Multiplexed transcriptome discovery of RNA-binding protein binding sites by antibody-barcode eCLIP.

Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.

Progressive Loading in a Strongman Following Distal Biceps Repair: Re-Thinking Load Progression - A Case Report.

Background: Rupture of the distal biceps is relatively rare and post-operative protocols are typically vague and are used on many patients, regardless of pre-morbid status. The primary objective is to share the progressive loading strategy used in the rehabilitation of a strongman athlete following a surgical repair of the distal biceps. An additional objective is to highlight the need for individualized protocols and progressions with respect to patient goals and sport demands, as well as the need for shared decision making (SDM) between the medical doctor, patient, and rehabilitation provider. Case Presentation: The subject is a 39-year-old strong man competitor who suffered a distal biceps rupture while doing a tire flip during training. After having it repaired, the post-operative recovery was unremarkable. The focus of the described intervention was establishing load during rehabilitation exercises that were unique to this individual based on his pre-morbid level of strength and training history as well as the unique demands of his sport. Outcomes: The patient achieved symmetrical isokinetic strength of the elbow flexors at 60°/second in supine at six months post-operative. Discussion: The case highlights a successful outcome in a strongman competitor with a distal biceps rupture repair. Typically, protocols are vague and lack specific standards for establishment of load for exercises. Often starting points and progressions are arbitrary and lack rationale tailored to individual needs and/or pre-morbid status. The case offers a framework for establishing and progressing load while also discussing how a shared decision-making model can lead to positive outcomes.

Can Clinician-Stabilization with Hand-Held Dynamometry Yield a Reliable Measure of Knee Flexion Torque?

Background: Assessment of knee flexion torque is a relevant clinical measure following various injuries and surgeries to determine progress in rehabilitation and inform decision making. A variety of methods using hand-held dynamometry have been shown to be reliable in obtaining this measure, and typically require a means of external fixation or stabilization. Clinically efficient methods of reliable clinician-stabilization are sparse in the literature. Hypothesis/Purpose: Determine inter and intra-rater reliability of two clinically efficient methods of assessing isometric knee flexion torque using hand-held dynamometry with clinician-stabilization. The hypothesis was that each method would yield good to excellent reliability. Study Design: Cross-Sectional Study. Methods: Twenty healthy individuals were assessed by two clinicians on two separate days. During each session, knee flexion torque was assessed with hand-held dynamometry with two methods: 1) in the seated position with the hip and knee flexed to 90 degrees while the clinician stabilized the dynamometer between the participant's leg and table and 2) in prone with the hip at 0 degrees and knee at 90 degrees while the clinician assumed a stride stance with elbows locked in extension to stabilize the dynamometer on the participant's leg. Inter and intra-rater reliability were determined for each method. Results: ICC values were 0.88-0.94 and 0.77-0.90 for inter and intra-rater reliability respectively with the seated method. The prone method yielded ICC values of 0.84-0.96 and 0.89-0.94 for inter and intra-rater reliability respectively. MDC values ranged from 30-62% with the seated method and 21-40% with the prone method. Conclusion: Inter and intra-rater reliability were good to excellent for assessing knee flexion torque with hand-held dynamometry using both the seated and prone methods with clinically efficient clinician-stabilization approaches. The prone method may be more sensitive to detecting change over time due to lower MDC values. Level of Evidence: 2b.

Conceptualising disaster social capital: what it is, why it matters, and how it can be enhanced.

Social capital discourse occupies an important place in disaster studies. Scholars have adopted various inflections of social capital to explain how those with greater amounts of this crucial resource are generally more resilient to disasters and experience speedier recovery. Disaster scholars have also discovered that people typically display altruistic tendencies in the wake of disasters and develop novel networks of mutual support, known as 'communitas', which is also seen to build resilience and boost recovery. In this paper, we use the work of Pierre Bourdieu to synthesise these literatures, conceptualising communitas as 'disaster social capital'. We offer a fleshed-out definition of disaster social capital to distinguish it from regular social capital and discuss the barriers to, and the enablers of, its formation. While primarily a conceptual discussion, we believe that it has practical and policy value for disaster scholars and practitioners interested in inclusive disaster risk reduction as well as full and just recoveries.

Blood Flow Restriction Training.

Muscle weakness and atrophy are common impairments after musculoskeletal injury. Blood flow restriction (BFR) training offers the ability to mitigate weakness and atrophy without overloading healing tissues. It appears to be a safe and effective approach to therapeutic exercise in sports medicine environments. This approach requires consideration of a wide range of factors, and the purpose of our article is to provide insights into proposed mechanisms of effectiveness, safety considerations, application guidelines, and clinical recommendations for BFR training after musculoskeletal injury. Whereas training with higher loads produces the most substantial increases in strength and hypertrophy, BFR training appears to be a reasonable option for bridging earlier phases of rehabilitation when higher loads may not be tolerated by the patient and later stages that are consistent with return to sport.

Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes.

RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.

A multi-centre, randomized, controlled trial on coaching and telemonitoring in patients with cystic fibrosis: conneCT CF.

BACKGROUND: The extend of lung disease remains the most important prognostic factor for survival in patients with cystic fibrosis (CF), and lack of adherence is the main reason for treatment failure. Early detection of deterioration in lung function and optimising adherence are therefore crucial in CF care. We implement a randomized controlled trial to evaluate efficacy of telemonitoring of adherence, lung function, and health condition in combination with behavior change interventions using innovative digital technologies. METHODS: This is a multi-centre, randomized, controlled, non-blinded trial aiming to include 402 patients ≥ 12 years-of-age with CF. A standard-of-care arm is compared to an arm receiving objective, continuous monitoring of adherence to inhalation therapies, weekly home spirometry using electronic devices with data transmission to patients and caring physicians combined with video-conferencing, a self-management app and professional telephone coaching. The duration of the intervention phase is 18 months. The primary endpoint is time to the first protocol-defined pulmonary exacerbation. Secondary outcome measures include number of and time between pulmonary exacerbations, adherence to inhalation therapy, changes in forced expiratory volume in 1 s from baseline, number of hospital admissions, and changes in health-related quality of life. CF-associated medical treatment and care, and health care related costs will be assessed by explorative analysis in both arms. DISCUSSION: This study offers the opportunity to evaluate the effect of adherence interventions using telemedicine capable devices on adherence and lung health, possibly paving the way for implementation of telemedicine in routine care for patients with CF. TRIAL REGISTRATION: This study has been registered with the German Clinical Trials Register (Identifier: DRKS00024642, date of registration 01 Mar 2021, URL: https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00024642 ).

A live-cell assay for the detection of pre-microRNA-protein interactions.

Recent efforts in genome-wide sequencing and proteomics have revealed the fundamental roles that RNA-binding proteins (RBPs) play in the life cycle and function of coding and non-coding RNAs. While these methodologies provide a systems-level view of the networking of RNA and proteins, approaches to enable the cellular validation of discovered interactions are lacking. Leveraging the power of bioorthogonal chemistry- and split-luciferase-based assay technologies, we have devised a conceptually new assay for the live-cell detection of RNA-protein interactions (RPIs), RNA interaction with Protein-mediated Complementation Assay, or RiPCA. As proof-of-concept, we utilized the interaction of the pre-microRNA, pre-let-7, with its binding partner, Lin28. Using this system, we have demonstrated the selective detection of the pre-let-7-Lin28 RPI in both the cytoplasm and nucleus. Furthermore, we determined that this technology can be used to discern relative affinities for specific sequences as well as of individual RNA binding domains. Thus, RiPCA has the potential to serve as a useful tool in supporting the investigation of cellular RPIs.

Frictions of implementing EU humanitarian aid in Greece (2016-2019)-the Emergency Support Instrument and its practical ramifications.

With the closure of the border with then-Macedonia in early 2016, it was foreseeable that Greece would become the "last station" for a large number of refugees. Flanked by the agreement between Turkey and the European Union of March 2016, Greece underwent a profound transformation from a transit country to a recipient country. Through a new regulation, the Emergency Support Instrument, initially activated by the European Commission 2016-2019, international humanitarian aid operations were supported for the first time in the EU. The article analyzes the resulting frictions on the basis of empirical field research and a broad literature review. While frictions similar to those in other non-European humanitarian operations exist, specific peculiarities due to the operation taking place in an austerity-ridden member state of the EU must also be noted.

CRITERIA-BASED RETURN TO SPRINTING PROGRESSION FOLLOWING LOWER EXTREMITY INJURY.

In the terminal phases of athletic rehabilitation, transitioning back to sport is a critical aspect to prepare an athlete for return to full participation. Numerous interval sport programs have been published in the literature and return to sports guidelines and criteria-based progressions for returning to sport have been published, but no such protocol exists for returning to the task of sprinting. Any field or court athlete must be able to sprint as part of his/her sport demands. Because of the absence of a specific progression, sports rehabilitation professionals lack knowledge about objective criteria to progress to sprinting as well as a progressive program to do so. Given that sports rehabilitation professionals have limited visits to complete rehabilitation or their athletes have limited financial resources to do so, it is imperative that a structured, criteria-based progression be available. The purpose of this clinical suggestion is to provide a criteria-based return to sprinting progression. LEVEL OF EVIDENCE: 5.

Direct RNA sequencing enables m6A detection in endogenous transcript isoforms at base-specific resolution.

Direct RNA sequencing holds great promise for the de novo identification of RNA modifications at single-coordinate resolution; however, interpretation of raw sequencing output to discover modified bases remains a challenge. Using Oxford Nanopore's direct RNA sequencing technology, we developed a random forest classifier trained using experimentally detected N6-methyladenosine (m6A) sites within DRACH motifs. Our software MINES (m6A Identification using Nanopore Sequencing) assigned m6A methylation status to more than 13,000 previously unannotated DRACH sites in endogenous HEK293T transcripts and identified more than 40,000 sites with isoform-level resolution in a human mammary epithelial cell line. These sites displayed sensitivity to the m6A writer, METTL3, and eraser, ALKBH5, respectively. MINES (https://github.com/YeoLab/MINES.git) enables m6A annotation at single coordinate-level resolution from direct RNA nanopore sequencing.

A click chemistry assay to identify natural product ligands for pre-microRNAs.

Despite the great diversity of structure and function and relevance to human health, RNA remains an underexploited area of drug discovery. A major bottleneck toward this goal has been the identification of probes and drug leads that are specific for select RNAs and methods that will facilitate such discovery efforts. Our laboratory has recently developed an innovative approach for assaying RNA-small molecule interactions, catalytic enzyme-linked click chemistry assay or cat-ELCCA, which is a functional assay that takes advantage of the power of catalytic signal amplification combined with the selectivity and bioorthogonality of click chemistry. Importantly, through application of this platform assay technology to the challenging problem of identifying selective inhibitors of pre-microRNA maturation, we identified natural products as a potential source of such compounds. Herein we describe this methodology in addition to the downstream pipeline toward the discovery of natural product ligands for pre-microRNAs. Through cat-ELCCA, our goal is to discover novel ligands to facilitate our investigation of RNA recognition by small molecules.

Click Chemistry-Mediated Complementation Assay for RNA-Protein Interactions.

Click chemistry-based assays are a growing class of biochemical assay for facilitating the discovery of modulators of important biological processes. To date, most have relied on the use of immobilized biomolecules, which increases the cost of the assay and decreases throughput because of the necessary washing steps. To overcome these challenges, we have developed a click chemistry-mediated complementation assay that retains many of the advantages of the previous technology, including catalytic signal amplification for assay robustness and applicability to full-length biomolecules, but that can be performed in a homogeneous format. As demonstration of this methodology, we have developed a new high-throughput screening method for RNA-protein interactions using the interaction of Lin28 with the pre-microRNA, prelet-7, as a model.

Tetracyclines as Inhibitors of Pre-microRNA Maturation: A Disconnection between RNA Binding and Inhibition.

In a high-throughput screening campaign, we recently discovered the rRNA-binding tetracyclines, methacycline and meclocycline, as inhibitors of Dicer-mediated processing of microRNAs. Herein, we describe our biophysical and biochemical characterization of these compounds. Interestingly, although direct, albeit weak, binding to the pre-microRNA hairpins was observed, the inhibitory activity of these compounds was not due to RNA binding. Through additional biochemical and chemical studies, we revealed that metal chelation likely plays a principle role in their mechanism of inhibition. By exploring the activity of other known RNA-binding scaffolds, we identified additional disconnections between direct RNA interaction and inhibition of Dicer processing. Thus, the results presented within provide a valuable case study in the complexities of targeting RNA with small molecules, particularly with weak binding and potentially promiscuous scaffolds.

Expansion of cat-ELCCA for the Discovery of Small Molecule Inhibitors of the Pre-let-7-Lin28 RNA-Protein Interaction.

Dysregulation of microRNA (miRNA) expression has been linked to many human diseases; however, because of the challenges associated with RNA-targeted drug discovery, additional approaches are needed for probing miRNA biology. The emerging regulatory role of miRNA-binding proteins in miRNA maturation presents such an alternative strategy. Exploiting our laboratory's click chemistry-based high-throughput screening (HTS) technology, catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a modular method by which to discover new chemical tools for manipulating pre-miRNA-miRNA-binding protein interactions. Using the pre-let-7d-Lin28 interaction as proof-of-concept, the results presented demonstrate how HTS using cat-ELCCA can enable the discovery of small molecules targeting RNA-protein interactions.

Enhanced CD8+ cytolytic T cell responses in the peripheral circulation of patients with sarcoidosis and non-Löfgren's disease.

BACKGROUND: The role of CD4+ T cells in the immunopathogenesis of pulmonary sarcoidosis is well-established, while less is known about the phenotype and function of CD8+ cytolytic T cells (CTLs). METHODS: CD8+ CTLs were explored in peripheral blood and bronchoalveolar lavage (BAL) samples obtained from up to 25 patients with sarcoidosis and 25 healthy controls. The proportion of CTLs was assessed by the expression of cytolytic effector molecules perforin, granzyme B and granulysin in CD8+ T cells, using flow cytometry. Cytolytic function in blood lymphocytes was assessed using a standard 51Cr-release assay. Patients with Löfgren´s syndrome (LS) and an acute disease onset, were compared to non-LS patients with an insidious onset. RESULTS: Higher proportions of peripheral CD8+ CTLs expressing perforin and granzyme B were observed in sarcoidosis compared to healthy controls. Blood CTLs from non-LS patients had significantly higher expression of perforin, granzyme B and granulysin compared to matched BAL, while LS patients maintained lower levels of effector molecules in both compartments. Mitogen-stimulated peripheral lymphocytes from sarcoidosis patients, particularly from the non-LS group, showed a higher target cell lysis compared to controls. CONCLUSION: These results demonstrated enhanced peripheral CD8+ CTL responses in sarcoidosis, especially in non-LS patients who have an increased risk of chronic disease. Further comprehensive clinical studies are warranted to increase our understanding of CD8+ CTL responses in sarcoidosis.

Development and Implementation of an HTS-Compatible Assay for the Discovery of Selective Small-Molecule Ligands for Pre-microRNAs.

microRNAs (miRNAs) are small gene regulatory RNAs, and their expression has been found to be dysregulated in a number of human diseases. To facilitate the discovery of small molecules capable of selectively modulating the activity of a specific miRNA, we have utilized new high-throughput screening technology targeting Dicer-mediated pre-miRNA maturation. Pilot screening of ~50,000 small molecules and ~33,000 natural product extract libraries against pre-miR-21 processing indicated the potential of our assay for this goal, yielding a campaign Z' factor of 0.52 and an average plate signal-to-background (S/B) ratio of 13. Using two-dimensional screening against a second pre-miRNA, pre-let-7d, we evaluated the selectivity of confirmed hits. The results presented demonstrate how high-throughput screening can be used to identify selective small molecules for a target RNA.

A click chemistry-based microRNA maturation assay optimized for high-throughput screening.

Catalytic enzyme-linked click-chemistry assays (cat-ELCCA) are an emerging class of biochemical assay. Herein we report on expanding the toolkit of cat-ELCCA to include the kinetically superior inverse-electron demand Diels-Alder (IEDDA) reaction. The result is a technology with improved sensitivity and reproducibility, enabling automated high-throughput screening.

CURRENT CONCEPTS IN PERIODIZATION OF STRENGTH AND CONDITIONING FOR THE SPORTS PHYSICAL THERAPIST.

UNLABELLED: The rehabilitation process is driven by the manipulation of training variables that elicit specific adaptations in order to meet established goals. Periodization is an overall concept of training that deals with the division of the training process into specific phases. Programming is the manipulation of the variables within these phases (sets, repetitions, load) that are needed to bring about the specific adaptations desired within that particular period. The current body of literature is very limited when it comes to how these variables are best combined in an injured population since most of the periodization research has been done in a healthy population. This manuscript explores what is currently understood about periodization, gives clinical guidelines for implementation, and provides the sports physical therapist with a framework to apply these principles in designing rehabilitation programs. LEVEL OF EVIDENCE: 5.

High-throughput platform assay technology for the discovery of pre-microrna-selective small molecule probes.

MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.