Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.

RAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.

Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening.

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient's immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and ß-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

Utrophin modulator drugs as potential therapies for Duchenne and Becker muscular dystrophies.

Utrophin is an autosomal paralogue of dystrophin, a protein whose deficit causes Duchenne and Becker muscular dystrophies (DMD/BMD). Utrophin is naturally overexpressed at the sarcolemma of mature dystrophin-deficient fibres in DMD and BMD patients as well as in the mdx Duchenne mouse model. Dystrophin and utrophin can co-localise in human foetal muscle, in the dystrophin-competent fibres from DMD/BMD carriers, and revertant fibre clusters in biopsies from DMD patients. These findings suggest that utrophin overexpression could act as a surrogate, compensating for the lack of dystrophin, and, as such, it could be used in combination with dystrophin restoration therapies. Different strategies to overexpress utrophin are currently under investigation. In recent years, many compounds have been reported to modulate utrophin expression efficiently in preclinical studies and ameliorate the dystrophic phenotype in animal models of the disease. In this manuscript, we discuss the current knowledge on utrophin protein and the different mechanisms that modulate its expression in skeletal muscle. We also include a comprehensive review of compounds proposed as utrophin regulators and, as such, potential therapeutic candidates for these muscular dystrophies.

The Experience of COPD Patients in Lockdown Due to the COVID-19 Pandemic.

Purpose: On March 16, 2020, the Spanish government declared a state of alarm due to the rapid spread of coronavirus disease 2019 (COVID-19). Patients with chronic obstructive pulmonary disease (COPD) were restricted to remain confined at home, and medical visits were cancelled for 3 months. The impact of this lockdown on the manifestations of COPD and the quality-of-life of these patients has not been explored. Patients and Methods: One hundred patients with COPD were interviewed by telephone from May 2-18, 2020. The interviews included questions about the lockdown, missed medical appointments, fears of the disease, possible COVID-19 infection, and exacerbations of COPD suffered during this period and their management. In addition, the COPD Assessment Test, the Hospital Anxiety and Depression, and the 5-Dimension Euro Quality-of-Life questionnaires were administered. Results: Sixty-four (64%) patients claimed to have strictly complied with the lockdown, and only 42 (42%) stated they had left home at least once during lockdown. Only one patient (1%) was hospitalized due to COVID-19, and 13 (13%) patients presented an exacerbation of COPD self-managed at home with no admissions due to exacerbation of COPD during this period. A medical consultation or complementary test was cancelled in 90% of the patients, but 61% had a medical telephone visit with a high degree of satisfaction (mean 9.3/10). Most patients declared that their feeling regarding lung disease and general health was similar or even better during lockdown (82% and 81%, respectively). Conclusion: Our results indicate that in general lockdown had a low impact on COPD patients. Only one patient was affected by COVID-19, but moderate exacerbations of COPD were not infrequent. Although many medical visits and test were cancelled, patients were very satisfied with the medical telephone visits.

An Overview of Alternative Splicing Defects Implicated in Myotonic Dystrophy Type I.

Myotonic dystrophy type I (DM1) is the most common form of adult muscular dystrophy, caused by expansion of a CTG triplet repeat in the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase (DMPK) gene. The pathological CTG repeats result in protein trapping by expanded transcripts, a decreased DMPK translation and the disruption of the chromatin structure, affecting neighboring genes expression. The muscleblind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) are two families of tissue-specific regulators of developmentally programmed alternative splicing that act as antagonist regulators of several pre-mRNA targets, including troponin 2 (TNNT2), insulin receptor (INSR), chloride channel 1 (CLCN1) and MBNL2. Sequestration of MBNL proteins and up-regulation of CELF1 are key to DM1 pathology, inducing a spliceopathy that leads to a developmental remodelling of the transcriptome due to an adult-to-foetal splicing switch, which results in the loss of cell function and viability. Moreover, recent studies indicate that additional pathogenic mechanisms may also contribute to disease pathology, including a misregulation of cellular mRNA translation, localization and stability. This review focuses on the cause and effects of MBNL and CELF1 deregulation in DM1, describing the molecular mechanisms underlying alternative splicing misregulation for a deeper understanding of DM1 complexity. To contribute to this analysis, we have prepared a comprehensive list of transcript alterations involved in DM1 pathogenesis, as well as other deregulated mRNA processing pathways implications.

How whole-body vibration can help our COPD patients. Physiological changes at different vibration frequencies.

OBJECTIVE: Evaluate cardiac, metabolic, and ventilatory changes during a training session with whole-body vibration training (WBVT) with 3 different frequencies in patients with chronic obstructive pulmonary disease (COPD). METHODS: This was a prospective, interventional trial in outpatients with severe COPD. Participants performed 3 vertical WBVT sessions once a week using frequencies of 35, 25 Hz and no vibration in squatting position (isometric). Cardiac, metabolic, and ventilator parameters were monitored during the sessions using an ergospirometer. Changes in oxygen pulse response (VO2/HR) at the different frequencies were the primary outcome of the study. RESULTS: Thirty-two male patients with a mean forced expiratory volume in 1 second of 39.7% completed the study. Compared to the reference of 35 Hz, VO2/HR at no vibration was 10.7% lower (P=0.005); however, no statistically significant differences were observed on comparing the frequencies of 35 and 25 Hz. The median oxygen uptake (VO2) at 25 Hz and no vibration was 9.43% and 13.9% lower, respectively, compared to that obtained at 35 Hz (both comparisons P<0.0001). The median expiratory volume without vibration was 9.43% lower than the VO2 at the end of the assessment at 35 Hz vibration (P=0.002). CONCLUSION: Vertical WBVT training sessions show greater cardiac, metabolic, and respiratory responses compared with the squat position. On comparing the 2 frequencies used, we observed that the frequency of 35 Hz provides higher cardiorespiratory adaptation.

Ischiocrural Strength May Be a Better Prognostic Marker Than Quadriceps Strength in COPD.

It is well known that chronic obstructive pulmonary disease (COPD) patients present with muscle dysfunction that may not correlate with the degree of severity of airflow obstruction. Historically, the strength deficit of the knee extensor musculature (quadriceps) has been described as an independent factor of mortality in COPD. We present the results of a retrospective study with longitudinal follow-up of 60 patients with severe COPD followed for 7 years. During follow-up 22 patients died, mainly of respiratory cause. We have observed that the strength of knee flexor muscles (ischiocrural) and dominant handgrip were independent predictors of mortality in severe COPD patients. However, knee extensor strength (quadriceps) was not an independent predictor of mortality. This is the first study which highlights the importance of knee flexor musculature as a prognostic factor in COPD.

Safety of Rehabilitation Program for COPD Patients / Seguridad de un programa de rehabilitación para pacientes con EPOC

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Effects of whole body vibration training in patients with severe chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: To determine if whole body vibration training (WBVT) improves muscular force and modifies functional capacity parameters in patients with severe chronic obstructive pulmonary disease (COPD). METHODS: We performed a randomized controlled trial in the outpatient Physical Medicine and Rehabilitation Department of a general hospital. Sixty stable male patients with COPD and mean forced expiratory volume in 1 s (FEV1) 34.3% (predicted) were enrolled with 51 patients completing the study. Participants were randomized into two groups: Whole Body Vibration Training Group (WBVTG), (n = 26) undergoing three sessions per week for a total of 6 weeks and a Control Group (CG) (n = 25) without intervention. The main outcome measures were isokinetic knee flexor and extensor testing in a concentric-concentric regime and exercise capacity measured by the 6-min walking test (6MWT). Secondary outcomes were pulmonary muscular assessment with maximum inspiratory pressure (MIP) and maximum expiratory pressure (MEP). RESULTS: No significant differences were observed between groups in maximum moment of isokinetic knee flexor/extensor testing force in a concentric-concentric regime. In contrast, WBVTG patients showed a significant increase in the 6MWT (81.2 ± 9.2) meters; mean ± SD; P < 0.001). There was also a significant decrease in maximum oxygen desaturation in the 6MWT after 6 weeks of training (3.1 ± 1.1; P = 0.01). There were significant differences between groups in MIP and MEP at the end of the study in favour of the WBVTG. CONCLUSIONS: WBVT provided significant improvements in functional capacity in severe COPD patients without changes in muscular force.