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.

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.

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.

Silencing of CD47 and SIRPα by Polypurine reverse Hoogsteen hairpins to promote MCF-7 breast cancer cells death by PMA-differentiated THP-1 cells.

BACKGROUND: In the context of tumor immunology, tumor cells have been shown to overexpress CD47, an anti-phagocytic signal directed to macrophages to escape from phagocytosis by interacting with Signal Regulatory Protein α SIRPα. In the present work, we designed Polypurine reverse Hoogsteen hairpins, PPRHs, to silence the expression of CD47 in tumor cells and SIRPα in macrophages with the aim to eliminate tumor cells by macrophages in co-culture experiments. METHODS: THP-1 cells were differentiated to macrophages with PMA. The mRNA levels of differentiation markers CD14 and Mcl-1 mRNA and pro-inflammatory cytokines (IL-1ß, IL-18, IL-6, IL-8 and TNF-α) were measured by qRT-PCR. The ability of PPRHs to silence CD47 and SIRPα was evaluated at the mRNA level by qRT-PCR and at the protein level by Western Blot. Macrophages were co-cultured with tumor cells in the presence of PPRHs to silence CD47 and/or SIRPα. Cell viability was assessed by MTT assays. RESULTS: THP-1 cells differentiated to macrophages with PMA showed an increase in macrophage surface markers (CD14, Mcl-1) and pro-inflammatory cytokines (IL-1ß, IL-18, IL-6, IL-8 and TNF-α). PPRHs were able to decrease both CD47 expression in MCF-7 cell line and SIRPα expression in macrophages at the mRNA and protein levels. In the presence of PPRHs, MCF-7 cells were eliminated by macrophages in co-culture experiments, whereas they survived in the absence of PPRHs. CONCLUSIONS: Our data support the usage of PPRHs to diminish CD47/SIRPα interaction by decreasing the expression of both molecules thus resulting in an enhanced killing of MCF-7 cells by macrophages, which might translate into beneficial effects in cancer therapy. These results indicate that PPRHs could represent a new approach with immunotherapeutic applications.

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.

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.

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.

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.

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.

Gene Correction of Point Mutations Using PolyPurine Reverse Hoogsteen Hairpins Technology.

Monogenic disorders are often the result of single point mutations in specific genes, leading to the production of non-functional proteins. Different blood disorders such as ß-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, and Hemophilia A and B are usually caused by point mutations. Gene editing tools including TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correct mutations responsible for different diseases. However, alternative molecular tools such as triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), not relying on nuclease activity, have also demonstrated their ability to correct mutations in the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins (PPRHs) technology, which can represent an alternative gene editing tool within this field. Repair-PPRHs are non-modified single-stranded DNA molecules formed by two polypurine mirror repeat sequences linked by a five-thymidine bridge, followed by an extended sequence at one end of the molecule which is homologous to the DNA sequence to be repaired but containing the corrected nucleotide. The two polypurine arms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between the purines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tracts located relatively near the target mutation in the dsDNA in a sequence-specific manner by Watson-Crick bonds, thus producing a triplex structure which stimulates recombination. This technology has been successfully employed to repair a collection of mutants of the dhfr and aprt genes within their endogenous loci in mammalian cells and could be suitable for the correction of mutations responsible for blood disorders.

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).

A novel DNA-binding motif in prostate tumor overexpressed-1 (PTOV1) required for the expression of ALDH1A1 and CCNG2 in cancer cells.

PTOV1 is a transcription and translation regulator and a promoter of cancer progression. Its overexpression in prostate cancer induces transcription of drug resistance and self-renewal genes, and docetaxel resistance. Here we studied PTOV1 ability to directly activate the transcription of ALDH1A1 and CCNG2 by binding to specific promoter sequences. Chromatin immunoprecipitation and electrophoretic mobility shift assays identified a DNA-binding motif inside the PTOV-A domain with similarities to known AT-hooks that specifically interacts with ALDH1A1 and CCNG2 promoters. Mutation of this AT-hook-like sequence significantly decreased the expression of ALDH1A1 and CCNG2 promoted by PTOV1. Immunohistochemistry revealed the association of PTOV1 with mitotic chromosomes in high grade prostate, colon, bladder, and breast carcinomas. Overexpression of PTOV1, ALDH1A1, and CCNG2 significantly correlated with poor prognosis in prostate carcinomas and with shorter relapse-free survival in colon carcinoma. The previously described interaction with translation complexes and its direct binding to ALDH1A1 and CCNG2 promoters found here reveal the PTOV1 capacity to modulate the expression of critical genes at multiple levels in aggressive cancers. Remarkably, the AT-hook motifs in PTOV1 open possibilities for selective targeting its nuclear and/or cytoplasmic activities.

Functional pharmacogenomics and toxicity of PolyPurine Reverse Hoogsteen hairpins directed against survivin in human cells.

PolyPurine Reverse Hoogsteen (PPRH) hairpins constitute a relatively new pharmacological agent for gene silencing that has been applied for a growing number of gene targets. Previously we reported that specific PPRHs against the antiapoptotic gene survivin were able to decrease viability of PC3 prostate cancer cells by increasing apoptosis, while not acting on HUVEC non-tumoral cells. These PPRHs were efficient both in vitro and in vivo. In the present work, we performed a functional pharmacogenomics study on the effects of specific and unspecific hairpins against survivin. Incubation of PC3 cells with the specific HpsPr-C-WT led to 244 differentially expressed genes when applying the p < 0.05, FC > 2, Benjamini-Hochberg filtering. Importantly, the unspecific or control Hp-WC did not originate differentially expressed genes using the same settings. Gene Set Enrichment Analysis (GSEA) revealed that the differentially expressed genes clustered very significantly within the gene sets of Regulation of cell proliferation, Cellular response to stress, Apoptosis and Prostate cancer. Network analyses using STRING identified important interacting gene-nodes within the response of PC3 cells to treatment with the PPRH against survivin, mainly POLR2G, PAK1IP1, SMC3, SF3A1, PPARGC1A, NCOA6, UGT2B7, ALG5, VAMP7 and HIST1H2BE, the former six present in the Gene Sets detected in the GSEA. Additionally, HepG2 and 786-O cell lines were used to carry out in vitro experiments of hepatotoxicity and nephrotoxicity, respectively. The unspecific hairpin did not cause toxicity in cell survival assays (MTT) and produced minor changes in gene expression for selected genes in RT-qPCR arrays specifically developed for hepatic and renal toxicity screening.

Cancer immunotherapy using PolyPurine Reverse Hoogsteen hairpins targeting the PD-1/PD-L1 pathway in human tumor cells.

Immunotherapy approaches stand out as innovative strategies to eradicate tumor cells. Among them, PD-1/PD-L1 immunotherapy is considered one of the most successful advances in the history of cancer immunotherapy. We used our technology of Polypurine reverse Hoogsteen hairpins (PPRHs) for silencing both genes with the aim to provoke the elimination of tumor cells by macrophages in co-culture experiments. Incubation of PPRHs against PD-1 and PD-L1 decreased the levels of mRNA and protein in THP-1 monocytes and PC3 prostate cancer cells, respectively. Viability of THP-1 cells and macrophages obtained by PMA-differentiation of THP-1 cells was not affected upon incubation with the different PPRHs. On the other hand, PC3 cell survival was partially decreased by PPRHs against PD-L1. The greatest effect in decreasing cell viability was obtained in macrophages/PC3 co-culture experiments by combining PPRHs against PD-1 and PD-L1. This effect was also observed in other cancer cell lines: HeLa, SKBR3 and to a minor extent in M21. Apoptosis was not detected when macrophages were treated with the different PPRHs. However, co-cultures of macrophages with the four cancer cell lines treated with PPRHs showed an increase in apoptosis. The order of fold-increase in apoptosis was HeLa > PC3 > SKBR3 > M21. This study demonstrates that PPRHs could be powerful pharmacological agents to use in immunotherapy approaches for the inhibition of PD-1 and PD-L1.

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.

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)

Transcatheter Aortic Valve Replacement: The Experience of One Brazilian Health Care Center.

OBJECTIVE: Transcatheter aortic valve replacement has been an alternative to invasive treatment for symptomatic severe aortic stenosis in high risk patients. The primary endpoint was 30-day and 1-year mortality from any cause. Secondary endpoints were to compare the clinical and echocardiographic variation pre-and post- transcatheter aortic valve replacement, and the occurrence of complications throughout a 4-year follow-up period. METHODS: This prospective cohort, nestled to a multicenter study (Registro Brasileiro de Implante de Bioprótese por Cateter), describes the experience of a public tertiary center in transcatheter aortic valve replacement. All patients who underwent this procedure between October 2011 and February 2016 were included. RESULTS: Fifty-eight patients underwent transcatheter aortic valve replacement. The 30-day all-cause mortality was 5.2% (n=3) and after 1 year was 17.2% (n=10). A significant improvement in New York Heart Association functional classification was observed when comparing pre-and post- transcatheter aortic valve replacement (III or IV 84.4% versus 5.8%; P<0.001). A decline in peak was observed (P<0.001) and mean (P<0.001) systolic transaortic gradient. The results of peak and mean post-implant transaortic gradient were sustained after one year (P=0.29 and P=0.36, respectively). Left ventricular ejection fraction did not change significantly during follow-up (P=0.41). The most frequent complications were bleeding (28.9%), the need for permanent pacemaker (27.6%) and acute renal injury (20.6%). CONCLUSION: Mortality and complications in this study were consistent with worldwide experience. Transcatheter aortic valve replacement had positive clinical and hemodynamic results, when comparing pre-and post-procedure, and the hemodynamic profile of the prosthesis was sustained throughout follow-up.

Polypurine Reverse Hoogsteen Hairpins as a Gene Silencing Tool for Cancer.

Polypurine reverse Hoogsteen (PPRH) molecules are DNA hairpins formed by two polypurine strands running in an antiparallel orientation and containing no nucleotide modifications. The two strands, linked by a pentathymidine loop, are bound through intramolecular reverse Hoogsteen bonds. Then, PPRHs can bind by Watson-Crick bonds to their corresponding polypyrimidine target in the dsDNA provoking a displacement of the polypurine strand of the duplex. We described the effect and mechanisms of action of PPRHs in cells using PPRHs designed against the template and coding strands of the dhfr gene. The proof of principle of PPRHs as a therapeutic tool was established using a PPRH against survivin in a xenograft prostate cancer tumor model. To improve the PPRHs effect, the influence of the length was studied obtaining a higher efficiency with longer molecules. To decrease the possible offtarget effect, when a purine interruption is found in the pyrimidine target, the PPRH sequence should contain both strands of the complementary base opposite to the interruption. Furthermore, the stability of PPRHs is higher than that of siRNAs, as evidenced by the longer halflife of the former in different types of serum and in PC3 cells. PPRHs do not induce the levels of the transcription factors nor the proinflammatory cytokines involved in the Toll-like Receptor pathway and they do not trigger the formation of the inflammasome complex. PPRHs can be used as therapeutic tools to target genes related to cancer progression, resistance to drugs or immunotherapy approaches.