A Feasibility Study of Using Headspace for Mindfulness Among Individuals Undergoing Surgical Repair of the Rotator Cuff

Mindfulness-based interventions (MBIs) have been found to help reduce psychological distress and pain in chronic musculoskeletal conditions. However, very limited evidence exists determining the impact of mindfulness on psychological distress and pain in acute musculoskeletal conditions including rotator cuff tears. Among individuals undergoing surgical repair of a rotator cuff tear, it is not clear how mindfulness may be combined with usual care, given the requirement of intense training as part of frequently used MBI protocols. The purpose of the present study was to determine if it was feasible to combine Headspace, a mobile application for mindfulness training that can be used anytime and anywhere, with the usual treatment for a single-tendon rotator cuff repair. One individual was recruited to use Headspace for three weeks, from two weeks before to one week after their rotator cuff surgery. Feasibility of using Headspace was measured in terms of satisfaction in using Headspace and changes in mindfulness across three time points (2 weeks before surgery, 1 week before surgery, and 1 week after surgery). Regarding satisfaction with using Headspace, four main themes emerged including the improved ability to focus and concentrate, manage pain, cope with life stressors and the ability to use the application anytime and anywhere. Regarding mindfulness, scores increased on one facet and decreased in the four other facets of FFMQ-SF. In addition, the participant reported becoming more mindful but still needed more practice with mindfulness. Based on our findings, we concluded that Headspace is an appropriate intervention to include in the treatment of rotator cuff repairs and can lead to the improved ability to concentrate, focus, manage pain, and cope with life stressors. However, given the short duration of the study, it is not clear how Headspace impacted mindfulness. Future studies should be conducted over a longer duration of time to examine the impact of Headspace on a person’s mindfulness from pre-surgery to the end of rehabilitation

Relatório de estágio em farmácia comunitária

Relatório de estágio realizado no âmbito do Mestrado Integrado em Ciências Farmacêuticas, apresentado à Faculdade de Farmácia da Universidade de Coimbr

Studies of OC-STAMP in Osteoclast Fusion: A New Knockout Mouse Model, Rescue of Cell Fusion, and Transmembrane Topology.

The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized cell fusion mechanisms

Diagram of OC-STAMP topology.

<p>Combining experimental results and topology predictions, the diagram shows current understanding of OC-STAMP topology with respect to the plasma membrane (PM). N- and C-termini are indicated, along with the position of the N-terminal FLAG and C-terminal GFP tags, and glycosylated residue N162. TM = transmembrane helix, IL = intracellular loop, EL = extracellular loop. A consensus mammalian core oligosaccharide is shown attached to N162: blue = GlcNac, green = mannose, yellow = galactose, red = Neu5Gc.</p

Viability, secretion, and gene expression.

<p><b>A.</b> Viability. BMMC from wild type (WT) or OCSt-KO (KO) were virally transduced, cultured with or without RANKL for 6 days as indicated, and MTT assays for cell viability were performed. Transduction with either GFP or OC-STAMP-GFP (OCSt:GFP) lentivirus had no effect on viability. Mean + s.d., n = 3. <b>B.</b> TRAP secretion. Supernatants from cells cultured as in <b>A</b> were analyzed for secreted TRAP enzyme. No significant difference in TRAP secretion was seen between wild type (WT) and OCSt-KO (KO) cells, whether transduced with GFP or with OC-STAMP fused to GFP (OCSt:GFP). Mean + s.d., n = 3. <b>C-H.</b> mRNA expression of osteoclast markers. Primary BMMC cells were virally transduced and were cultured for 0, 3, and 6 days in the presence of RANKL, and Q-PCR analyses for osteoclast mRNAs were performed, as indicated. Note that no OC-STAMP mRNA was detected in OCSt-KO (KO) cells at any time point unless the viral vector encoded OC-STAMP (OCSt:GFP), whereas no other osteoclast markers were significantly affected; n = 3.</p

Glycosylation of OC-STAMP.

<p><b>A</b>. HEK293 cells were transfected with V5-tagged wild type (WT) OC-STAMP or glycosylation-deficient (N162D) OC-STAMP. Wild type-transfected cell extracts were either untreated or digested with N-glycanase (N-Glyc’). Extracts were blotted and probed with anti-V5 antibody. Arrowheads indicate 2 bands, an upper, glycosylated form approximately 3kDa higher than the lower band at 50 kDa. Both N-glycanase-treated and the N >D mutation show only the low molecular weight form. Some of the overexpressed WT protein escapes glycosylation, giving 2 bands. <b>B</b>. BMMC from OCSt-KO mice were transduced with lentiviral vectors encoding wild type (WT) or (N162D) OC-STAMP fused to GFP. Cells were cultured for 6 days in RANKL, and extracts were blotted and probed with anti-GFP (upper panel) or anti-α-tubulin (lower panel). The glycosylated WT OC-STAMP appears to have much greater stability. <b>C</b>-<b>F.</b> TRAP enzyme cytochemistry. Wild type (WT) or OCSt-KO BMMCs were differentiated with RANKL for 6 days in 96-well plates. They were transduced with either GFP (<b>C, D</b>), OC-STAMP-GFP fusion protein (OCSt:GFP; <b>E</b>), or OC-STAMP-GFP fusion carrying the N162 mutation D (OC-StN>D; <b>F</b>). Fusion was rescued in the knockout cells by either the glycosylated or the non-glycosylated form of OC-STAMP. Bar in D = 200 μm.</p

TRAP-positive osteoclast measurements on bone sections.

<p>Images were obtained with a 5X objective of distal femora of WT and OCSt-KO mice at 6 weeks post-partum and TRAP-positive total area, number of TRAP-positive sites, and mean area of those sites were measured. <b>A</b>, A typical whole image of distal femur metaphysis, stained histochemically for TRAP (red) without counterstain. The region outlined in <b>A</b> is enlarged in <b>B. C</b>. shows the same area of the section after color matching was performed to select TRAP-positive sites in the section (now black). Finally, the images were made binary (<b>D</b>), leaving only TRAP-positive (black) and TRAP-negative (white) areas for analysis. Bar in A = 100 μM, bar in B = 50 μm in B, C, and D. E. The mean total TRAP-positive area per micrograph differed significantly between WT and OCSt-KO (left; *<i>P</i>< 0.05). The total count of TRAP-positive sites was not different between genotypes (middle); however, the mean area of each TRAP-positive sites was significantly lower in OCSt-KO (*<i>P</i> < 0.02). Results from 3 or 4 sections per animal, and 3 animals per genotype were pooled for analysis.</p

Peripheral localization of OC-STAMP and topology of N- and C-termini.

<p><b>A-C.</b> HEK293T cells were transfected with either GFP or OC-STAMP-GFP vectors (OCSt:GFP). <b>A.</b> Western blot with anti-GFP antibody shows expected molecular weights (marker positions indicated at left). <b>B</b> and <b>C,</b> GFP fluorescence. In <b>B</b>, endogenous GFP fluorescence is seen throughout the cell, as expected for a cytoplasmic protein. In <b>C</b>, OC-STAMP-GFP signal is mainly confined to the cell periphery, as expected for a plasma membrane-localized transmembrane protein. Scale bar = 50μm. <b>D-F.</b> HEK 293 cells were transfected with tagged constructs indicated at top, permeabilized as indicated at left (none, plasma membrane selectively with digitonin, or all membranes with Triton X-100), and probed with anti-tag antibodies. With membranes intact, anti-FLAG antibody could not access the N-terminus, nor could anti-GFP access the C-terminus, whereas both antibodies could access their antigens when the plasma membrane or all membranes were permeabilized. For the GFP tag, separate green and red channels are also shown in small panels. GFP endogenous signal (green) vs. that for anti-GFP (red) varied in intensity in some cells, but the same cells were positive for both. Scale bars = 10 μm.</p

Topology of mouse OC-STAMP.

<p>Transmembrane orientation of mouse OC-STAMP (GenBank accession # NP_083297.1) predicted by consensus of 5 algorithms utilized by the TOPCONS server (<a href="http://topcons.cbr.su.se/" target="_blank">http://topcons.cbr.su.se/</a>: SCAMPI-seq, SCAMPI-msa, PRODIV, PRO, and OCTOPUS). Also, we have shown in this report that the N- and C-termini are intracellular and that N162 (bold, underlined) is glycosylated, supporting those predictions.</p><p>Topology of mouse OC-STAMP.</p

Low and high power histology of adjacent sections (A, B and C, D) of OCst-KO (A, B, E, F) and WT (C D, G, H) 6-week-old mouse distal femur.

<p>Glycol methacrylate, 3 μm sections were stained histochemically for TRAP (<b>B, D, E, G</b>) and some were counterstained with toluidine blue (<b>A, C, F. H</b>). At low power, overall appearance was highly similar, with growth plates normal (purple, wavy band in <b>A</b> and <b>C,</b> asterisks in <b>B</b> and <b>D</b>), similar trabecular size and thickness in both the primary and secondary ossification centers, and open marrow space (<i>m</i>) in the diaphysis. TRAP stains (<b>B, D</b>) show overall similar distribution of osteoclasts, including along the chondroosseous junction, among the trabeculae of the primary ossification center, and notably, along the periosteal surface (arrows in B and D), where the bone is being removed to maintain the flared shape of the metaphysis. At higher power (<b>E, F, G, H</b>), only mononuclear osteoclasts are present in OCSt-KO mice. A row of such cells is indicated in <b>A</b> by arrows, attached to trabecular bone (<i>B</i>) in the primary spongiosa. In contrast, wild type mice had typical, multinucleated osteoclasts in this area, and individual nuclei (visible by lack of overlying TRAP label in <b>C</b>), are indicated by asterisks. Blinded observers consistently identified OCSt-KO and WT sections based solely on the presence or absence of multinucleated osteoclasts. Bar in A = 500 μm in A, B, C, D. Bar in E = 10 μm in E and G; 7.3 μm in F and H. <i>B</i> = bone, <i>C</i> = cartilage core.</p