The novel pre-rRNA detection workflow "Riboprobing" allows simple identification of undescribed RNA species.

Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, Northern Blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with Primer Extension, both approaches often use radioactivity and are time consuming and costly. Here we present "Riboprobing" a reverse transcription-based workflow extended by linker ligation for easy and fast detection and characterization of various pre-rRNA species and their 5` as well as 3` ends. Using standard molecular biology lab equipment, our technique allows reliable discrimination of pre-rRNA species not resolved by Northern Blotting (e.g.: 27SA2, 27SA3 and 27SB). The method can be successfully used for analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA, arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that is lacking most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS for the assembly process.

Occupational therapy for persons with cognitive impairments.

Background: Damage to the central nervous system can occur in adulthood, for example, due to stroke, trauma, tumours, or chronic diseases. After damage to the central nervous system, cognitive impairments occur in addition to physical limitations. Occupational therapy is most often prescribed for neurological diagnoses, including stroke and traumatic brain injury. Methods: The health technology assessment (HTA) report this HTA article is based on investigates the clinical effectiveness, cost-effectiveness, and patient-related, social and ethical aspects of occupational therapy for patients with cognitive impairments compared to no occupational therapy. In addition, the effects of different occupational therapy interventions with and without cognitive components were compared in an explorative overview. Patients with moderate or severe dementia are excluded from the assessment. Systematic overviews, that is, systematic reviews of systematic reviews, were conducted. Results: For the evaluation of clinical effectiveness, a total of nine systematic reviews were included. No systematic review was identified for the assessment of costs or cost-effectiveness. Five systematic reviews were included for the assessment of patient and social aspects. For the assessment of clinical effectiveness compared with no occupational therapy, five systematic reviews comprising 20 randomised controlled trials with a total of 1,316 subjects reported small positive effects for the outcomes "global cognitive function" and "activities of daily living" as well as a non-quantified positive effect on the outcomes "health-related quality of life" and "behavioural control". No effect was found for individual components of cognition and measures of perception. The quality of the evidence for all outcomes is low due to a high risk of bias. In the supplementary presentations, no positive effects could be demonstrated on the basis of the available evidence. The quality of this evidence was not assessed. For the assessment of patient and social aspects, five systematic reviews on patients with a stroke or a traumatic brain injury - without specification regarding cognitive deficits or studies with their relatives - were included. It was reported that patients and family caregivers go through different phases of rehabilitation in which the discharge home is a decisive turning point. The discharge home represents a crucial breaking point. Regaining an active, self-determining role is a process that requires therapists to find the right level of support for patients and relatives. For the assessment of ethical aspects, nine documents were included. We identified ethical problem-solving models for occupational therapy and 16 ethical aspects in occupational therapy for cognitive deficits. The central theme of the analysis is the limited autonomy due to the consequences of the disease as well as the resulting tensions with those treating the patient. Conclusions: Based on this systematic overview, it can neither be proven nor excluded with certainty that occupational therapy for cognitive impairment is an effective therapy for adult patients with central nervous system injuries compared to no occupational therapy. There is a lack of randomised trials with sufficient sample size, well-defined interventions, and comparable concomitant therapies in the control groups, but there is also a lack of well-designed observational studies in routine care and health economic studies. The identified systematic reviews on patient and social aspects provide information on the needs of patients after stroke or traumatic brain injury and their relatives, but there is a lack of studies on this aspect in German-speaking countries. For the ethical assessment, in addition to the identified theoretical models for solving ethical conflicts in occupational therapy, more empirical studies on ethical aspects with patients with cognitive deficits and their relatives as well as occupational therapists are needed.

Visualizing maturation factor extraction from the nascent ribosome by the AAA-ATPase Drg1.

The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large ribosomal subunit. Drg1 releases the shuttling maturation factor Rlp24 from pre-60S particles shortly after nuclear export, a strict requirement for downstream maturation. The molecular mechanism of release remained elusive. Here, we report a series of cryo-EM structures that captured the extraction of Rlp24 from pre-60S particles by Saccharomyces cerevisiae Drg1. These structures reveal that Arx1 and the eukaryote-specific rRNA expansion segment ES27 form a joint docking platform that positions Drg1 for efficient extraction of Rlp24 from the pre-ribosome. The tips of the Drg1 N domains thereby guide the Rlp24 C terminus into the central pore of the Drg1 hexamer, enabling extraction by a hand-over-hand translocation mechanism. Our results uncover substrate recognition and processing by Drg1 step by step and provide a comprehensive mechanistic picture of the conserved modus operandi of AAA-ATPases.

Clinical management and primary prevention of suspected coronary artery disease guided by computed tomography.

AIMS: Despite the well established role of coronary computed tomography angiography (CCTA) as a diagnostic gatekeeper, the yield of subsequent invasive coronary angiographies (ICA) remains low. We evaluated the adherence of CCTA integration in clinical management and primary prevention therapy. METHODS: We retrospectively analyzed patients referred for ICA after CCTA without known coronary artery disease (CAD) or structural cardiac pathologies. Based on computed tomography (CT) findings, patients were classified as appropriately or inappropriately referred to ICA, equaling Coronary Artery Disease - Reporting and Data System (CAD-RADS) categories 0-2 (<50% stenosis) and 3-5 (>50% stenosis), respectively. CT exams were compared regarding invasive findings and revascularizations. Integration of CT results into primary prevention measures was analyzed and compared to measures taken after ICA. RESULTS: Of 1005 patients referred for ICA, 81 (8.1%) had no obstructive CT findings and therefore no ICA indication. ICA inappropriate patients did not differ in symptom characteristics, but had a significantly lower revascularization rate (3.7% vs. 42.1%, P < 0.0001) compared with patients appropriately referred to ICA. In patients with indication for lipid-lowering therapy after the CCTA statin rate was 53.1% and significantly increased after ICA to 76.4% (P < 0.0001). In CCTA, obstructive findings in proximal-only lesions did not increase the revascularization rate (45.6% vs. 42.1%, P = 0.11) but missed nonproximal relevant stenoses (15.0% vs. 2.5%, P < 0.0001) compared with obstructive findings in all segments. CONCLUSION: The overall rate of inappropriateness was low, but there is relevant statin underutilization in eligible patients due to a lack of CT findings integration. Both ICA referrals and primary preventive therapy could be improved by the implementation of CT results based on CAD-RADS recommendations.

From Snapshots to Flipbook-Resolving the Dynamics of Ribosome Biogenesis with Chemical Probes.

The synthesis of ribosomes is one of the central and most resource demanding processes in each living cell. As ribosome biogenesis is tightly linked with the regulation of the cell cycle, perturbation of ribosome formation can trigger severe diseases, including cancer. Eukaryotic ribosome biogenesis starts in the nucleolus with pre-rRNA transcription and the initial assembly steps, continues in the nucleoplasm and is finished in the cytoplasm. From start to end, this process is highly dynamic and finished within few minutes. Despite the tremendous progress made during the last decade, the coordination of the individual maturation steps is hard to unravel by a conventional methodology. In recent years small molecular compounds were identified that specifically block either rDNA transcription or distinct steps within the maturation pathway. As these inhibitors diffuse into the cell rapidly and block their target proteins within seconds, they represent excellent tools to investigate ribosome biogenesis. Here we review how the inhibitors affect ribosome biogenesis and discuss how these effects can be interpreted by taking the complex self-regulatory mechanisms of the pathway into account. With this we want to highlight the potential of low molecular weight inhibitors to approach the dynamic nature of the ribosome biogenesis pathway.

Inhibiting eukaryotic ribosome biogenesis: Mining new tools for basic research and medical applications.

The formation of new ribosomes is a fundamental cellular process for each living cell and is tightly interwoven with cell cycle control and proliferation. Minimal disturbances of this pathway can result in ribosomopathies including an increased risk for certain cancer types. Thus, targeting ribosome biogenesis is an emerging strategy in cancer therapy. However, due to its complex nature, we are only at the beginning to understand the dynamics of the ribosome biogenesis pathway. One arising approach that will help us to embrace the tight timely cascade of events that is needed to form a new ribosome is the use of targeted chemical inhibition. However, only very few specific chemical inhibitors of the ribosome biogenesis pathway have been identified so far. Here we review our recently published screen to identify novel inhibitors of the ribosome biogenesis pathway in yeast (Awad et al., 2019, BMC Biology). These inhibitors can provide novel tools for basic research and can serve as starting-points to develop new chemotherapeutics.

Inhibiting eukaryotic ribosome biogenesis.

BACKGROUND: Ribosome biogenesis is a central process in every growing cell. In eukaryotes, it requires more than 250 non-ribosomal assembly factors, most of which are essential. Despite this large repertoire of potential targets, only very few chemical inhibitors of ribosome biogenesis are known so far. Such inhibitors are valuable tools to study this highly dynamic process and elucidate mechanistic details of individual maturation steps. Moreover, ribosome biogenesis is of particular importance for fast proliferating cells, suggesting its inhibition could be a valid strategy for treatment of tumors or infections. RESULTS: We systematically screened ~ 1000 substances for inhibitory effects on ribosome biogenesis using a microscopy-based screen scoring ribosomal subunit export defects. We identified 128 compounds inhibiting maturation of either the small or the large ribosomal subunit or both. Northern blot analysis demonstrates that these inhibitors cause a broad spectrum of different rRNA processing defects. CONCLUSIONS: Our findings show that the individual inhibitors affect a wide range of different maturation steps within the ribosome biogenesis pathway. Our results provide for the first time a comprehensive set of inhibitors to study ribosome biogenesis by chemical inhibition of individual maturation steps and establish the process as promising druggable pathway for chemical intervention.