A modified wire hanging apparatus for small animal muscle function testing

Wire hang tests are simple and cheap methods to assess muscle performance in small rodents, but do not always yield consistent results. We describe a simple wire hang apparatus that comprises a continuous rolling loop. Wire hang times measured using the rolling wire provide consistent and reliable data that more accurately reflect the output of a continuous physical effort. As such data obtained in mice using a rolling wire are more representative of the physical changes in the mouse muscle and less susceptible to individual mouse behaviour and differences in animal handling

γ-Secretase Dependent Nuclear Targeting of Dystroglycan

Dystroglycan is frequently lost in adenocarcinoma. α‐dystroglycan is known to become hypoglycosylated due to transcriptional silencing of LARGE, whereas β‐dystroglycan is proteolytically cleaved and degraded. The mechanism and proteases involved in the cleavage events affecting β‐dystroglycan are poorly understood. Using LNCaP prostate cancer cells as a model system, we have investigated proteases and tyrosine phosphorylation affecting β‐dystroglycan proteolysis and nuclear targeting. Cell density or phorbol ester treatment increases dystroglycan proteolysis, whereas furin or γ‐secretase inhibitors decreased dystroglycan proteolysis. Using resveratrol treatment of LNCaP cells cultured at low cell density in order to up‐regulate notch and activate proteolysis, we identified significant increases in the levels of a 26 kDa β‐dystroglycan fragment. These data, therefore, support a cell density‐dependent γ‐secretase and furin mediated proteolysis of β‐dystroglycan, which could be notch stimulated, leading to nuclear targeting and subsequent degradatio

Dystrophin and utrophin: the missing links!

AbstractThere is considerable sequence homology between dystrophin and utrophin, both at the protein and DNA level, and consequently it was assumed that their domain structures and functions would be similar. As more of the detailed biochemical and cell biological properties of these two proteins become known, so it becomes clear that there are subtle if not significant differences between them. We review recent findings and present new hypotheses into the structural and functional properties of the actin-binding domain, central coiled-coil region and regulatory/membrane protein-binding regions of dystrophin and utrophin

Dasatinib as a treatment for Duchenne muscular dystrophy

Identification of a systemically acting and universal small molecule therapy for Duchenne muscular dystrophy would be an enormous advance for this condition. Based on evidence gained from studies on mouse genetic models, we have identified tyrosine phosphorylation and degradation of β-dystroglycan as a key event in the aetiology of Duchenne muscular dystrophy. Thus, preventing tyrosine phosphorylation and degradation of β-dystroglycan presents itself as a potential therapeutic strategy. Using the dystrophic sapje zebrafish, we have investigated the use of tyrosine kinase and other inhibitors to treat the dystrophic symptoms in this model of Duchenne muscular dystrophy. Dasatinib, a potent and specific Src tyrosine kinase inhibitor, was found to decrease the levels of β-dystroglycan phosphorylation on tyrosine and to increase the relative levels of non-phosphorylated β-dystroglycan in sapje zebrafish. Furthermore, dasatinib treatment resulted in the improved physical appearance of the sapje zebrafish musculature and increased swimming ability as measured by both duration and distance of swimming of dasatinib-treated fish compared with control animals. These data suggest great promise for pharmacological agents that prevent the phosphorylation of β-dystroglycan on tyrosine and subsequent steps in the degradation pathway as therapeutic targets for the treatment of Duchenne muscular dystrophy

ChatGPT sits the DFPH exam: large language model performance and potential to support public health learning

Background Artificial intelligence-based large language models, like ChatGPT, have been rapidly assessed for both risks and potential in health-related assessment and learning. However, their applications in public health professional exams have not yet been studied. We evaluated the performance of ChatGPT in part of the Faculty of Public Health’s Diplomat exam (DFPH). Methods ChatGPT was provided with a bank of 119 publicly available DFPH question parts from past papers. Its performance was assessed by two active DFPH examiners. The degree of insight and level of understanding apparently displayed by ChatGPT was also assessed. Results ChatGPT passed 3 of 4 papers, surpassing the current pass rate. It performed best on questions relating to research methods. Its answers had a high floor. Examiners identified ChatGPT answers with 73.6% accuracy and human answers with 28.6% accuracy. ChatGPT provided a mean of 3.6 unique insights per question and appeared to demonstrate a required level of learning on 71.4% of occasions. Conclusions Large language models have rapidly increasing potential as a learning tool in public health education. However, their factual fallibility and the difficulty of distinguishing their responses from that of humans pose potential threats to teaching and learning

A role for dystroglycan in the pathophysiology of acute leukemic cells.

AIMS: Previous reports have demonstrated that alterations or reduced expression of Dystroglycan (Dg) complex (αDg and βDg subunits) are related to progression and severity of neoplastic solid tissues. Therefore we determined the expression pattern and subcellular distribution of Dg complex in Acute Myeloid Leukemia (AML) primary blasts (M1, M2, and M3 phenotypes), as well as HL-60 and Kasumi-1 leukemia cell lines. Additionally, we evaluated the relative expression of the main enzymes controlling α-Dg glycosylation to ascertain the post-translational modifications in the leukemia cell phenotype. MAIN METHODS: Primary leukemia blasts and leukemia cell lines were processed by confocal analysis to determine the subcellular distribution of α-Dg, β-Dg, and phosphorylated β-Dg (Y892), to evaluate the expression pattern of the different Dg species we performed Western Blot (WB) assays, while the messenger RNA (mRNA) expression of enzymes involved in α-Dg glycosylation, such as POMGnT1, POMT1, POMT2, LARGE, FKTN, and FKRP, were evaluated by qualitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Finally, in an attempt to ameliorate the leukemia cell phenotype, we transfected leukemia cells with a plasmid expressing the Dg complex. KEY FINDINGS: The Dg complex was altered in leukemia cells, including decreased mRNA, protein, and α-Dg glycosylated levels, mislocalization of β-Dg, and a diminution of mRNA expression of LARGE in patients leukemia blasts and in cell lines. Interestingly, the exogenous expression of Dg complex promoted filopodial formation, differentiation, and diminished proliferation, attenuating some HL-60 and Kasumi cells characteristics. SIGNIFICANCE: Dg complex integrity and balance are required for a proper hematopoietic cell function, in that its disruption might contribute to leukemia pathophysiology

How the central domain of dystrophin acts to bridge F-actin to sarcolemmal lipids

Dystrophin is a large intracellular protein that prevents sarcolemmal ruptures by providing a mechanical link between the intracellular actin cytoskeleton and the transmembrane dystroglycan complex. Dystrophin deficiency leads to the severe muscle wasting disease Duchenne Muscular Dystrophy and the milder allelic variant, Becker Muscular Dystrophy (DMD and BMD). Previous work has shown that concomitant interaction of the actin binding domain 2 (ABD2) comprising spectrin like repeats 11 to 15 (R11-15) of the central domain of dystrophin, with both actin and membrane lipids, can greatly increase membrane stiffness. Based on a combination of SAXS and SANS measurements, mass spectrometry analysis of cross-linked complexes and interactive low-resolution simulations, we explored in vitro the molecular properties of dystrophin that allow the formation of ABD2-F-actin and ABD2-membrane model complexes. In dystrophin we identified two subdomains interacting with F-actin, one located in R11 and a neighbouring region in R12 and another one in R15, while a single lipid binding domain was identified at the C-terminal end of R12. Relative orientations of the dystrophin central domain with F-actin and a membrane model were obtained from docking simulation under experimental constraints. SAXS-based models were then built for an extended central subdomain from R4 to R19, including ABD2. Overall results are compatible with a potential F-actin/dystrophin/membrane lipids ternary complex. Our description of this selected part of the dystrophin associated complex bridging muscle cell membrane and cytoskeleton opens the way to a better understanding of how cell muscle scaffolding is maintained through this essential protein

Control of nuclear beta-dystroglycan content is crucial for the maintenance of nuclear envelope integrity and function

β-Dystroglycan (β-DG) is a plasma membrane protein that has ability to target to the nuclear envelope (NE) to maintain nuclear architecture. Nevertheless, mechanisms controlling β-DG nuclear localization and the physiological consequences of a failure of trafficking are largely unknown. We show that β-DG has a nuclear export pathway in myoblasts that depends on the recognition of a nuclear export signal located in its transmembrane domain, by CRM1. Remarkably, NES mutations forced β-DG nuclear accumulation resulting in mislocalization and decreased levels of emerin and lamin B1 and disruption of various nuclear processes in which emerin (centrosome-nucleus linkage and β-catenin transcriptional activity) and lamin B1 (cell cycle progression and nucleoli structure) are critically involved. In addition to nuclear export, the lifespan of nuclear β-DG is restricted by its nuclear proteasomal degradation. Collectively our data show that control of nuclear β-DG content by the combination of CRM1 nuclear export and nuclear proteasome pathways is physiologically relevant to preserve proper NE structure and activity

The molecular basis and biologic significance of the β-dystroglycan-emerin interaction

β-dystroglycan (β-DG) assembles with lamins A/C and B1 and emerin at the nuclear envelope (NE) to maintain proper nuclear architecture and function. To provide insight into the nuclear function of β-DG, we characterized the interaction between β-DG and emerin at the molecular level. Emerin is a major NE protein that regulates multiple nuclear processes and whose deficiency results in Emery–Dreifuss muscular dystrophy (EDMD). Using truncated variants of β-DG and emerin, via a series of in vitro and in vivo binding experiments and a tailored computational analysis, we determined that the β-DG–emerin interaction is mediated at least in part by their respective transmembrane domains (TM). Using surface plasmon resonance assays we showed that emerin binds to β-DG with high affinity (KD in the nanomolar range). Remarkably, the analysis of cells in which DG was knocked out demonstrated that loss of β-DG resulted in a decreased emerin stability and impairment of emerin-mediated processes. β-DG and emerin are reciprocally required for their optimal targeting within the NE, as shown by immunofluorescence, western blotting and immunoprecipitation assays using emerin variants with mutations in the TM domain and B-lymphocytes of a patient with EDMD. In summary, we demonstrated that β-DG plays a role as an emerin interacting partner modulating its stability and function

Macrophage delivered HSV1716 is active against triple negative breast cancer

Oncolytic viruses (OV) promote anti-tumour responses through the initiation of immunogenic cancer cell death which activates the host’s systemic anti-tumour immunity. We have previously shown that intravenously administered HSV1716 is an effective treatment for mammary cancer. However, intravenous administration of a virus has the potential to result in neutralization and sequestration of the virus which may reduce efficacy. Here, we show that the oncolytic virus HSV1716 can be administered within a cellular carrier (macrophages). PyMT and 4T1 murine mammary cancer cell lines were implanted into immuno-competent murine models (orthotopic primary, early metastatic and brain metastasis models). HSV1716 or macrophages armed with HSV1716 (M-HSV1716) were administered intravenously, and tumour size was quantified using caliper measurement or bioluminescence imaging. Administration of M-HSV1716 led to tumour shrinkage and increased the survival of animals. Furthermore, these results were achieved with a 100-fold lower viral load, which has the potential for decreased toxicity. Our results demonstrate that M-HSV1716 is associated with activity against murine mammary cancers and provides an alternative platform for the systemic delivery of OV