Absolute Protein Amounts and Relative Abundance of Volume-regulated Anion Channel (VRAC) LRRC8 Subunits in Cells and Tissues Revealed by Quantitative Immunoblotting

The volume-regulated anion channel (VRAC) plays an important role in osmotic cell volume regulation. In addition, it is involved in various physiological processes such as insulin secretion, glia-neuron communication and purinergic signaling. VRAC is formed by hetero-hexamers of members of the LRRC8 protein family, which consists of five members, LRRC8A-E. LRRC8A is an essential subunit for physiological functionality of VRAC. Its obligate heteromerization with at least one of its paralogues, LRRC8B-E, determines the biophysical properties of VRAC. Moreover, the subunit composition is of physiological relevance as it largely influences the activation mechanism and especially the substrate selectivity. However, the endogenous tissue-specific subunit composition of VRAC is unknown. We have now developed and applied a quantitative immunoblot study of the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using recombinant protein for signal calibration. We found tissue-specific expression patterns of the subunits, and generally relative low expression of the essential LRRC8A subunit. Immunoprecipitation of LRRC8A also co-precipitates an excess of the other subunits, suggesting that non-LRRC8A subunits present the majority in hetero-hexamers. With this, we can estimate that in the tested cell lines, the number of VRAC channels per cell is in the order of 10,000, which is in agreement with earlier calculations from the comparison of single-channel and whole-cell currents

Absolute Protein Amounts and Relative Abundance of Volume-regulated Anion Channel (VRAC) LRRC8 Subunits in Cells and Tissues Revealed by Quantitative Immunoblotting

The volume-regulated anion channel (VRAC) plays an important role in osmotic cell volume regulation. In addition, it is involved in various physiological processes such as insulin secretion, glia-neuron communication and purinergic signaling. VRAC is formed by hetero-hexamers of members of the LRRC8 protein family, which consists of five members, LRRC8A-E. LRRC8A is an essential subunit for physiological functionality of VRAC. Its obligate heteromerization with at least one of its paralogues, LRRC8B-E, determines the biophysical properties of VRAC. Moreover, the subunit composition is of physiological relevance as it largely influences the activation mechanism and especially the substrate selectivity. However, the endogenous tissue-specific subunit composition of VRAC is unknown. We have now developed and applied a quantitative immunoblot study of the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using recombinant protein for signal calibration. We found tissue-specific expression patterns of the subunits, and generally relative low expression of the essential LRRC8A subunit. Immunoprecipitation of LRRC8A also co-precipitates an excess of the other subunits, suggesting that non-LRRC8A subunits present the majority in hetero-hexamers. With this, we can estimate that in the tested cell lines, the number of VRAC channels per cell is in the order of 10,000, which is in agreement with earlier calculations from the comparison of single-channel and whole-cell currents

RECOVER-E - a mobile app for patients undergoing total knee or hip replacement: study protocol.

Stauber A, SchuSSler N, Palmdorf S, et al. RECOVER-E - a mobile app for patients undergoing total knee or hip replacement: study protocol. BMC musculoskeletal disorders. 2020;21(1): 71.BACKGROUND: Total knee replacement (TKR) or total hip replacement (THR) are common and effective procedures in patients with osteoarthritis (OA) to restore physical function and reduce joint related pain. Patient education plays an important role in the treatment process aiming to develop necessary self-management skills to facilitate recovery and ensure long-term success. We have developed a mobile app (RECOVER-E) for iOS and Android smartphones which provides important information on the preoperative phase, surgery and recovery. The concomitant study will determine the efficacy of RECOVER-E on patients' surgical outcomes.; METHODS/DESIGN: This study is a non-randomized, multi-centre (4 sites), double-arm, controlled trial with 1:1 assignment. 160 patients undergoing primary TKR or THR will be recruited from January until October 2019 in 4 German hospitals. Both groups will receive standard care. Additionally, the intervention group will use the app RECOVER-E. Measurements will be taken 4-6weeks before surgery, on the day of admission to the hospital, on the first and 7th postoperative day and 3months post-surgery. Primary outcome will be self-reported physical function measured on the activities of daily living (ADL) subscale of the Knee injury and Osteoarthritis Outcome Score (KOOS) and the Hip disability and Osteoarthritis Outcome Score (HOOS) for patients with knee and hip osteoarthritis, respectively. Secondary outcomes include the subscales for pain, symptoms, function in sport and recreation and knee/hip-related quality of life of the HOOS and KOOS, preoperative anxiety, measured by the Hospital Anxiety and Depression Scale (HADS), as well as, pain at rest and pain during activity measured by a numerical rating scale (NRS). Primary endpoint is 3months post-surgery.; DISCUSSION: Mobile Health (mHealth) has become increasingly important in patient-centred health care aiming to enhance patient involvement and self-management capabilities. To our knowledge this is the first study to investigate the effect of an evidence-based mobile app on patient reported outcomes after joint replacement. This study should provide evidence supporting the use of mHealth to facilitate recovery and open up new possibilities for patient care in joint replacement.; TRIAL REGISTRATION: DRKS Data Management retrospectively registered.; DRKS-ID: DRKS00012744

Extrinsic Macrophages Protect While Tendon Progenitors Degrade: Insights from a Tissue Engineered Model of Tendon Compartmental Crosstalk

Tendons are among the most mechanically stressed tissues of the body, with a functional core of type-I collagen fibers maintained by embedded stromal fibroblasts known as tenocytes. The intrinsic load-bearing core compartment of tendon is surrounded, nourished, and repaired by the extrinsic peritendon, a synovial-like tissue compartment with access to tendon stem/progenitor cells as well as blood monocytes. In vitro tendon model systems generally lack this important feature of tissue compartmentalization, while in vivo models are cumbersome when isolating multicellular mechanisms. To bridge this gap, an improved in vitro model of explanted tendon core stromal tissue (mouse tail tendon fascicles) surrounded by cell-laden collagen hydrogels that mimic extrinsic tissue compartments is suggested. Using this model, CD146+ tendon stem/progenitor cell and CD45+ F4/80+ bone-marrow derived macrophage activity within a tendon injury-like niche are recapitulated. It is found that extrinsic stromal progenitors recruit to the damaged core, contribute to an overall increase in catabolic ECM gene expression, and accelerate the decrease in mechanical properties. Conversely, it is found that extrinsic bone-marrow derived macrophages in these conditions adopt a proresolution phenotype that mitigates rapid tissue breakdown by outwardly migrated tenocytes and F4/80+ "tenophages" from the intrinsic tissue core. Keywords: crosstalk; ex vivo tissue; macrophages; progenitors; tendonsTendons are among the most mechanically stressed tissues of the body, with a functional core of type-I collagen fibers maintained by embedded stromal fibroblasts known as tenocytes. The intrinsic load-bearing core compartment of tendon is surrounded, nourished, and repaired by the extrinsic peritendon, a synovial-like tissue compartment with access to tendon stem/progenitor cells as well as blood monocytes. In vitro tendon model systems generally lack this important feature of tissue compartmentalization, while in vivo models are cumbersome when isolating multicellular mechanisms. To bridge this gap, an improved in vitro model of explanted tendon core stromal tissue (mouse tail tendon fascicles) surrounded by cell-laden collagen hydrogels that mimic extrinsic tissue compartments is suggested. Using this model, CD146$^{+}$ tendon stem/progenitor cell and CD45$^{+}$ F4/80$^{+}$ bone-marrow derived macrophage activity within a tendon injury-like niche are recapitulated. It is found that extrinsic stromal progenitors recruit to the damaged core, contribute to an overall increase in catabolic ECM gene expression, and accelerate the decrease in mechanical properties. Conversely, it is found that extrinsic bone-marrow derived macrophages in these conditions adopt a proresolution phenotype that mitigates rapid tissue breakdown by outwardly migrated tenocytes and F4/80$^{+}$ "tenophages" from the intrinsic tissue core