Analysis of the determinants of Pol II pausing

Pausing of transcribing RNA polymerase II (Pol II) has emerged as a general feature of gene expression in human cells. Many transcription factors, DNA sequences and chromatin characteristics have been implicated in inducing transcriptional pausing. However, it is unclear what are the relative contributions of these factors on the observed Pol II pausing. Furthermore, research in metazoans has mainly focused on Pol II promoter-proximal pausing, leaving the causes of pausing outside of this region unknown. To reliably detect real transcriptional pausing sites and advance the understanding of the causes of this phenomenon, we developed a pausing detection algorithm for nucleotide-resolution Pol II occupancy data. We scrutinized the characteristics and potential shortcomings of Native Elongating Transcript sequencing (NET-seq), which is one of the high-resolution methods of Pol II profiling, and we used our observations to improve the NET-seq processing pipeline. Leveraging the improved processing pipeline and the developed pausing detection algorithm revealed widespread genome-wide Pol II pausing at a nucleotide resolution in human cells. Next, we set out to identify the determinants of Pol II pausing in an unbiased manner based on the underlying DNA sequence. To predict the predisposition of a genomic site to evoke Pol II pausing, we applied a range of machine learning approaches using previously identified high-confidence pausing sites. For each of the sites, we created a large number of features, including both factors that were previously linked to transcriptional pausing and factors that were not yet implicated in invoking pausing. Our analysis revealed DNA sequence properties underlying widespread Pol II pausing including a new pausing motif. Interestingly, key sequence determinants of RNA polymerase pausing are shared by human cells and bacteria. Our study indicates that transcriptional pausing in human cells is sequence-induced and that the determinants of Pol II pausing might be evolutionary conserved.Ein allgemeines Merkmal der Genexpression in menschlichen Zellen ist das Pausieren der RNA Polymerase II (Pol II). Verschiedene Aspekte wie Transkriptionsfaktoren, DNA Sequenzen und Eigenschaften des Chromatins werden mit dem Prozess in Verbindung gebracht. Der relative Beitrag dieser Faktoren zur Entstehung der beobachteten Pausen ist unbekannt. Darüber hinaus hat sich die bisherige Forschung bei Metazoen hauptsächlich auf Pol II Pausen während der frühen Elongationsphase, im promoter-proximalen Bereich, konzentriert. Die Ursachen für das Pausieren außerhalb dieser Regionen sind unbekannt. Um das Verständnis der Ursachen von Transkriptionspausen zu verbessern, haben wir einen Algorithmus entwickelt, der Pol II Signale verarbeitet und Pausen präzise bis auf ein einzelnes Nukleotid lokalisiert. Die Pol II Signalmessungen werden mithilfe von NET-seq (Native Elongating Transcript Sequencing), einer hochauflösenden Methode, erstellt. Bei der Untersuchung der Methode identifizierten wir systematische Fehler in den Messdaten, welche zur Anpassung bei der Datenverarbeitung führte. Diese algorithmischen Verbesserungen zeigten, dass Pol II Pausen in menschlichen Zellen weit verbreitet sind und verteilt über das gesamte Genom, an einzelnen Nukleotiden, beobachtet werden können. Für eine unvoreingenommene Identifizierung der Sequenzspezifischen Faktoren, die zum Pausieren der Pol II beitragen, wurden eine Reihe von Methoden des maschinellen Lernens angewandt. Mit hoher Sicherheit detektierte Transkriptionspausen wurden genutzt, um Prädispositionen in DNA-Abschnitten zu lernen und vorherzusagen. Für jedes dieser Beispiel Regionen werden beschreibende Merkmale erstellt. Darunter befinden sich Faktoren, die zuvor mit Transkriptionspausen in Verbindung gebracht wurden, sowie Merkmale ohne bekannte Assoziation. Unsere Analyse identifiziert ein neues DNA Sequenzmotiv und andere relevante Sequenzeigenschaften, welche dem pausieren der Pol II zugrunde liegen. Interessanterweise sind die identifizierten Sequenzeigenschaften sowohl in menschlichen Zellen als auch in Bakterien zu finden. Unsere Studie deutet darauf hin, dass Transkriptionspausen in menschlichen Zellen sequenzabhängig und evolutionär konserviert sind

IsoTV: processing and visualizing functional features of translated transcript isoforms

Despite the continuous discovery of new transcript isoforms, fueled by the recent increase in accessibility and accuracy of long-read RNA sequencing data, functional differences between isoforms originating from the same gene often remain obscure. To address this issue and enable researchers to assess potential functional consequences of transcript isoform variation on the proteome, we developed IsoTV. IsoTV is a versatile pipeline to process, predict and visualize the functional features of translated transcript isoforms. Attributes such as gene and isoform expression, transcript composition and functional features are summarized in an easy-to-interpret visualization. IsoTV is able to analyze a variety of data types from all eukaryotic organisms, including short- and long-read RNA-seq data. Using Oxford Nanopore long read data, we demonstrate that IsoTV facilitates the understanding of potential protein isoform function in different cancer cell types

Management of stress incontinence in older women

Introduction: Stress incontinence is a problem increasingly affecting older people. This discomfort has an impact on women as a greater extent. Stress urinary incontinence is described as involuntary leakage of urine during even minor efforts: sneezing, coughing, rapid gait. At the beginning inconspicuous loss of a few drops of urine is often not alarming for women. The problem is significant because women often go to a specialist very late and the reason for this is shame. Many people think that this is an accident of old age and it can’t be stopped. Nothing could be more wrong, it can be dealt with. Material and methods: Articles in the EBSCO database have been analysed using keywords: stress incontinence, problems of old age, physiotherapy in urology, older women. Results: The incidence of stress incontinence in women increases with age. The main reason is muscular weakness, which is caused, among other things by pregnancies and labours. Another reason may be hormonal disorders and genetic predisposition. Statistics show that obese women are more likely to suffer from SUI. The progress of medicine and pharmacology is also increasingly effective in the treatment of stress urinary incontinence. First, after finding the problem, conservative treatment is introduced. Treatment is adapted to each patient individually. Age, existing diseases, weight are important factors in the process of treatment. In pharmacology Duloxetine is used. The use of this drug does not completely eliminate the symptoms of stress urinary incontinence. Due to the possibility of side effects i.e. nausea, the drug is used very carefully. In recent years, physiotherapy has been highly valued in the treatment of stress urinary incontinence. The most important is kinesitherapy here. Strengthening the pelvic floor muscles brings the most beneficial effects. Active exercises are supported by: electrostimulation, magnetotherapy and vibro-therapy. The final form of SUI treatment is surgical treatment. However, this do not always bring the expected results. In order to increase the effectiveness of therapy for patients with SUI, the interdisciplinary cooperation of the medical team should be used. Treatment of stress urinary incontinence primarily leads to improved quality of life for patients. Conclusions: Urinary incontinence is a social disease. Women struggle with this problem twice as often than men. Stress incontinence accounts for 63% of all forms of urinary incontinence in women in Poland. The incidence of incontinence increases with age. Problems with stress urinary incontinence become a reason for isolation from society. The patients are not aware of the treatment possibilities, which often results in late inclusion of treatment and rehabilitation. There is a wide range of SUI treatment options. Therefore it is necessary to personalise rehabilitation process to best fit to each patient. Keywords: stress incontinence, problems of old age, physiotherapy in urology, older women

A rehabilitation approach to the falls of geriatric patients

Abstract: Background: Falls of the elderly are not only in themselves one of the most common problems of geriatric patients, but also the cause of many complications that affect permanently or for a shorter period of time affect the quality of life of people over 65 years of age. These changes affect not only the efficiency and motility of the elderly but also may reduce self-esteem, limit social life or cause loss of self-confidence. Material and methods: Analysis of available literature, articles in the Google Scholar database and PubMed using keywords: falls, geriatrics, aging process, problem Results: In clinical practice, many tests and scales are used to prevent the fall of older people and related complications. These include: Timed Up & Go (TUG), Tinetti scale, Dynamic Gait Index (DGI), One Leg Standing (OLS), Four Square Step Test (FSST), Berg test or Functional Reach (FR). Preventing falls can primarily be achieved by the use of appropriate rehabilitation methods, which include, among others, kinesitherapy and properly selected orthopedic equipment. The aim of individually selected kinesitherapy is to restore or maintain the patient's full fitness through the use of physical exercises to improve the balance, increasing mobility and efficiency. In the case of orthopedic supplies, the most common are baby walkers (without wheels, walker with wheels, using a triangular and quadrangular base) and orthopedic balls. Conclusions: Prophylaxis to prevent destructive effects of falls of geriatric patients should be carried out in a comprehensive manner and cover a wide range of activities. There is a further need for research and discussion on the effectiveness of forms of rehabilitation to prevent the falls of older peopl

Sarcopenia as a problem of old age - a form of rehabilitation

Background: Sarkopenia as a disease has relatively recently become the object of research and the work of the academic community. Despite this, sarcopenia is an increasingly serious social problem, it can be seen in a large number of statistics and epidemiological studies. Among geriatric patients, along with their multiformity and the associated use of a large amount of medicines, it causes many negative effects related to the functioning of the whole organism as well as the quality of life of the patient himself. Material and methods: Analysis of available literature, articles in the Google Scholar and PubMed database using keywords: sarcopenia, geriatrics, rehabilitation, loss of muscle mass. Results: Among the studies and recommendations concerning sarcopenia, a continuous lack of clear, transparent and confirmed diagnostic criteria can be stated. At the diagnosis of this disease, an X-ray or a bioelectrical impedance analysis is used. Other methods used are functional tests, including a six-minute walk test or "Timed-Up and Go" test (TUG). In the case of rehabilitation of people with sarcopenia, the most effective form of kinesitherapy is individually selected resistive training, which has a positive effect not only on muscle mass growth, but also the body's protein metabolism and the improvement of the endocrine system. Physiotherapy is the most commonly used method of physical therapy, among other methods of rehabilitation there are also therapeutic massage, aqua aerobics, vibratory training, and Nordic Walking and diet supplementation. Conclusions: Sarcopenia in geriatric patients, especially with ineffective treatment and rehabilitation, has a destructive impact on both the physical and mental sphere of the patient. There is a great need for more extensive research, both on the whole disease process and the effectiveness of forms of assistance to patients

Ten simple rules for implementing open and reproducible research practices after attending a training course

Open, reproducible, and replicable research practices are a fundamental part of science. Training is often organized on a grassroots level, offered by early career researchers, for early career researchers. Buffet style courses that cover many topics can inspire participants to try new things; however, they can also be overwhelming. Participants who want to implement new practices may not know where to start once they return to their research team. We describe ten simple rules to guide participants of relevant training courses in implementing robust research practices in their own projects, once they return to their research group. This includes (1) prioritizing and planning which practices to implement, which involves obtaining support and convincing others involved in the research project of the added value of implementing new practices; (2) managing problems that arise during implementation; and (3) making reproducible research and open science practices an integral part of a future research career. We also outline strategies that course organizers can use to prepare participants for implementation and support them during this process

Conserved DNA sequence features underlie pervasive RNA polymerase pausing

Pausing of transcribing RNA polymerase is regulated and creates opportunities to control gene expression. Research in metazoans has so far mainly focused on RNA polymerase II (Pol II) promoter-proximal pausing leaving the pervasive nature of pausing and its regulatory potential in mammalian cells unclear. Here, we developed a pause detecting algorithm (PDA) for nucleotide-resolution occupancy data and a new native elongating transcript sequencing approach, termed nested NET-seq, that strongly reduces artifactual peaks commonly misinterpreted as pausing sites. Leveraging PDA and nested NET-seq reveal widespread genome-wide Pol II pausing at single-nucleotide resolution in human cells. Notably, the majority of Pol II pauses occur outside of promoter-proximal gene regions primarily along the gene-body of transcribed genes. Sequence analysis combined with machine learning modeling reveals DNA sequence properties underlying widespread transcriptional pausing including a new pause motif. Interestingly, key sequence determinants of RNA polymerase pausing are conserved between human cells and bacteria. These studies indicate pervasive sequence-induced transcriptional pausing in human cells and the knowledge of exact pause locations implies potential functional roles in gene expression

Ten simple rules for implementing open and reproducible research practices after attending a training course

Training in robust research practices is becoming increasingly common. However, many course participants may encounter challenges in implementation of what they learned after returning to their research groups. In this piece, we summarize insights and "lessons learned" from a group of former course participants. We offer practical tips on implementation and cultural change that may be useful for researchers at any career stage. In addition, we provide a list of considerations for course instructors to help them support course attendees after training is over