Remarkably High Repeat Content in the Genomes of Sparrows: The Importance of Genome Assembly Completeness for Transposable Element Discovery.

Transposable elements (TE) play critical roles in shaping genome evolution. Highly repetitive TE sequences are also a major source of assembly gaps making it difficult to fully understand the impact of these elements on host genomes. The increased capacity of long-read sequencing technologies to span highly repetitive regions promises to provide new insights into patterns of TE activity across diverse taxa. Here we report the generation of highly contiguous reference genomes using PacBio long-read and Omni-C technologies for three species of Passerellidae sparrow. We compared these assemblies to three chromosome-level sparrow assemblies and nine other sparrow assemblies generated using a variety of short- and long-read technologies. All long-read based assemblies were longer (range: 1.12 to 1.41 Gb) than short-read assemblies (0.91 to 1.08 Gb) and assembly length was strongly correlated with the amount of repeat content. Repeat content for Bell's sparrow (31.2% of genome) was the highest level ever reported within the order Passeriformes, which comprises over half of avian diversity. The highest levels of repeat content (79.2% to 93.7%) were found on the W chromosome relative to other regions of the genome. Finally, we show that proliferation of different TE classes varied even among species with similar levels of repeat content. These patterns support a dynamic model of TE expansion and contraction even in a clade where TEs were once thought to be fairly depauperate and static. Our work highlights how the resolution of difficult-to-assemble regions of the genome with new sequencing technologies promises to transform our understanding of avian genome evolution.

Color-Flu Fluorescent Reporter Influenza A Viruses Allow for In Vivo Studies of Innate Immune Function in Zebrafish.

Influenza virus infection can cause severe respiratory disease and is estimated to cause millions of illnesses annually. Studies on the contribution of the innate immune response to influenza A virus (IAV) to viral pathogenesis may yield new antiviral strategies. Zebrafish larvae are useful models for studying the innate immune response to pathogens, including IAV, in vivo. Here, we demonstrate how Color-flu, four fluorescent IAV strains originally developed for mice, can be used to study the host response to infection by simultaneously monitoring infected cells, neutrophils, and macrophages in vivo. Using this model, we show how the angiotensin-converting enzyme inhibitor, ramipril, and mitophagy inhibitor, MDIVI-1, improved survival, decreased viral burden, and improved the respiratory burst response to IAV infection. The Color-flu zebrafish larvae model of IAV infection is complementary to other models where the dynamics of infection and the response of innate immune cells can be visualized in a transparent host in vivo.

Multi-spectral Fluorescent Reporter Influenza A Viruses Allow for in vivo Studies of Innate Immune Function in Zebrafish.

Influenza virus infection can cause severe respiratory disease and is estimated to cause millions of illnesses annually. Studies of the contribution of the innate immune response to influenza A virus (IAV) to viral pathogenesis may yield new antiviral strategies. Zebrafish larvae are useful models to study the innate immune response to pathogens, including IAV, in vivo. Here, we demonstrate how Color-flu, four fluorescent IAV strains originally developed for mice, can be used to study host-virus interactions by simultaneously monitoring virus particles, neutrophils, and macrophages in vivo. Using this model, we show how the angiotensin-converting enzyme inhibitor, ramipril, and mitophagy inhibitor, MDIVI-1, improved survival, decreased viral burden, and improved the respiratory burst response to IAV infection. The Color-flu zebrafish model of IAV infection is complementary to other models as it is the only model where interactions between virus particles and host cells in an intact vertebrate can be visualized in vivo.

Paracentral acute middle maculopathy presenting as a sign of impending central retinal artery occlusion: a case report.

BACKGROUND: To report a case of paracentral acute middle maculopathy (PAMM) that progressed to central retinal artery occlusion (CRAO) on spectral domain-optical coherence tomography (SD-OCT). CASE PRESENTATION: A 63-year-old male presented with a paracentral scotoma that began several days ago. His past medical history consisted of third-degree atrioventricular heart block requiring a pacemaker. Giant cell arteritis was unlikely given the patient's labs, demographics and review of systems. SD-OCT revealed a characteristic hyperreflective band in the inner nuclear layer consistent with PAMM in his left eye. Fluorescein angiography was obtained and was unremarkable. Five days later, the patient developed no light perception in the left eye. SD-OCT showed a diffuse inner retinal hyperreflectivity consistent with CRAO. CONCLUSION: PAMM can be a harbinger event for complete CRAO. Complete stroke evaluation should be performed to prevent a cerebrovascular event or progression to complete blindness in the involved eye.

A New Phenotype in Candida-Epithelial Cell Interaction Distinguishes Colonization- versus Vulvovaginal Candidiasis-Associated Strains.

Vulvovaginal candidiasis (VVC) affects nearly 3/4 of women during their lifetime, and its symptoms seriously reduce quality of life. Although Candida albicans is a common commensal, it is unknown if VVC results from a switch from a commensal to pathogenic state, if only some strains can cause VVC, and/or if there is displacement of commensal strains with more pathogenic strains. We studied a set of VVC and colonizing C. albicans strains to identify consistent in vitro phenotypes associated with one group or the other. We find that the strains do not differ in overall genetic profile or behavior in culture media (i.e., multilocus sequence type [MLST] profile, rate of growth, and filamentation), but they show strikingly different behaviors during their interactions with vaginal epithelial cells. Epithelial infections with VVC-derived strains yielded stronger fungal proliferation and shedding of fungi and epithelial cells. Transcriptome sequencing (RNA-seq) analysis of representative epithelial cell infections with selected pathogenic or commensal isolates identified several differentially activated epithelial signaling pathways, including the integrin, ferroptosis, and type I interferon pathways; the latter has been implicated in damage protection. Strikingly, inhibition of type I interferon signaling selectively increases fungal shedding of strains in the colonizing cohort, suggesting that increased shedding correlates with lower interferon pathway activation. These data suggest that VVC strains may intrinsically have enhanced pathogenic potential via differential elicitation of epithelial responses, including the type I interferon pathway. Therefore, it may eventually be possible to evaluate pathogenic potential in vitro to refine VVC diagnosis. IMPORTANCE Despite a high incidence of VVC, we still have a poor understanding of this female-specific disease whose negative impact on women's quality of life has become a public health issue. It is not yet possible to determine by genotype or laboratory phenotype if a given Candida albicans strain is more or less likely to cause VVC. Here, we show that Candida strains causing VVC induce more fungal shedding from epithelial cells than strains from healthy women. This effect is also accompanied by increased epithelial cell detachment and differential activation of the type I interferon pathway. These distinguishing phenotypes suggest it may be possible to evaluate the VVC pathogenic potential of fungal isolates. This would permit more targeted antifungal treatments to spare commensals and could allow for displacement of pathogenic strains with nonpathogenic colonizers. We expect these new assays to provide a more targeted tool for identifying fungal virulence factors and epithelial responses that control fungal vaginitis.

Not Just Surface Energy: The Role of Bis(pentafluorophenoxy) Silicon Phthalocyanine Axial Functionalization and Molecular Orientation on Organic Thin-Film Transistor Performance.

Understanding the effect of surface chemistry on the dielectric-semiconductor interface, thin-film morphology, and molecular alignment enables the optimization of organic thin-film transistors (OTFTs). We explored the properties of thin films of bis(pentafluorophenoxy) silicon phthalocyanine (F10-SiPc) evaporated onto silicon dioxide (SiO2) surfaces modified by self-assembled monolayers (SAMs) of varying surface energies and by weak epitaxy growth (WEG). The total surface energy (γtot), dispersive component of the total surface energy (γd), and polar component of the total surface energy (γp) were calculated using the Owens-Wendt method and related to electron field-effect mobility of devices (µe), and it was determined that minimizing γp and matching γtot yielded films with the largest relative domain sizes and highest resulting µe. Subsequent analyses were completed using atomic force microscopy (AFM) and grazing-incidence wide-angle X-ray scattering (GIWAXS) to relate surface chemistry to thin-film morphology and molecular order at the surface and semiconductor-dielectric interface, respectively. Films evaporated on n-octyltrichlorosilane (OTS) yielded devices with the highest average µe of 7.2 × 10-2 cm2·V-1·s-1 that we attributed to it having both the largest domain length, which were extracted from power spectral density function (PSDF) analysis, and a subset of molecules with a pseudo edge-on orientation relative to the substrate. Films of F10-SiPc with the mean molecular orientation of the π-stacking direction being more edge-on relative to the substrate also generally resulted in OTFTs with a lower average VT. Unlike conventional MPcs, F10-SiPc films fabricated by WEG experienced no macrocycle in an edge-on configuration. These results demonstrate the critical role of the F10-SiPc axial groups on WEG, molecular orientation, and film morphology as a function of surface chemistry and the choice of SAMs.

Aortobifemoral reconstruction in open abdominal aortic aneurysm repair is associated with increased morbidity and mortality.

OBJECTIVE: Much attention has been given to the influence of anatomic and technical factors, such as maximum abdominal aortic aneurysm diameter and proximal clamp position, in open abdominal aortic aneurysm repair (OSR). However, no studies have rigorously examined the correlation between site of distal anastomosis and OSR outcomes despite conventional wisdom that more proximal sites of anastomosis are preferrable when technically feasible. This study aimed to test the association between sites of distal anastomosis and clinical outcomes for patients undergoing primary elective OSR. METHODS: Our study included 5683 patients undergoing primary elective OSR at 233 centers from 2014 to 2020. Using a variety of statistical methods to account for potential confounders, including multivariable logistic regression and Cox proportional hazards modeling, as well as subgroup analysis, we examined the association between site of distal anastomosis and clinical outcomes in elective OSR. Primary outcomes were major in-hospital complication rate, 30-day mortality, and long-term survival. RESULTS: Patients undergoing elective aortobifemoral reconstruction (n = 672) exhibited significantly increased rates of smoking, chronic obstructive pulmonary disease, and peripheral artery disease in comparison to patients undergoing elective OSR with distal anastomosis to the aorta (n = 2298), common iliac artery (n = 2163), or external iliac artery (n = 550). Patients undergoing aorto-aortic tube grafting were significantly less likely to exhibit iliac aneurysmal disease and significantly more likely to be undergoing elective OSR with a suprarenal or supraceliac proximal clamp position. Using multivariable logistic regression and Cox proportional hazards analysis to control for important confounders, such as age, smoking status, and medical history, we found that distal anastomosis to the common femoral artery was associated with increased odds of major in-hospital complications (adjusted odds ratio, 1.79; 95% confidence interval, 1.46-2.18; P < .001) and reduced long-term survival (adjusted hazard ratio, 1.44; 95% confidence interval, 1.09-1.89; P = .010). We observed no significant differences in 30-day mortality across sites of distal anastomosis in our study population. CONCLUSIONS: It is generally accepted that more proximal sites of distal anastomosis should be selected in OSR when technically feasible. Our findings support this hypothesis by demonstrating that distal anastomosis to the common femoral artery is associated with increased perioperative morbidity and reduced long-term survival. Careful diligence regarding optimization of preoperative health status, perioperative care, and long-term follow-up should be applied to mitigate major complications in this patient population.

Tautomerisation Mechanisms in the Adenine-Thymine Nucleobase Pair during DNA Strand Separation.

The adenine-thymine tautomer (A*-T*) has previously been discounted as a spontaneous mutagenesis mechanism due to the energetic instability of the tautomeric configuration. We study the stability of A*-T* while the nucleobases undergo DNA strand separation. Our calculations indicate an increase in the stability of A*-T* as the DNA strands unzip and the hydrogen bonds between the bases stretch. Molecular Dynamics simulations reveal the time scales and dynamics of DNA strand separation and the statistical ensemble of opening angles present in a biological environment. Our results demonstrate that the unwinding of DNA, an inherently out-of-equilibrium process facilitated by helicase, will change the energy landscape of the adenine-thymine tautomerization reaction. We propose that DNA strand separation allows the stable tautomerization of adenine-thymine, providing a feasible pathway for genetic point mutations via proton transfer between the A-T bases.

Postoperative Echography for Optimization of Radiation Dosimetry in Patients with Uveal Melanoma Treated with Plaque Brachytherapy.

PURPOSE: (1) To describe the technique of postoperative echography to confirm the intended treatment dose to the tumor apex in patients with uveal melanoma treated with plaque brachytherapy. (2) To describe the local tumor control rate and visual outcomes with the brachytherapy strategies used at our institution. DESIGN: Retrospective review. SUBJECTS: Three hundred seventy-two consecutive patients with uveal melanoma (small, medium, and large) treated with plaque brachytherapy at the University of Iowa from August 2008 to February 2019. METHODS: Patient demographics and tumor characteristics were recorded for each patient. Patients with posterior tumors treated with plaque brachytherapy (n = 355) underwent intraoperative ultrasound to confirm plaque placement, and additional postoperative ultrasound on day 1 to 3 postplaque insertion. In cases where intratumor/episcleral plaque edema or hemorrhage shifted the dose to the prescription point to < 85 Gray (Gy), the duration of plaque brachytherapy was increased to compensate. Statistical analysis was performed to compare variables associated with the need for plaque adjustment. MAIN OUTCOMES MEASURES: Variables associated with plaque dose needing to be recalculated, local tumor control, and visual acuity outcomes. RESULTS: In 31 (8.3%) cases, postoperative echography showed that the tumor apex had shifted outside the 85 Gy isodose curve, requiring adjustment of the duration of brachytherapy (28 cases) or repositioning of the plaque (3 cases). Collaborative Ocular Melanoma Study tumor size was significantly associated with need to adjust the plaque prescription dose (P = 0.03), with large tumors having the highest rate of adjustment. Tumor thickness was larger in cases requiring plaque adjustment compared with those that were not adjusted (median 4.9 mm vs. 3.0 mm, P < 0.01). Local tumor control was 99% (95% confidence interval, 97%-100%) at 5 years and 99% (95% confidence interval, 97%-100%) at 10 years. The percentage of patients who had experienced a visual acuity decline of ≥ 3 lines of vision or had < 20/200 acuity was 14.9% at 1 year after brachytherapy, 35.3% at 3 years, and 51.6% at 5 years. CONCLUSIONS: Postoperative ultrasound performed on postoperative day 1 to 3 after plaque insertion for patients undergoing brachytherapy for uveal melanoma may result in improved local tumor control, particularly in the setting of thicker or larger tumors. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Chronic Kidney Disease Induces Proarrhythmic Remodeling.

BACKGROUND: Patients with chronic kidney disease (CKD) are at increased risk of developing cardiac arrhythmogenesis and sudden cardiac death; however, the basis for this association is incompletely known. METHODS: Here, using murine models of CKD, we examined interactions between kidney disease progression and structural, electrophysiological, and molecular cardiac remodeling. RESULTS: C57BL/6 mice with adenine supplemented in their diet developed progressive CKD. Electrocardiographically, CKD mice developed significant QT prolongation and episodes of bradycardia. Optical mapping of isolated-perfused hearts using voltage-sensitive dyes revealed significant prolongation of action potential duration with no change in epicardial conduction velocity. Patch-clamp studies of isolated ventricular cardiomyocytes revealed changes in sodium and potassium currents consistent with action potential duration prolongation. Global transcriptional profiling identified dysregulated expression of cellular stress response proteins RBM3 (RNA-binding motif protein 3) and CIRP (cold-inducible RNA-binding protein) that may underlay the ion channel remodeling. Unexpectedly, we found that female sex is a protective factor in the progression of CKD and its cardiac sequelae. CONCLUSIONS: Our data provide novel insights into the association between CKD and pathologic proarrhythmic cardiac remodeling. Cardiac cellular stress response pathways represent potential targets for pharmacologic intervention for CKD-induced heart rhythm disorders.

Increasing Diversity of Patients in Radiation Oncology Clinical Trials.

Radiation oncology clinical trials lack full representation of the ethnic and racial diversity present in the general United States and in the cancer patient population. There are low rates of both recruitment and enrollment of individuals from underrepresented ethnic and racial backgrounds, especially Black and Hispanic patients, people with disabilities, and patients from underrepresented sexual and gender groups. Even if approached for enrollment, barriers such as mistrust in medical research stemming from historical abuse and contemporary biased systems, low socioeconomic status, and lack of awareness prohibit historically marginalized populations from participating in clinical trials. In this review, we reflect on these specific barriers and detail approaches to increase diversity of the patient population in radiation oncology clinical trials to better reflect the communities we serve. We hope that implementation of these approaches will increase the diversity of clinical trials patient populations in not only radiation oncology but also other medical specialties.

Intravitreal Carboplatin as Salvage Treatment for Progressive Vitreous Disease in Retinoblastoma: A Phase I Clinical Trial.

PURPOSE: To determine the safety and toxicity profile of intravitreal carboplatin as salvage treatment for retinoblastoma with vitreous disease. DESIGN: Single-institution, interventional prospective clinical trial. PARTICIPANTS: Patients with progressive or recurrent vitreous seeds after completion of primary treatment for intraocular retinoblastoma. METHODS: Eligible eyes received an intravitreal injection of carboplatin every 14 to 21 days with simultaneous focal therapy (laser, thermotherapy, and brachytherapy) provided at the discretion of the ocular oncologist. The evaluation with examination under anesthesia, ultrasound biomicroscopy, and electroretinography (ERG) were performed before each injection to assess for tumor response and drug-related toxicity. A serious adverse event resulted in dose recalculation and ultimately early closure of the study. MAIN OUTCOME MEASURES: Regression pattern of vitreous disease and incidence of dose-limiting toxicities. RESULTS: Four patients were enrolled at an initial dose of 0.3 mg. Complete regression of vitreous seeds was noted in all patients after 5, 2, 2, and 1 injections (respectively). Two patients developed recurrent vitreous disease at 3 and 25 months after complete regression and ultimately required enucleation. A serious adverse event occurred in 1 patient who developed acute vision loss with extinguished ERG response 72 hours after the second injection; ultimately, this eye developed a cataract and required enucleation. After temporary suspension and dose modification, 3 patients were enrolled at an injection dose of 3 µg and treated with a total of 5, 2, and 1 injections, respectively. Complete regression of vitreous disease was not achieved in any patient though ERG amplitudes remained stable. After removal from protocol, all 3 patients had a complete response to intravitreal melphalan. Concern for dose escalation and further toxicity in the setting of an effective and safe alternative (melphalan) led to the termination of the study. CONCLUSIONS: Intravitreal carboplatin may be effective in treating progressive vitreous seeding at higher doses, but permanent retinal toxicity was observed. Other alternative agents should be considered. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Assessing appropriate panel size and optimization of quality metrics for ambulatory care pharmacists in the primary care setting.

PURPOSE: Chronic disease state management utilizing pharmacists improves quality metrics, allows providers to focus on acute issues, and decreases physician burnout risk. Minimal data exist on pharmacist panel size and its impact. This study aimed to determine appropriate pharmacist panel size based on workload, quality metrics, and patient access. METHODS: This study was a retrospective, multiclinic cohort analysis of patients with diabetes managed by pharmacists at 7 outpatient clinics. The primary objective calculated panel size per full-time equivalent (FTE) utilizing the National Health Interview Survey. Secondary objectives calculated the ideal FTE based on provider to pharmacist ratio and determined the impact of pharmacist panel size on patient access and quality metrics. RESULTS: A total of 4,399 patients were analyzed from 2017 through 2019, with age (range, 57.4 to 62.6 years), sex (52.5% to 63.5% female), race (41.2% to 93.7% African American), insurance type (13.3% to 41% Medicaid), and mean number of medications (13.1 to 20.3) significantly different between sites. Primary outcome results showed that actual panel sizes were less than calculated. However, secondary outcomes indicated that each site was understaffed (actual 0.2 to 0.5 FTE vs calculated 2.52 to 7.34 FTEs) and overbooked (95% to 122% capacity, 17 to 54.2 days for time to third next available appointment). Patients met the composite quality metric 35.1% to 56.3% of the time across sites. CONCLUSION: This study supports the use of patient access data to determine appropriate pharmacist panel size. Utilizing provider panel size to pharmacist ratio and time to third next available appointment is preferable for determining appropriate pharmacist panel size. Further research is needed to evaluate return times to help determine an ideal pharmacist panel size.

RS-FISH: precise, interactive, fast, and scalable FISH spot detection.

Fluorescent in-situ hybridization (FISH)-based methods extract spatially resolved genetic and epigenetic information from biological samples by detecting fluorescent spots in microscopy images, an often challenging task. We present Radial Symmetry-FISH (RS-FISH), an accurate, fast, and user-friendly software for spot detection in two- and three-dimensional images. RS-FISH offers interactive parameter tuning and readily scales to large datasets and image volumes of cleared or expanded samples using distributed processing on workstations, clusters, or the cloud. RS-FISH maintains high detection accuracy and low localization error across a wide range of signal-to-noise ratios, a key feature for single-molecule FISH, spatial transcriptomics, or spatial genomics applications.

Retraction Notice to: Modulation of TNFα Activity by the microRNA Let-7 Coordinates Zebrafish Heart Regeneration.

[This retracts the article DOI: 10.1016/j.isci.2019.06.017.].

Retraction Notice to: Modulation of TNFa Activity by the microRNA Let-7 Coordinates Zebrafish Heart Regeneration.

[This retracts the article DOI: 10.1016/j.isci.2019.04.009.].

Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation.

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.

Rearrangement in the Hypervariable Region of JC Polyomavirus Genomes Isolated from Patient Samples and Impact on Transcription Factor-Binding Sites and Disease Outcomes.

JC polyomavirus (JCPyV) is the causative agent of the fatal, incurable, neurological disease, progressive multifocal leukoencephalopathy (PML). The virus is present in most of the adult population as a persistent, asymptotic infection in the kidneys. During immunosuppression, JCPyV reactivates and invades the central nervous system. A main predictor of disease outcome is determined by mutations within the hypervariable region of the viral genome. In patients with PML, JCPyV undergoes genetic rearrangements in the noncoding control region (NCCR). The outcome of these rearrangements influences transcription factor binding to the NCCR, orchestrating viral gene transcription. This study examines 989 NCCR sequences from patient isolates deposited in GenBank to determine the frequency of mutations based on patient isolation site and disease status. The transcription factor binding sites (TFBS) were also analyzed to understand how these rearrangements could influence viral transcription. It was determined that the number of TFBS was significantly higher in PML samples compared to non-PML samples. Additionally, TFBS that could promote JCPyV infection were more prevalent in samples isolated from the cerebrospinal fluid compared to other locations. Collectively, this research describes the extent of mutations in the NCCR that alter TFBS and how they correlate with disease outcome.

Medical Treatment of Benign Prostatic Hyperplasia.

In this article, we review the primary medical treatments of BPH-associated male LUTS, with a focus on physiology, indications, and side effects. We cover selective alpha-1 antagonists, anticholinergics, PDE5 inhibitors, 5-alpha reductase inhibitors, and beta-3 agonists. Through a review of the literature, we provide our clinical pathway for the medical management of BPH, generally starting with tamsulosin in most men and progressing to combination therapy based on patient's response and symptoms.

Beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in the zebrafish model of Duchenne muscular dystrophy.

Neuromuscular electrical stimulation (NMES) allows activation of muscle fibers in the absence of voluntary force generation. NMES could have the potential to promote muscle homeostasis in the context of muscle disease, but the impacts of NMES on diseased muscle are not well understood. We used the zebrafish Duchenne muscular dystrophy (dmd) mutant and a longitudinal design to elucidate the consequences of NMES on muscle health. We designed four neuromuscular stimulation paradigms loosely based on weightlifting regimens. Each paradigm differentially affected neuromuscular structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES improves muscle and neuromuscular junction morphology, swimming, and survival. Heme oxygenase and integrin alpha7 are required for eNMES-mediated improvement. Our data indicate that neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.