replication stress (WEE1 and CHK1). Furthermore, PPRHs have the ability to target the complementary strand of a G-quadruplex motif as a regulatory element of the TYMS gene. PPRHs have also the potential to correct point mutations in the DNA as shown in two collections of CHO cell lines bearing mutations in either the dhfr or aprt loci. Finally, based on the capability of PPRHs to form triplexes, they have been incorporated as probes in biosensors for the determination of the DNA methylation status of PAX-5 in cancer and the detection of mtLSU rRNA for the diagnosis of Pneumocystis jirovecii. Of note, PPRHs have high stability and do not present immunogenicity, hepatotoxicity or nephrotoxicity in vitro. Overall, PPRHs constitute a new economical biotechnological tool with multiple biomedical applications.

Cardiovascular Responses during and after Maximal Walking in Men and Women with Symptomatic Peripheral Artery Disease.

BACKGROUND: Walking is recommended for patients with peripheral arterial disease (PAD). It has been shown that patients with PAD present sharper increases in blood pressure (BP) and heart rate (HR) during maximal walking when compared with healthy subjects. Additionally, women with PAD present a worse physiological profile, and it is possible that they may present higher cardiovascular load during and after a bout of maximal walking than men. Thus, the objective of this study was to compare cardiovascular and autonomic responses during and after maximal walking between men and women with PAD and intermittent claudication (IC). METHODS: Forty patients with PAD and IC (20 men and 20 women) underwent, in random order, 2 sessions: control (standing on treadmill) and exercise (maximal treadmill walking test with Gardner's protocol). During the exercise, HR and BP were measured. Before and after the sessions, cardiovascular variables (BP HR, cardiac output, peripheral vascular resistance, and stroke volume) and autonomic modulation (HR and BP variabilities and baroreflex sensitivity) were assessed. In addition, an ambulatory BP monitoring was recorded after each session. RESULTS: Men and women presented similar maximal walking capacity. During the walking test, HR and systolic BP increased similarly in men and women. After the maximal walking, cardiovascular and autonomic responses did not differ between the genders. In addition, postintervention ambulatory BP parameters were also similar in men and women. Therefore, in men and women, maximal walking similarly reduced clinic systolic BP and stroke volume, and increased HR and total power of HR variability during the recovery period. CONCLUSIONS: Men and women with PAD and IC present similar cardiovascular and autonomic responses during and after maximal walking.

Experiência de um Centro Brasileiro com Crioablação para Isolamento Elétrico das Veias Pulmonares na Fibrilação Atrial Paroxística e Persistente - Resultados Preliminares no Brasil / Experience in a Brazilian Center with Cryoablation for Electric Isolation of the Pulmonary Veins in Paroxysmal and Persistent Atrial Fibrillation - Preliminary Results in Brazil

Resumo Fundamento O isolamento elétrico das veias pulmonares é reconhecidamente base fundamental para o tratamento não farmacológico da fibrilação atrial (FA) e, portanto, tem sido recomendado como passo inicial na ablação de FA em todas as diretrizes. A técnica com balão de crioenergia, embora amplamente utilizada na América do Norte e Europa, ainda se encontra em fase inicial em muitos países em desenvolvimento, como o Brasil. Objetivo Avaliar o sucesso e a segurança da técnica de crioablação em nosso serviço, em pacientes com FA paroxística e persistente. Métodos Cento e oito pacientes consecutivos com FA sintomática e refratária ao tratamento farmacológico foram submetidos à crioablação para isolamento das veias pulmonares. Os pacientes foram separados em dois grupos, de acordo com a classificação convencional da FA paroxística (duração de até sete dias) e persistente (FA por mais de sete dias). Dados de recorrência e segurança do procedimento foram analisados respectivamente como desfechos primário e secundário. O nível de significância adotado foi de 5%. Resultados Cento e oito pacientes, com idade média de 58±13 anos, 84 do sexo masculino (77,8%), foram submetidos ao procedimento de crioablação de FA. Sessenta e cinco pacientes apresentavam FA paroxística (60,2%) e 43, FA persistente (39,2%). O tempo médio do procedimento foi de 96,5±29,3 minutos e o tempo médio de fluoroscopia foi de 29,6±11,1 minutos. Foram observadas cinco (4,6%) complicações, nenhuma fatal. Considerando a evolução após os 3 meses iniciais, foram observadas 21 recorrências (19,4%) em período de um ano de seguimento. As taxas de sobrevivência livre de recorrência nos grupos paroxístico e persistente foram de 89,2% e 67,4%, respectivamente. Conclusão A crioablação para isolamento elétrico das veias pulmonares é um método seguro e eficaz para tratamento da FA. Nossos resultados estão consoantes com demais estudos, que sugerem que a tecnologia pode ser utilizada como abordagem inicial, mesmo nos casos de FA persistente. (Arq Bras Cardiol. 2020; [online].ahead print, PP.0-0)

Abstract Background Electrical isolation of the pulmonary veins is recognized as the cornerstone of non-pharmacological treatment of Atrial Fibrillation (AF), and therefore, has been recommended as the first step in AF ablation according to all guidelines. Even though the cryoballoon technology is widely used in North America and Europe, this experience is still incipient in many developing countries such as Brazil. Objective To evaluate initial results regarding success and safety of the new technology in patients with persistent and paroxysmal AF. Methods One hundred and eight consecutive patients with symptomatic AF refractory to pharmacological treatment were submitted to cryoablation for isolation of the pulmonary veins. Patients were separated into two groups according to AF classification: persistent (AF for over one week); or paroxysmal (shorter episodes). Recurrence and procedural safety data were analyzed respectively as primary and secondary outcomes. The level of significance was 5%. Results One hundred and eight patients, with mean age 58±13 years, 84 males (77.8%), underwent cryoablation. Sixty-five patients had paroxysmal AF (60.2%) and 43 had persistent AF (39.2%). The mean time of the procedure was 96.5±29.3 minutes and the mean fluoroscopy time was 29.6±11.1 minutes. Five (4.6%) complications were observed, none fatal. Considering a blanking period of 3 months, 21 recurrences (19.4%) were observed in a one-year follow-up period. The recurrence-free survival rates of AF in the paroxysmal and persistent groups were 89.2% and 67.4%, respectively. Conclusion Cryoablation for electrical isolation of the pulmonary veins is a safe and effective method for the treatment of AF. Our results are consistent with other studies suggesting that this technology can be used as an initial technique even in cases of persistent AF.

Experience in a Brazilian Center with Cryoablation for Electric Isolation of the Pulmonary Veins in Paroxysmal and Persistent Atrial Fibrillation - Preliminary Results in Brazil. / Experiência de um Centro Brasileiro com Crioablação para Isolamento Elétrico das Veias Pulmonares na Fibrilação Atrial Paroxística e Persistente ­ Resultados Preliminares no Brasil.

BACKGROUND: Electrical isolation of the pulmonary veins is recognized as the cornerstone of non-pharmacological treatment of Atrial Fibrillation (AF), and therefore, has been recommended as the first step in AF ablation according to all guidelines. Even though the cryoballoon technology is widely used in North America and Europe, this experience is still incipient in many developing countries such as Brazil. OBJECTIVE: To evaluate initial results regarding success and safety of the new technology in patients with persistent and paroxysmal AF. METHODS: One hundred and eight consecutive patients with symptomatic AF refractory to pharmacological treatment were submitted to cryoablation for isolation of the pulmonary veins. Patients were separated into two groups according to AF classification: persistent (AF for over one week); or paroxysmal (shorter episodes). Recurrence and procedural safety data were analyzed respectively as primary and secondary outcomes. The level of significance was 5%. RESULTS: One hundred and eight patients, with mean age 58±13 years, 84 males (77.8%), underwent cryoablation. Sixty-five patients had paroxysmal AF (60.2%) and 43 had persistent AF (39.2%). The mean time of the procedure was 96.5±29.3 minutes and the mean fluoroscopy time was 29.6±11.1 minutes. Five (4.6%) complications were observed, none fatal. Considering a blanking period of 3 months, 21 recurrences (19.4%) were observed in a one-year follow-up period. The recurrence-free survival rates of AF in the paroxysmal and persistent groups were 89.2% and 67.4%, respectively. CONCLUSION: Cryoablation for electrical isolation of the pulmonary veins is a safe and effective method for the treatment of AF. Our results are consistent with other studies suggesting that this technology can be used as an initial technique even in cases of persistent AF.

FUNDAMENTO: O isolamento elétrico das veias pulmonares é reconhecidamente base fundamental para o tratamento não farmacológico da fibrilação atrial (FA) e, portanto, tem sido recomendado como passo inicial na ablação de FA em todas as diretrizes. A técnica com balão de crioenergia, embora amplamente utilizada na América do Norte e Europa, ainda se encontra em fase inicial em muitos países em desenvolvimento, como o Brasil. OBJETIVO: Avaliar o sucesso e a segurança da técnica de crioablação em nosso serviço, em pacientes com FA paroxística e persistente. MÉTODOS: Cento e oito pacientes consecutivos com FA sintomática e refratária ao tratamento farmacológico foram submetidos à crioablação para isolamento das veias pulmonares. Os pacientes foram separados em dois grupos, de acordo com a classificação convencional da FA paroxística (duração de até sete dias) e persistente (FA por mais de sete dias). Dados de recorrência e segurança do procedimento foram analisados respectivamente como desfechos primário e secundário. O nível de significância adotado foi de 5%. RESULTADOS: Cento e oito pacientes, com idade média de 58±13 anos, 84 do sexo masculino (77,8%), foram submetidos ao procedimento de crioablação de FA. Sessenta e cinco pacientes apresentavam FA paroxística (60,2%) e 43, FA persistente (39,2%). O tempo médio do procedimento foi de 96,5±29,3 minutos e o tempo médio de fluoroscopia foi de 29,6±11,1 minutos. Foram observadas cinco (4,6%) complicações, nenhuma fatal. Considerando a evolução após os 3 meses iniciais, foram observadas 21 recorrências (19,4%) em período de um ano de seguimento. As taxas de sobrevivência livre de recorrência nos grupos paroxístico e persistente foram de 89,2% e 67,4%, respectivamente. CONCLUSÃO: A crioablação para isolamento elétrico das veias pulmonares é um método seguro e eficaz para tratamento da FA. Nossos resultados estão consoantes com demais estudos, que sugerem que a tecnologia pode ser utilizada como abordagem inicial, mesmo nos casos de FA persistente. (Arq Bras Cardiol. 2020; [online].ahead print, PP.0-0).

Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins.

Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5'UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS.

Correction of the aprt Gene Using Repair-Polypurine Reverse Hoogsteen Hairpins in Mammalian Cells.

In this study, we describe the correction of single-point mutations in mammalian cells by repair-polypurine reverse Hoogsteen hairpins (repair-PPRHs). These molecules consist of (1) a PPRH hairpin core that binds to a polypyrimidine target sequence in the double-stranded DNA (dsDNA), producing a triplex structure, and (2) an extension sequence homologous to the DNA sequence to be repaired but containing the wild-type nucleotide instead of the mutation and acting as a donor DNA to correct the mutation. We repaired different point mutations in the adenosyl phosphoribosyl transferase (aprt) gene contained in different aprt-deficient Chinese hamster ovary (CHO) cell lines. Because we had previously corrected mutations in the dihydrofolate reductase (dhfr) gene, in this study, we demonstrate the generality of action of the repair-PPRHs. Repaired cells were analyzed by DNA sequencing, mRNA expression, and enzymatic activity to confirm the correction of the mutation. Moreover, whole-genome sequencing analyses did not detect any off-target effect in the repaired genome. We also performed gel-shift assays to show the binding of the repair-PPRH to the target sequence and the formation of a displacement-loop (D-loop) structure that can trigger a homologous recombination event. Overall, we demonstrate that repair-PPRHs achieve the permanent correction of point mutations in the dsDNA at the endogenous level in mammalian cells without off-target activity.