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Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin (MSTN)-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein. Myogenesis assay and immunofluorescence staining were performed to evaluate muscle differentiation, while CCK8 assay, cell cycle assay, and 5-ethynyl-2'-deoxyuridine staining were used to measure muscle proliferation. Reverse transcription-polymerase chain reaction and Western blotting were also performed to examine cellular signaling. Myogenic factors (including myosin heavy chain, MSTN, myocardin, and serum response factor) increased significantly after day 5 during myogenesis. MSTN was efficiently silenced after transfecting the dCas9 RNP complex, which significantly promoted more myotube formation and a higher fusion index for L6 cells. In cellular signaling, MSTN repression enhanced the expression of MyoG and MyoD, phosphorylation of Smad2, and the activity of Wnt1/GSK-3β/β-catenin pathway. Moreover, MSTN repression accelerated L6 cell growth with a higher cell proliferation index as well as a higher expression of cyclin D1 and cyclin E. Nonviral delivery of the dCas9 RNP complex significantly promoted myoblast differentiation and proliferation, providing a promising approach to improve muscle regeneration for SUI and PFD. Further characterization and validation of this approach in vivo are needed.

BackgroundThe mechanisms of the microenergy acoustic pulse (MAP) therapy on restoring structure and function of pelvic floor muscles (PFM) after simulated birth injury are not well understood.MethodsA total 24 female Sprague-Dawley rats were randomly grouped into sham control (sham), vaginal balloon dilation and ovariectomy (VBDO), VBDO + β-aminopropionitrile (BAPN, an irreversible LOX inhibitor), and VBDO + BAPN and treated with MAP (n=6 in each group). The MAP therapy was administered 2 times per week for 4 weeks with 1-week washout, the functional and histological studies were conducted in all 24 rats. The viscoelastic behavior of the PFM, including iliococcygeus (IC) and pubococcygeus (PC), was examined with a biomechanical assay. The structure of the PFM was assessed by immunofluorescence and Masson's trichrome staining.ResultsThe leak point pressure (LPP) assay demonstrated that the MAP therapy group had higher LPPs compared to that of VBDO and BAPN groups. In the sham group, the muscular stiffness (K) of IC muscle was significantly higher than that of PC muscle while the pelvic floor muscle rebound activity (MRA) of PC muscle was stronger than that of IC muscle (291.26±45.33 and 241.18±14.23 N/cm2, respectively). Both VBDO and BAPN decreased the MRA and increased the K in both IC and PC. Histologic examination revealed increased fibrous tissue (collagen) and degeneration of muscle fibers in both VBDO and BAPN groups. MAP therapy significantly reduced the collagen content and improved the architecture of muscle fibers.ConclusionsMAP appears to restore the structure and function of PFM by regenerating muscular fibers and improving biomechanical properties in an animal model of simulated birth injury.

Background: Erectile dysfunction and atherosclerosis are common cardiovascular complications in diseases. Clinical associations between erectile dysfunction and atherosclerosis have been noticed, but the specific mechanisms are not illustrated adequately. Objectives: The aim of the study was to further mine associated pathological mechanisms and genetic alterations of atherosclerosis in diabetes mellitus‐related erectile dysfunction. Materials and methods: Significant atherosclerosis‐related genes were identified from transcriptome data of diabetes mellitus‐related erectile dysfunction and atherosclerosis‐related gene sets from DisGeNET and GeneCard databases. Functional enrichment and immune infiltration analyses were performed to clarify the biological roles and pathways as well as immune responses of significant atherosclerosis‐related gene sets. A protein–protein interaction network was constructed, and gene clusters were performed. Then, data of diabetic plaques and high‐glucose cavernosum endothelial cells were analyzed for validation. And hub atherosclerosis‐related gene sets were identified. Finally, expressed pattern of hub atherosclerosis‐related gene sets were explored by single‐cell profiling and immune analysis. Results: In total, 202 significant atherosclerosis‐related gene sets including 100 upregulated and 102 downregulated genes were identified. These genes were related to endothelial cell migration, inflammatory response, regulation of oxidative stress, and immune response. In immune infiltration, immature dendritic cells and monocytes showed differential expression between the diabetes mellitus‐related erectile dysfunction and control groups, A protein–protein interaction network containing 135 nodes was constructed. A hub atherosclerosis‐related gene set signature consisting of HBEGF, LOX, NQO1, and VLDLR was obtained by multi‐omics validation. In addition, Functional enrichment analysis revealed that hub atherosclerosis‐related gene sets were involved in oxidoreductase activity and extracellular matrix organization. Discussion and conclusion: We explored atherosclerosis‐related genetic changes and signaling pathways in diabetes mellitus‐related erectile dysfunction. HBEGF, LOX, NQO1, and VLDLR were identified as hub atherosclerosis‐related gene sets. These may serve as potential biomarkers for the clinical management of atherosclerosis and preventing further cardiovascular risks in diabetes mellitus‐related erectile dysfunction. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has had a profound psychological impact on our frontline healthcare workers. Throughout the entire second COVID-19 wave at one major tertiary hospital in Melbourne Australia, longitudinal qualitative data between perioperative staff members, and analyses of intrapersonal changes were reported. Inductive analysis of three open-ended questions generated four major themes: Organisational Response to the Pandemic, Psychological Impact, Changes in Feelings of Support Over Time and Suggestions for Changes. Understanding the challenges, perception and suggestions from this longitudinal study allows us to provide a range of support services and interventions to minimise the long-term negative psychological impact and be better prepared should another similar situation arises again. [ABSTRACT FROM AUTHOR]