The E3 ubiquitin ligase Nedd4L preserves skeletal muscle stem cell quiescence by inhibiting their activation.

Adult stem cells play a critical role in tissue repair and maintenance. In tissues with slow turnover, including skeletal muscle, these cells are maintained in a mitotically quiescent state yet remain poised to re-enter the cell cycle to replenish themselves and regenerate the tissue. Using a panomics approach we show that the PAX7/NEDD4L axis acts against muscle stem cell activation in homeostatic skeletal muscle. Our findings suggest that PAX7 transcriptionally activates the E3 ubiquitin ligase Nedd4L and that the conditional genetic deletion of Nedd4L impairs muscle stem cell quiescence, with an upregulation of cell cycle and myogenic differentiation genes. Loss of Nedd4L in muscle stem cells results in the expression of doublecortin (DCX), which is exclusively expressed during their in vivo activation. Together, these data establish that the ubiquitin proteasome system, mediated by Nedd4L, is a key contributor to the muscle stem cell quiescent state in adult mice.

Intracorporeal evaluation of hyaluronic acid fillers with varied rheological properties and correlations with aesthetic outcomes.

BACKGROUND: Understanding the differences in soft tissue filler rheology and how these properties can impact clinical results is a fundamental concepts for any injector. This study aimed to assess the tissue integration characteristics of hyaluronic acid (HA) fillers manufactured with different technologies (Non-Animal Stabilized HA [HA-N] or Optimal Balance Technology [HA-O]) using ultra-high-frequency ultrasound. METHODS: Twelve female participants with mild-to-moderate midface volume loss and temporal hollowing were enrolled and treated with HA-N and/or HA-O. Participants were seen at five visits (screening/baseline [treatment], and Weeks 1 [optional touch-up], 4, 6, and 8 [follow-up visits]). Ultrasound was used to evaluate the degree of product integration. RESULTS: On ultrasound, HA-N presented with distinct borders, minimal tissue integration, and a capacity to displace tissues. Conversely, HA-O tended to spread horizontally within the same tissue plane and integrated within tissues. The volumizing capacity of the HA-O fillers was dependent on particle size. CONCLUSION: HA-N is suited for deep injections in areas such as the upper lateral cheek and under the muscle of the temporal region when a lifting effect is desired; HA-O is best suited for subcutaneous injections, in areas of dynamic movement or for patients with thin skin; and can be injected subcutaneously or supraperiosteally when a volumizing effect is desired.

Subcellular fractionation of brain tumor stem cells.

Brain tumor stem cells (BTSCs) are a rare population of self-renewing stem cells that are cultured as spheres and are often slow growing compared to other mammalian cell lines. Analysis of BTSC proteome requires careful handling as well as techniques that can be applied to small quantities of cell material. Subcellular fractionation is a widely used technique to assess protein localization. Although proteins are often destined to a defined cell compartment via a signal peptide such as mitochondrial or nuclear localization signals, the recruitment of a protein from one compartment to another can occur as a result of post-translational modification and/or structural variations in response to intracellular and extracellular stimuli. These events assign different functions to a protein making the study of protein localization a useful approach for better understanding of its role in disease progression. Sequential centrifugation remains a simple and versatile fractionation method for proteomic analysis. It can also be applied for diverse downstream applications such as multi-omics using pure nuclear fractions or metabolomic studies on isolated mitochondria. In this chapter, we describe our optimized protocol for subcellular fractionation of BTSC spheres in which we use a commercially available kit with additional centrifugation steps. We provide details on BTSC maintenance and handling, fractionation protocol and evaluation of fraction purity.

In situ detection of protein-protein interaction by proximity ligation assay in patient derived brain tumor stem cells.

Improper or aberrant protein-protein interactions can lead to severe human diseases including cancer. Here, we describe an adapted proximity ligation assay (PLA) protocol for the assessment of galectin-1-HOXA5 interaction in brain tumor stem cells (BTSCs). We detail the steps for culturing and preparation of BTSCs followed by PLA and detection of protein interactions in situ using fluorescent microscopy. This PLA protocol is optimized specifically for BTSCs and includes key controls for effective result analysis. For complete details on the use and execution of this protocol, please refer to Sharanek et al. (2021).