Proteomic analysis identifies key differences in the cardiac interactomes of dystrophin and micro-dystrophin

ΔR4-R23/ΔCT micro-dystrophin (μDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, μDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of μDys, we compared by mass spectrometry the composition of purified dystrophin and μDys protein complexes in the mouse heart. We report that compared to dystrophin, μDys has altered associations with α1- and β2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavins −1 and − 4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx^{5cv} mice,a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx^{5cv} mouse heart. Expression of μDys in mdx^{5cv} mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients

Cyclic Peptides as Non-carboxyl-terminal Ligands of Syntrophin PDZ Domains

Syntrophins, a family of intracellular peripheral membrane proteins of the dystrophin-associated protein complex (DAPC), each contain a single PDZ domain. Syntrophin PDZ domains bind C-terminal peptide sequences with the consensus R/K-E-S/T-X-V-COOH, an interaction that mediates association of skeletal muscle sodium channels with the DAPC. Here, we have isolated cyclic peptide ligands for syntrophin PDZ domains from a library of combinatorial peptides displayed at the N terminus of protein III of bacteriophage M13. Affinity selection from a library of X10C peptides yielded ligands with the consensus X-(R/K)-E-T-C-L/M-A-G-X-Psi-C, where Psi represents any hydrophobic amino acid. These peptides contain residues (underlined) similar to the C-terminal consensus sequence for binding to syntrophin PDZ domains and bind to the same site on syntrophin PDZ domains as C-terminal peptides, but do not bind to other closely related PDZ domains. PDZ binding is dependent on the formation of an intramolecular disulfide bond in the peptides, since treatment with dithiothreitol, or substitution of either of the two cysteines with alanines, eliminated this activity. Furthermore, amino acid replacements revealed that most residues in the phage-selected peptides are required for binding. Our results define a new mode of binding to PDZ domains and suggest that proteins containing similar conformationally constrained sequences may be ligands for PDZ domains

Skeletal Muscle NADPH Oxidase Is Increased and Triggers Stretch-Induced Damage in the mdx Mouse

Recent studies have shown that oxidative stress contributes to the pathogenesis of muscle damage in dystrophic (mdx) mice. In this study we have investigated the role of NADPH oxidase as a source of the oxidative stress in these mice. The NADPH oxidase subunits gp91phox, p67phox and rac 1 were increased 2–3 fold in tibilais anterior muscles from mdx mice compared to wild type. Importantly, this increase occurred in 19 day old mice, before the onset of muscle necrosis and inflammation, suggesting that NADPH oxidase is an important source of oxidative stress in mdx muscle. In muscles from 9 week old mdx mice, gp91phox and p67phox were increased 3–4 fold and NADPH oxidase superoxide production was 2 times greater than wild type. In single fibers from mdx muscle NADPH oxidase subunits were all located on or near the sarcolemma, except for p67phox,which was expressed in the cytosol. Pharmacological inhibition of NADPH oxidase significantly reduced the intracellular Ca2+ rise following stretched contractions in mdx single fibers, and also attenuated the loss of muscle force. These results suggest that NADPH oxidase is a major source of reactive oxygen species in dystrophic muscle and its enhanced activity has a stimulatory effect on stretch-induced Ca2+ entry, a key mechanism for muscle damage and functional impairment

α-Syntrophin Modulates Myogenin Expression in Differentiating Myoblasts

α-Syntrophin is a scaffolding protein linking signaling proteins to the sarcolemmal dystrophin complex in mature muscle. However, α-syntrophin is also expressed in differentiating myoblasts during the early stages of muscle differentiation. In this study, we examined the relationship between the expression of α-syntrophin and myogenin, a key muscle regulatory factor.The absence of α-syntrophin leads to reduced and delayed myogenin expression. This conclusion is based on experiments using muscle cells isolated from α-syntrophin null mice, muscle regeneration studies in α-syntrophin null mice, experiments in Sol8 cells (a cell line that expresses only low levels of α-syntrophin) and siRNA studies in differentiating C2 cells. In primary cultured myocytes isolated from α-syntrophin null mice, the level of myogenin was less than 50% that from wild type myocytes (p<0.005) 40 h after differentiation induction. In regenerating muscle, the expression of myogenin in the α-syntrophin null muscle was reduced to approximately 25% that of wild type muscle (p<0.005). Conversely, myogenin expression is enhanced in primary cultures of myoblasts isolated from a transgenic mouse over-expressing α-syntrophin and in Sol8 cells transfected with a vector to over-express α-syntrophin. Moreover, we find that myogenin mRNA is reduced in the absence of α-syntrophin and increased by α-syntrophin over-expression. Immunofluorescence microscopy shows that α-syntrophin is localized to the nuclei of differentiating myoblasts. Finally, immunoprecipitation experiments demonstrate that α-syntrophin associates with Mixed-Lineage Leukemia 5, a regulator of myogenin expression.We conclude that α-syntrophin plays an important role in regulating myogenesis by modulating myogenin expression

COVID-19 and the Global Impact on Colorectal Practice and Surgery

Background: The novel severe acute respiratory syndrome coronavirus 2 virus that emerged in December 2019 causing coronavirus disease 2019 (COVID-19) has led to the sudden national reorganization of health care systems and changes in the delivery of health care globally. The purpose of our study was to use a survey to assess the global effects of COVID-19 on colorectal practice and surgery. Materials and Methods: A panel of International Society of University Colon and Rectal Surgeons (ISUCRS) selected 22 questions, which were included in the questionnaire. The questionnaire was distributed electronically to ISUCRS fellows and other surgeons included in the ISUCRS database and was advertised on social media sites. The questionnaire remained open from April 16 to 28, 2020. Results: A total of 287 surgeons completed the survey. Of the 287 respondents, 90% were colorectal specialists or general surgeons with an interest in colorectal disease. COVID-19 had affected the practice of 96% of the surgeons, and 52% were now using telemedicine. Also, 66% reported that elective colorectal cancer surgery could proceed but with perioperative precautions. Of the 287 respondents, 19.5% reported that the use of personal protective equipment was the most important perioperative precaution. However, personal protective equipment was only provided by 9.1% of hospitals. In addition, 64% of surgeons were offering minimally invasive surgery. However, 44% reported that enough information was not available regarding the safety of the loss of intra-abdominal carbon dioxide gas during the COVID-19 pandemic. Finally, 61% of the surgeons were prepared to defer elective colorectal cancer surgery, with 29% willing to defer for ≤ 8 weeks. Conclusion: The results from our survey have demonstrated that, globally, COVID-19 has affected the ability of colorectal surgeons to offer care to their patients. We have also discussed suggestions for various practical adaptation strategies for use during the recovery period. We have presented the results of a survey used to assess the global impact of coronavirus disease 2019 (COVID-19) on the delivery of colorectal surgery. Despite accessible guidance information, our results have demonstrated that COVID-19 has significantly affected the ability of colorectal surgeons to offer care to patients. We have also discussed practical adaptation strategies for use during the recovery phase

A Common Carcinogen Benzo[a]pyrene Causes Neuronal Death in Mouse via Microglial Activation

BACKGROUND: Benzo[a]pyrene (B[a]P) belongs to a class of polycyclic aromatic hydrocarbons that serve as micropollutants in the environment. B[a]P has been reported as a probable carcinogen in humans. Exposure to B[a]P can take place by ingestion of contaminated (especially grilled, roasted or smoked) food or water, or inhalation of polluted air. There are reports available that also suggests neurotoxicity as a result of B[a]P exposure, but the exact mechanism of action is unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using neuroblastoma cell line and primary cortical neuron culture, we demonstrated that B[a]P has no direct neurotoxic effect. We utilized both in vivo and in vitro systems to demonstrate that B[a]P causes microglial activation. Using microglial cell line and primary microglial culture, we showed for the first time that B[a]P administration results in elevation of reactive oxygen species within the microglia thereby causing depression of antioxidant protein levels; enhanced expression of inducible nitric oxide synthase, that results in increased production of NO from the cells. Synthesis and secretion of proinflammatory cytokines were also elevated within the microglia, possibly via the p38MAP kinase pathway. All these factors contributed to bystander death of neurons, in vitro. When administered to animals, B[a]P was found to cause microglial activation and astrogliosis in the brain with subsequent increase in proinflammatory cytokine levels. CONCLUSIONS/SIGNIFICANCE: Contrary to earlier published reports we found that B[a]P has no direct neurotoxic activity. However, it kills neurons in a bystander mechanism by activating the immune cells of the brain viz the microglia. For the first time, we have provided conclusive evidence regarding the mechanism by which the micropollutant B[a]P may actually cause damage to the central nervous system. In today's perspective, where rising pollution levels globally are a matter of grave concern, our study throws light on other health hazards that such pollutants may exert

Autoantibodies to Agrin in Myasthenia Gravis Patients

To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ

A new simplified comorbidity score as a prognostic factor in non-small-cell lung cancer patients: description and comparison with the Charlson's index

Treatment of non-small-cell lung cancer (NSCLC) might take into account comorbidities as an important variable. The aim of this study was to generate a new simplified comorbidity score (SCS) and to determine whether or not it improves the possibility of predicting prognosis of NSCLC patients. A two-step methodology was used. Step 1: An SCS was developed and its prognostic value was compared with classical prognostic determinants in the outcome of 735 previously untreated NSCLC patients. Step 2: the SCS reliability as a prognostic determinant was tested in a different population of 136 prospectively accrued NSCLC patients with a formal comparison between SCS and the classical Charlson comorbidity index (CCI). Prognosis was analysed using both univariate and multivariate (Cox model) statistics. The SCS summarised the following variables: tobacco consumption, diabetes mellitus and renal insufficiency (respective weightings 7, 5 and 4), respiratory, neoplastic and cardiovascular comorbidities and alcoholism (weighting=1 for each item). In step 1, aside from classical variables such as age, stage of the disease and performance status, SCS was a statistically significant prognostic variable in univariate analyses. In the Cox model weight loss, stage grouping, performance status and SCS were independent determinants of a poor outcome. There was a trend towards statistical significance for age (P=0.08) and leucocytes count (P=0.06). In Step 2, both SCS and well-known prognostic variables were found as significant determinants in univariate analyses. There was a trend towards a negative prognostic effect for CCI. In multivariate analysis, stage grouping, performance status, histology, leucocytes, lymphocytes, lactate dehydrogenase, CYFRA 21-1 and SCS were independent determinants of a poor prognosis. CCI was removed from the Cox model. In conclusion, the SCS, constructed as an independent prognostic factor in a large NSCLC patient population, is validated in another prospective population and appears more informative than the CCI in predicting NSCLC patient outcome