Statistical analysis plan for a randomized controlled trial examining pedometer-based walking intervention in patients with heart failure with reduced ejection fraction: the WATCHFUL trial.

BACKGROUND: Physical activity is an effective management strategy for heart failure with reduced ejection fraction, but patients' compliance is challenging. Walking is a suitable form of physical activity due to its convenience and sustainability, and it can potentially improve functional capacity in heart failure patients. OBJECTIVES: The WATCHFUL trial aims to determine whether a pedometer-based walking intervention combined with face-to-face sessions and regular telephone contact improves functional capacity in heart failure patients. METHODS: The WATCHFUL trial is a 6-month multicenter, parallel-group, randomized, controlled, superiority trial with a 6-month follow-up. A total of 202 patients were recruited for the trial. The primary analysis will evaluate the change in distance walked during the 6-min walk test from baseline to 6 months based on the intention-to-treat population; the analysis will be performed using a linear mixed-effect model adjusted for baseline values. Missing data will be imputed using multiple imputations, and the impact of missing data will be assessed using a sensitivity analysis. Adverse events are monitored and recorded throughout the trial period. DISCUSSION: The trial has been designed as a pragmatic trial with a scalable intervention that could be easily translated into routine clinical care. The trial has been affected by the COVID-19 pandemic, which slowed patients' recruitment and impacted their physical activity patterns. CONCLUSIONS: The present publication provides details of the planned statistical analyses for the WATCHFUL trial to reduce the risks of reporting bias and erroneous data-driven results. TRIAL REGISTRATION: ClinicalTrials.gov (identifier: NCT03041610, registered: 3/2/2017).

Variability of Inverted Repeats in All Available Genomes of Bacteria.

Noncanonical secondary structures in nucleic acids have been studied intensively in recent years. Important biological roles of cruciform structures formed by inverted repeats (IRs) have been demonstrated in diverse organisms, including humans. Using Palindrome analyser, we analyzed IRs in all accessible bacterial genome sequences to determine their frequencies, lengths, and localizations. IR sequences were identified in all species, but their frequencies differed significantly across various evolutionary groups. We detected 242,373,717 IRs in all 1,565 bacterial genomes. The highest mean IR frequency was detected in the Tenericutes (61.89 IRs/kbp) and the lowest mean frequency was found in the Alphaproteobacteria (27.08 IRs/kbp). IRs were abundant near genes and around regulatory, tRNA, transfer-messenger RNA (tmRNA), and rRNA regions, pointing to the importance of IRs in such basic cellular processes as genome maintenance, DNA replication, and transcription. Moreover, we found that organisms with high IR frequencies were more likely to be endosymbiotic, antibiotic producing, or pathogenic. On the other hand, those with low IR frequencies were far more likely to be thermophilic. This first comprehensive analysis of IRs in all available bacterial genomes demonstrates their genomic ubiquity, nonrandom distribution, and enrichment in genomic regulatory regions. IMPORTANCE Our manuscript reports for the first time a complete analysis of inverted repeats in all fully sequenced bacterial genomes. Thanks to the availability of unique computational resources, we were able to statistically evaluate the presence and localization of these important regulatory sequences in bacterial genomes. This work revealed a strong abundance of these sequences in regulatory regions and provides researchers with a valuable tool for their manipulation.

Interaction of C-terminal p53 isoforms depends strongly upon DNA sequence and topology.

The p53 protein is a key tumor suppressor and the most commonly mutated and down-regulated protein in human tumors. It functions mainly through interaction with DNA, and p53 acts as a transcription factor that recognizes the so-called p53 target sites on the promoters of various genes. P53 has been shown to exist as many isoforms, including three C-terminal isoforms that are produced by alternative splicing. Because the C-terminal domain is responsible for sequence-nonspecific binding and regulation of p53 binding, we have analyzed DNA recognition by these C-terminal isoforms. Using atomic force microscopy, we show for the first time that all C-terminal isoforms recognize superhelical DNA. It is particularly noteworthy that a sequence-specific p53 consensus binding site is bound by p53α and ß isoforms with similar affinities, whilst p53α shows higher binding to a quadruplex sequence than both p53ß and p53γ, and p53γ loses preferential binding to both the consensus binding sequence and the quadruplex-forming sequence. These results show the important role of the variable p53 C-terminal amino acid sequences for DNA recognition.

R-Loop Tracker: Web Access-Based Tool for R-Loop Detection and Analysis in Genomic DNA Sequences.

R-loops are common non-B nucleic acid structures formed by a three-stranded nucleic acid composed of an RNA-DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. Because the aberrant R-loop formation leads to increased mutagenesis, hyper-recombination, rearrangements, and transcription-replication collisions, it is regarded as important in human diseases. Therefore, its prevalence and distribution in genomes are studied intensively. However, in silico tools for R-loop prediction are limited, and therefore, we have developed the R-loop tracker tool, which was implemented as a part of the DNA Analyser web server. This new tool is focused upon (1) prediction of R-loops in genomic DNA without length and sequence limitations; (2) integration of R-loop tracker results with other tools for nucleic acids analyses, including Genome Browser; (3) internal cross-evaluation of in silico results with experimental data, where available; (4) easy export and correlation analyses with other genome features and markers; and (5) enhanced visualization outputs. Our new R-loop tracker tool is freely accessible on the web pages of DNA Analyser tools, and its implementation on the web-based server allows effective analyses not only for DNA segments but also for full chromosomes and genomes.

Analyses of viral genomes for G-quadruplex forming sequences reveal their correlation with the type of infection.

G-quadruplexes contribute to the regulation of key molecular processes. Their utilization for antiviral therapy is an emerging field of contemporary research. Here we present comprehensive analyses of the presence and localization of putative G-quadruplex forming sequences (PQS) in all viral genomes currently available in the NCBI database (including subviral agents). The G4Hunter algorithm was applied to a pool of 11,000 accessible viral genomes representing 350 Mbp in total. PQS frequencies differ across evolutionary groups of viruses, and are enriched in repeats, replication origins, 5'UTRs and 3'UTRs. Importantly, PQS presence and localization is connected to viral lifecycles and corresponds to the type of viral infection rather than to nucleic acid type; while viruses routinely causing persistent infections in Metazoa hosts are enriched for PQS, viruses causing acute infections are significantly depleted for PQS. The unique localization of PQS identifies the importance of G-quadruplex-based regulation of viral replication and life cycle, providing a tool for potential therapeutic targeting.

Tracing dsDNA Virus-Host Coevolution through Correlation of Their G-Quadruplex-Forming Sequences.

The importance of gene expression regulation in viruses based upon G-quadruplex may point to its potential utilization in therapeutic targeting. Here, we present analyses as to the occurrence of putative G-quadruplex-forming sequences (PQS) in all reference viral dsDNA genomes and evaluate their dependence on PQS occurrence in host organisms using the G4Hunter tool. PQS frequencies differ across host taxa without regard to GC content. The overlay of PQS with annotated regions reveals the localization of PQS in specific regions. While abundance in some, such as repeat regions, is shared by all groups, others are unique. There is abundance within introns of Eukaryota-infecting viruses, but depletion of PQS in introns of bacteria-infecting viruses. We reveal a significant positive correlation between PQS frequencies in dsDNA viruses and corresponding hosts from archaea, bacteria, and eukaryotes. A strong relationship between PQS in a virus and its host indicates their close coevolution and evolutionarily reciprocal mimicking of genome organization.

G-Quadruplexes in the Archaea Domain.

The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in Hadesarchaea archeon (strikingly, more than 50% of the Hadesarchaea archaeon isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the Parvarchaeota phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea.

G4Killer web application: a tool to design G-quadruplex mutations.

MOTIVATION: G-quadruplexes (G4) are important regulatory non-B DNA structures with therapeutic potential. A tool for rational design of mutations leading to decreased propensity for G4 formation should be useful in studying G4 functions. Although tools exist for G4 prediction, no easily accessible tool for the rational design of G4 mutations has been available. RESULTS: We developed a web-based tool termed G4Killer that is based on the G4Hunter algorithm. This new tool is a platform-independent and user-friendly application to design mutations crippling G4 propensity in a parsimonious way (i.e., keeping the primary sequence as close as possible to the original one). The tool is integrated into our DNA analyzer server and allows for generating mutated DNA sequences having the desired lowered G4Hunter score with minimal mutation steps. AVAILABILITY AND IMPLEMENTATION: The G4Killer web tool can be accessed at: http://bioinformatics.ibp.cz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Divergent distributions of inverted repeats and G-quadruplex forming sequences in Saccharomyces cerevisiae.

The importance of DNA structure in the regulation of basic cellular processes is an emerging field of research. Among local non-B DNA structures, inverted repeat (IR) sequences that form cruciforms and G-rich sequences that form G-quadruplexes (G4) are found in all prokaryotic and eukaryotic organisms and are targets for regulatory proteins. We analyzed IRs and G4 sequences in the genome of the most important biotechnology microorganism, S. cerevisiae. IR and G4-prone sequences are enriched in specific genomic locations and differ markedly between mitochondrial and nuclear DNA. While G4s are overrepresented in telomeres and regions surrounding tRNAs, IRs are most enriched in centromeres, rDNA, replication origins and surrounding tRNAs. Mitochondrial DNA is enriched in both IR and G4-prone sequences relative to the nuclear genome. This extensive analysis of local DNA structures adds to the emerging picture of their importance in genome maintenance, DNA replication and transcription of subsets of genes.

The Presence and Localization of G-Quadruplex Forming Sequences in the Domain of Bacteria.

The role of local DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, the significance of G-quadruplexes was demonstrated in the last decade, and their presence and functional relevance has been demonstrated in many genomes, including humans. In this study, we analyzed the presence and locations of G-quadruplex-forming sequences by G4Hunter in all complete bacterial genomes available in the NCBI database. G-quadruplex-forming sequences were identified in all species, however the frequency differed significantly across evolutionary groups. The highest frequency of G-quadruplex forming sequences was detected in the subgroup Deinococcus-Thermus, and the lowest frequency in Thermotogae. G-quadruplex forming sequences are non-randomly distributed and are favored in various evolutionary groups. G-quadruplex-forming sequences are enriched in ncRNA segments followed by mRNAs. Analyses of surrounding sequences showed G-quadruplex-forming sequences around tRNA and regulatory sequences. These data point to the unique and non-random localization of G-quadruplex-forming sequences in bacterial genomes.

G4Hunter web application: a web server for G-quadruplex prediction.

MOTIVATION: Expanding research highlights the importance of guanine quadruplex structures. Therefore, easy-accessible tools for quadruplex analyses in DNA and RNA molecules are important for the scientific community. RESULTS: We developed a web version of the G4Hunter application. This new web-based server is a platform-independent and user-friendly application for quadruplex analyses. It allows retrieval of gene/nucleotide sequence entries from NCBI databases and provides complete characterization of localization and quadruplex propensity of quadruplex-forming sequences. The G4Hunter web application includes an interactive graphical data representation with many useful options including visualization, sorting, data storage and export. AVAILABILITY AND IMPLEMENTATION: G4Hunter web application can be accessed at: http://bioinformatics.ibp.cz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Selective coronary angiography, percutaneous coronary intervention and asymptomatic peri-procedural myocardial injury.

BACKGROUND: While there have been a number of studies reporting the incidence and implications of elevated troponin levels after percutaneous coronary intervention (PCI), the body of information about the incidence, associations, and implications of elevated troponin levels following coronary angiography (CAG) is limited. MATERIALS AND METHODS: A total of 220 consecutive patients with stable coronary artery disease or intermediate or low-risk acute coronary syndrome without persistent ST-segment elevation (NSTE-ACS) were included in our study. High-sensitivity cardiac troponin I (hs-cTnI) levels were measured before and after coronary angiography (CAG) in patients with or without PCI and correlated with a number of clinical variables. RESULTS: Hs-cTnI elevations above the 99th percentile upper reference limit (URL), or above 20% of the initially positive, yet already declining values, were found in 60 (37.2%) patients after CAG and in 45 (76.2%) patients undergoing PCI. Significant correlations of hs-cTnI elevation were found with the following variables: volume of contrast, fluoroscopy time, dose-area product, amount of contrast agent injected directly into the coronary arteries, total time of balloon dilation and the number and total length of implanted stents (P<0.001 for all). CONCLUSION: While an asymptomatic elevation of hs-cTnI is a common finding after PCI, it does occur, quite surprisingly, also after CAG. Despite contradictory views regarding the clinical relevance of asymptomatic post-procedural elevated hs-cTnI levels, it is generally believed that a mild elevation is not associated with an increased risk. Still, it may pose a diagnostic quandary following a successful interventional procedure and even more so after an uncomplicated CAG. TRIAL REGISTRATION: Clinicaltrials.gov - NCT02960321.

[Palliative care in cardiology]. / Paliativní péce v kardiologii.

Chronic heart failure is a progressive disease with an increasing prevalence. In spite of all medical progress (or thanks to it), it is finally one of the most common causes of death. Palliative care is an approach aimed to improve the quality of life of patients and their families in cases of life-threatening illness. Therefore, the use of palliative care in cardiology is entirely relevant. Estimating the course and prognosis of a patient with chronic heart failure is difficult despite many prognostic tools. This makes it difficult to find the moment when palliative care is to begin. In general, gradual accentuation of treatment is recommended, focusing on the symptoms of the chronic progressive disease trajectory, and a reassessment of the benefit and potential adverse effects of conventional therapy. The main aspects of palliative care in cardiology are: 1. long-term communication with the patient and the family and their continuous education; 2. symptom-based treatment; 3. planning of advanced care; 4. A multidisciplinary team trying to meet all the patient's personality needs. Accepting palliative care as a part of complex care is a great challenge for the future of cardiology in the Czech Republic.Key words: heart failure, palliative care, cardiology.

Effect of a 6-month pedometer-based walking intervention on functional capacity in patients with chronic heart failure with reduced (HFrEF) and with preserved (HFpEF) ejection fraction: study protocol for two multicenter randomized controlled trials.

BACKGROUND: Regular physical activity is recommended for patients with chronic heart failure to improve their functional capacity, and walking is a popular, effective, and safe form of physical activity. Pedometers have shown potential to increase the amount of walking across a range of chronic diseases, but it is unknown whether a pedometer-based intervention improves functional capacity and neurohumoral modulation in heart failure patients. METHODS: Two multicenter randomized controlled trials will be conducted in parallel: one in patients with chronic heart failure with reduced ejection fraction (HFrEF), the other in patients with chronic heart failure with preserved ejection fraction (HFpEF). Each trial will consist of a 6-month intervention with an assessment at baseline, at 3 months, at the end of the intervention, and 6 months after completing the intervention. Each trial will aim to include a total of 200 physically inactive participants with chronic heart failure who will be randomly assigned to intervention or control arms. The 6-month intervention will consist of an individualized pedometer-based walking program with weekly step goals, behavioral face-to-face sessions with a physician, and regular telephone calls with a research nurse. The intervention will be based on effective behavioral principles (goal setting, self-monitoring, personalized feedback). The primary outcome is the change in 6-min walk distance at the end of the 6-month intervention. Secondary outcomes include changes in serum biomarkers levels, pulmonary congestion assessed by ultrasound, average daily step count measured by accelerometry, anthropometric measures, symptoms of depression, health-related quality of life, self-efficacy, and MAGGIC risk score. DISCUSSION: To our knowledge, these are the first studies to evaluate a pedometer-based walking intervention in patients with chronic heart failure with either reduced or preserved ejection fraction. The studies will contribute to a better understanding of physical activity promotion in heart failure patients to inform future physical activity recommendations and heart failure guidelines. Trial registration The trials are registered in ClinicalTrials.gov, identifiers: NCT03041610, registered 29 January 2017 (HFrEF), NCT03041376, registered 1 February 2017 (HFpEF).

Association of genetic variability in selected regions in visfatin (NAMPT) gene with anthropometric parameters and dietary composition in obese and non-obese Central-European population.

AIMS: Visfatin (NAMPT/PBEF) is a recently identified adipocytokine which harbors strong insulin-mimetic activity and was reported to be associated with obesity. However, nothing is known about whether visfatin is related to specific nutritional behavior which may result in obesity development. This is the first study focusing on genetic variability of the visfatin gene and its association with circulating visfatin, anthropometric parameters and dietary composition. MATERIALS AND METHODS: We analyzed a total of 11 exons and adjacent non-coding regions of the NAMPT gene in 20 extremely obese Czech individuals (mean BMI 52.2±5.0 SD) using direct sequencing and a frequency of rs2302559 was established in the validation cohort of another 605 individuals with completed 7-day food records and complex anthropometric measurements. Serum levels of visfatin, leptin and leptin-receptor were measured in all sequenced individuals and in part of the validation cohort. RESULTS: Three common polymorphisms were identified, two in non-coding regions (rs78411774 A/C, rs71564769 A/C) and one synonymous SNP in exon 7 (rs2302559 A/G). The rs2302559 showed significant correlation with visfatin serum level throughout the entire study cohort (p<0.001); there was a significant tendency toward higher visfatin levels in G allele carriers with GG homozygotes having the highest visfatin serum levels. Furthermore, a negative correlation was observed between visfatin and leptin serum level (p=0.01). No association between investigated SNPs and anthropometric parameters or native dietary composition was observed. CONCLUSION: This is the first study to demonstrate that the rs2302559 polymorphism in the PBEF gene is related to circulating levels of visfatin. As the SNP is synonymous, we hypothesize it might be linked to another SNP in the PBEF gene which controls visfatin serum levels.

Visfatin and its role in obesity development.

Visfatin, a product of PBEF gene, is an adipocytokine that harbours strong insulin-mimetic activity and it has been reported previously to associate with obesity. Recent reports also provide evidence that Visfatin has also important intracellular effects as it is homologous with nicotinamide phosphoribosyltransferase (NAMPT). In this review, we summarize the main documented effects of Visfatin on metabolism in humans, with special emphasis put on the pathways associated with obesity.