Proteomic profiling of the brain from the wobbler mouse model of amyotrophic lateral sclerosis reveals elevated levels of the astrogliosis marker glial fibrillary acidic protein.

The wobbler mouse is a widely used model system of amyotrophic lateral sclerosis and exhibits progressive neurodegeneration and neuroinflammation in association with skeletal muscle wasting. This study has used wobbler brain preparations for the systematic and mass spectrometric determination of proteome-wide changes. The proteomic characterization of total protein extracts from wobbler specimens was carried out with the help of an Orbitrap mass spectrometer and revealed elevated levels of glia cell marker proteins, i.e., glial fibrillary acidic protein and the actin-binding protein coronin. In contrast, the abundance of the actin-binding protein neurabin and the scaffolding protein named piccolo of the presynaptic cytomatrix were shown to be reduced. The increased abundance of glial fibrillary acidic protein, which is frequently used in neuropathological studies as a marker protein of glial scar formation, was confirmed by immunoblotting. In analogy, the proteomic profiling of the brain from another established murine model of motor neuron disease, the SOD1mouse, also showed increased levels of this intermediate filament protein. This suggests that neurodegenerative processes are associated with astrogliosis in both the wobbler and SOD1 brain.

Barriers and enablers for safe medication administration in adult and neonatal intensive care units mapped to the behaviour change wheel.

BACKGROUND: Intensive care settings have high rates of medication administration errors. Medications are often administered by nurses and midwives using a specified process (the '5 rights'). Understanding where medication errors occur, the contributing factors and how best practice is delivered may assist in developing interventions to improve medication safety. AIMS: To identify medication administration errors and context specific barriers and enablers for best practice in an adult and a neonatal intensive care unit. Secondary aims were to identify intervention functions (through the Behaviour Change Wheel). STUDY DESIGN: A dual methods exploratory descriptive study was conducted (May to June 2021) in a mixed 56-bedded adult intensive care unit and a 6-bedded neonatal intensive care unit in Sydney, Australia. Incident monitoring data were examined. Direct semi-covert observational medication administration audits using the 5 rights (n = 39) were conducted. Brief interviews with patients, parents and nurses were conducted. Data were mapped to the Behaviour Change Wheel. RESULTS: No medication administration incidents were recorded. Audits (n = 3) for the neonatal intensive care unit revealed no areas for improvement. Adult intensive care unit nurses (n = 36) performed checks for the right medication 35 times (97%) and patient identity 25 times (69%). Sixteen administrations (44%) were interrupted. Four themes were synthesized from the interview data: Trust in the nursing profession; Availability of policies and procedures; Adherence to the '5 rights' and departmental culture; and Adequate staffing. The interventional functions most likely to bring about behaviour change were environmental restructuring, enablement, restrictions, education, persuasion and modelling. CONCLUSIONS: This study reveals insights about the medication administration practices of nurses in intensive care. Although there were areas for improvement there was widespread awareness among nurses regarding their responsibilities to safely administer medications. Interview data indicated high levels of trust among patients and parents in the nurses. RELEVANCE TO CLINICAL PRACTICE: This novel study indicated that nurses in intensive care are aware of their responsibilities to safely administer medications. Mapping of contextual data to the Behaviour Change Wheel resulted in the identification of Intervention functions most likely to change medication administration practices in the adult intensive care setting that is environmental restructuring, enablement, restrictions, education, persuasion and modelling.

Losing your touch? Sustained inattentional numbness for dynamic tactile events.

It is now well-known that a lack of attention can leave people unaware of clearly-noticeable, long-lasting and dynamic stimuli, such as a visible person dressed as a gorilla or an audible person claiming to be a gorilla. However, the question of whether touch can ever be susceptible to such extreme inattentional effects remains open. Here, we present evidence across two experiments that the absence of attention can leave people "numb" to the presence of a tactile stimulus that lasts for 3.5 s and moves across six different skin locations, establishing the new phenomenon of "sustained inattentional numbness." The effect is particularly surprising in light of claims that tactile information processing is more direct than auditory or visual processing, which would suggest that tactile awareness might not be open to attentional modulation of the type that we observe here. The findings also have important applied implications given the increasing prevalence of tactile warnings in everyday information delivery systems. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

DIGE Analysis of ProteoMiner™ Fractionated Serum/Plasma Samples.

The discovery of clinically relevant biomarkers using gel-based proteomics has proven extremely challenging, principally because of the large dynamic range of protein abundances in biofluids such as blood and the fact that only a small number of proteins constitute the vast majority of total blood protein mass. Various separation, depletion, enrichment, and quantitative developments coupled with improvements in gel-based protein quantification technologies, specifically fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), have contributed to significant improvements in the detection and identification of lower abundance proteins. One of these enrichment technologies, ProteoMiner, is the focus of this chapter. The ProteoMiner technology utilizes hexapeptide bead library with huge diversity to bind and enrich low-abundance proteins but at the same time suppresses the concentration of high-abundance proteins in subsequent analysis.

Protein Digestion for 2D-DIGE Analysis.

In-gel digestion of protein spots derived from two-dimensional gels and their subsequent identification by mass spectrometry is involved in a multitude of mass spectrometry-driven proteomic experiments, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). This type of proteomic methodology has been involved in the establishment of comparative proteome maps and in the identification of differentially expressed proteins and their isoforms in health and disease. Most in-gel digestion protocols follow a number of common steps including excision of the protein spots of interest, destaining, reduction and alkylation (for silver-stained gels), and dehydration and overnight digestion with the proteolytic enzyme of choice. While trypsin has been a mainstay of peptide digestion for many years, it does have its shortcomings, particularly related to incomplete peptide digestion, and this has led to a rise in popularity for other proteolytic enzymes either used alone or in combination. This chapter discusses the alternative enzymes available and describes the process of in-gel digestion using the enzyme trypsin.

Subcellular Fractionation for DIGE-Based Proteomics.

Mass spectrometry-based protein methodologies have revolutionized the field of analytical biochemistry and enable the identification of hundreds to thousands of proteins in biological fluids, cell lines, and tissue. This methodology requires the initial separation of a protein constellation, and this has been successfully achieved using gel-based techniques, particularly that of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). However, given the complexity of the proteome, fractionation techniques may be required to optimize the detection of low-abundance proteins, which are often underrepresented but which may represent important players in health and disease. Such subcellular fractionation protocols typically utilize density-gradient centrifugation and have enabled the enrichment of crude microsomes, the cytosol, the plasmalemma, the nuclei, and the mitochondria. In this chapter, we describe the experimental steps involved in the enrichment of crude microsomes from the skeletal muscle using differential centrifugation and subsequent verification of enrichment by gel electrophoresis and immunoblotting, prior to comparative 2D-DIGE analysis.

Bioinformatic Analysis of the Subproteomic Profile of Cardiomyopathic Tissue.

Following large-scale protein separation by two-dimensional gel electrophoresis or liquid chromatography, mass spectrometry-based proteomics can be used for the swift identification and characterization of cardiac proteins and their various proteoforms. Comparative cardiac proteomics has been widely applied for the systematic analysis of heart disease and the establishment of novel diagnostic protein biomarkers. The X-linked neuromuscular disorder Duchenne muscular dystrophy is a multisystemic disease that is characterized by late-onset cardiomyopathy. This chapter outlines the bioinformatic analysis of the subproteomic profile of cardiac tissue from wild-type versus the dystrophic mdx-4cv mouse model of dystrophinopathy.

The role and contribution of family carers accompanying community-living older people with cognitive impairment to the emergency department: An interview study.

BACKGROUND: Older people with cognitive impairment may have lack of understanding of their health and ability to co-ordinate care needs. Family carers play a key role in supporting older people but the role of family carers in emergency discharge has not been explored well. OBJECTIVE: To explore and describe the role and contribution of family carers accompanying the older person with cognitive impairment to the emergency department. DESIGN: A exploratory descriptive study was conducted in which telephone interviews were performed. SETTING: Three emergency departments located in metropolitan Sydney, Australia; two major tertiary referral centres and one district hospital. The EQUATOR research checklist (COnsolidated criteria for REporting Qualitative research) (COREQ) was used to report the qualitative study. SUBJECTS: Participants were family carers accompanying people aged ≥64 years with cognitive impairment; with non-urgent triage classifications; English speaking and eligible for discharge home. Semi-structured telephone interviews were conducted 2 to 4 weeks after the older person was discharged. Data were reflexively thematically analysed in NVIVO independently by three researchers. RESULTS: Twenty-eight interviews were conducted. Three key themes were evident: (1) 'communicating knowledge of the older person's health status and usual behaviour'; (2) 'providing advocacy, translation, surrogacy and care co-ordination'; and (3) 'ensuring safe transition from the emergency department to home'. CONCLUSIONS: Study findings detailed how family carers created an important safety net while the older person was in emergency, through advocacy and the communication of vital health information. More importantly, their knowledge influenced the healthcare management of the older person and ensured safe discharge and co-ordination of care in the community. RELEVANCE TO CLINICAL PRACTICE: The study identified for older persons with cognitive impairment a safe stay in the ED and transition home from hospital was supported by family carers and assisted to ensure that discharge information was understood and adhered to optimise wellbeing and prevent adverse outcomes. The findings of this study can inform discharge processes for nurses, nurse practitioners and doctors. Additionally, processes to support family carer engagement would optimise older person compliance and better inform healthcare decision-making and choice for older peoples, family carers. The findings of the study should inform discharge processes to minimise risk of readmission, financial burden and harm.

Histopathology of Duchenne muscular dystrophy in correlation with changes in proteomic biomarkers.

Duchenne muscular dystrophy is an inherited disorder of early childhood that affects multiple systems in the body. Besides late-onset cardio-respiratory syndrome and various body-wide pathophysiological changes, X-linked muscular dystrophy is primarily classified as a disorder of the skeletal musculature. This is reflected by severe histopathological alterations in voluntary contractile tissues, including progressive fibre degeneration, fat substitution, reactive myofibrosis and chronic inflammation. The underlying cause for dystrophinopathy are genetic abnormalities in the DMD gene, which can result in the almost complete loss of the membrane cytoskeletal protein dystrophin, which triggers the collapse of the dystrophin-associated glycoprotein complex and disintegration of sarcolemmal integrity. This in turn results in an increased frequency of membrane micro-rupturing and abnormal calcium ion fluxes through the impaired plasmalemma, which renders muscle fibres more susceptible to enhanced proteolytic degradation and necrosis. This review focuses on the complexity of skeletal muscle changes in X-linked muscular dystrophy and outlines cell biological and histological alterations in correlation to proteome-wide variations as judged by mass spectrometric analyses. This includes a general outline of sample handling, subcellular fraction protocols and modern proteomic approaches using gel electrophoretic and liquid chromatographic methods for efficient protein separation prior to mass spectrometry. The proteomic profiling of the dystrophic and highly fibrotic diaphragm muscle is described as an example to swiftly identify novel proteomic markers of complex histopathological changes during skeletal muscle degeneration. The potential usefulness of new protein markers is examined in relation to key histopathological hallmarks for establishing improved diagnostic, prognostic and therapy-monitoring approaches in the field of dystrophinopathy.

Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2.

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNß or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

The Dystrophin Node as Integrator of Cytoskeletal Organization, Lateral Force Transmission, Fiber Stability and Cellular Signaling in Skeletal Muscle.

The systematic bioanalytical characterization of the protein product of the DMD gene, which is defective in the pediatric disorder Duchenne muscular dystrophy, led to the discovery of the membrane cytoskeletal protein dystrophin. Its full-length muscle isoform Dp427-M is tightly linked to a sarcolemma-associated complex consisting of dystroglycans, sarcoglyans, sarcospan, dystrobrevins and syntrophins. Besides these core members of the dystrophin-glycoprotein complex, the wider dystrophin-associated network includes key proteins belonging to the intracellular cytoskeleton and microtubular assembly, the basal lamina and extracellular matrix, various plasma membrane proteins and cytosolic components. Here, we review the central role of the dystrophin complex as a master node in muscle fibers that integrates cytoskeletal organization and cellular signaling at the muscle periphery, as well as providing sarcolemmal stabilization and contractile force transmission to the extracellular region. The combination of optimized tissue extraction, subcellular fractionation, advanced protein co-purification strategies, immunoprecipitation, liquid chromatography and two-dimensional gel electrophoresis with modern mass spectrometry-based proteomics has confirmed the composition of the core dystrophin complex at the sarcolemma membrane. Importantly, these biochemical and mass spectrometric surveys have identified additional members of the wider dystrophin network including biglycan, cavin, synemin, desmoglein, tubulin, plakoglobin, cytokeratin and a variety of signaling proteins and ion channels.

Family members' perceptions of older person discharge from emergency departments.

BACKGROUND: People aged over 64 years account for approximately 20% of adult emergency presentations, with up to 60% of people discharged home from emergency departments (EDs). Many older people discharged home are supported by family. OBJECTIVES: The objective of this study was to explore the family members' perspectives of older people's discharge from ED to inform new alternative or innovative models of care. METHODS: The design was a descriptive exploratory study. A convenience sample of family members was recruited from three EDs across Sydney, New South Wales. Telephone interviews were conducted over a six-month period and data were analysed using statistics or thematic analysis. RESULTS: Interviews were conducted with 133 family members of whom the majority were female (n = 80, 60%) with a median age of 70 years (IQR 91-35). Over 87% of family members were satisfied with ED care and discharge processes that were provided to the older person. The majority (n = 129, 97%) of family members reported that they understood the treatment and perceived that the older person's condition was well managed (n = 119, 86%). The majority (n = 114, 86%) of family members reported being informed of the medical diagnosis and were confident (87%, n = 115) to continue care of the older person at home. Three themes emerged from qualitative data: (a) a sense of time-moving through ED; (b) giving voice to the impact of clinician communication; and (c) the delivery of comfort and basic care. DISCUSSION: Family members reported that they were engaged in and satisfied with the older person's ED treatment and discharge. However, family members suggested that there was opportunity to improve communication consistency for ED discharge and managing the wait. CONCLUSION: Clinicians need to engage with family members to optimise quality and safety. Clinicians need to understand that family members considered comfort and fundamentals of care to be an important dimension of the older person's ED management plan.

c-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis.

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-ß1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.

Emerging proteomic biomarkers of X-linked muscular dystrophy.

Introduction: Progressive skeletal muscle wasting is the manifesting symptom of Duchenne muscular dystrophy, an X-linked inherited disorder triggered by primary abnormalities in the DMD gene. The almost complete loss of dystrophin isoform Dp427 causes a multi-system pathology that features in addition to skeletal muscle weakness also late-onset cardio-respiratory deficiencies, impaired metabolism and abnormalities in the central nervous system. Areas covered: This review focuses on the mass spectrometry-based proteomic characterization of X-linked muscular dystrophy with special emphasis on the identification of novel biomarker candidates in skeletal muscle tissues, as well as non-muscle tissues and various biofluids. Individual sections focus on molecular and cellular aspects of the pathogenic changes in dystrophinopathy, proteomic workflows used in biomarker research, the proteomics of the dystrophin-glycoprotein complex and the potential usefulness of newly identified protein markers involved in fibre degeneration, fibrosis and inflammation. Expert opinion: The systematic application of large-scale proteomic surveys has identified a distinct cohort of both tissue- and biofluid-associated protein species with considerable potential for improving diagnostic, prognostic and therapy-monitoring procedures. Novel proteomic markers include components involved in fibre contraction, cellular signalling, ion homeostasis, cellular stress response, energy metabolism and the immune response, as well as maintenance of the cytoskeletal and extracellular matrix.

Proteomic identification of elevated saliva kallikrein levels in the mdx-4cv mouse model of Duchenne muscular dystrophy.

Dystrophinopathies are multi-system disorders that affect the skeletal musculature, the cardio-respiratory system and the central nervous system. The systematic screening of suitable biofluids for released or altered proteins promises new insights into the highly complex pathophysiology of X-linked muscular dystrophy. However, standard detection approaches using antibody-based assays often fail to reproducibly detect low-abundance protein isoforms in dilute biological fluids. In contrast, mass spectrometric screening approaches enable the proteome-wide identification of minor protein changes in biofluids. This report describes the findings from the comparative proteomic analysis of whole saliva samples from wild type versus the established mdx-4cv mouse model of highly progressive muscular dystrophy, focusing on the kallikrein protein family. Kallikrein-1 (Klk1) and 13 Klk1-related peptidases were identified in saliva and serum from normal mice. Comparative proteomics revealed elevated saliva levels of the Klk1-related peptidases Klk1-b1, Klk1-b5 and Klk-b22, as well as an increased Klk-1 concentration, which agrees with higher Klk-1 levels in serum from mdx-4cv mice. This indicates altered cellular signaling, extracellular matrix remodeling and an altered immune response in the mdx-4cv mouse, and establishes liquid biopsy procedures as suitable bioanalytical tools for the systematic survey of complex pathobiochemical changes in animal models of muscular dystrophy.

Proteomic profiling of the mouse diaphragm and refined mass spectrometric analysis of the dystrophic phenotype.

The diaphragm is a crucial muscle involved in active inspiration and whole body homeostasis. Previous biochemical, immunochemical and cell biological investigations have established the distribution and fibre type-specific expression of key diaphragm proteins. Building on these findings, it was of interest to establish the entire experimentally assessable diaphragm proteome and verify the presence of specific protein isoforms within this specialized subtype of skeletal muscle. A highly sensitive Orbitrap Fusion Tribrid mass spectrometer was used for the systematic identification of the mouse diaphragm-associated protein population. Proteomics established 2925 proteins by high confidence peptide identification. Bioinformatics was used to determine the distribution of the main protein classes, biological processes and subcellular localization within the diaphragm proteome. Following the establishment of the respiratory muscle proteome with special emphasis on protein isoform expression in the contractile apparatus, the extra-sarcomeric cytoskeleton, the extracellular matrix and the excitation-contraction coupling apparatus, the mass spectrometric analysis of the diaphragm was extended to the refined identification of proteome-wide changes in X-linked muscular dystrophy. The comparative mass spectrometric profiling of the dystrophin-deficient diaphragm from the mdx-4cv mouse model of Duchenne muscular dystrophy identified 289 decreased and 468 increased protein species. Bioinformatics was employed to analyse the clustering of changes in protein classes and potential alterations in interaction patterns of proteins involved in metabolism, the contractile apparatus, proteostasis and the extracellular matrix. The detailed pathoproteomic profiling of the mdx-4cv diaphragm suggests highly complex alterations in a variety of crucial cellular processes due to deficiency in the membrane cytoskeletal protein dystrophin.

Proteomic profiling of giant skeletal muscle proteins.

INTRODUCTION: Distinct subtypes of contractile fibres are highly diverse in their proteomic profile and greatly adaptable to physiological or pathological challenges. A striking biochemical feature of heterogeneous skeletal muscle tissues is the presence of a considerable number of extremely large protein species, which often present a bioanalytical challenge for the systematic separation and identification of muscle proteomes during large-scale screening surveys. Areas covered: This review outlines the proteomic characterization of skeletal muscles with a special focus on giant proteins of the sarcomere, the cytoskeleton and the sarcoplasmic reticulum. This includes an overview of the involvement of large muscle proteins, such as titin, nebulin, obscurin, plectin, dystrophin and the ryanodine receptor calcium release channel, during normal muscle functioning, swift adaptations to changed physiological demands and changes in relation to pathobiochemical insults. Expert commentary: The proteomic screening and characterization of total muscle extracts and various subcellular fractions has confirmed the critical role of large skeletal muscle proteins in the regulation of ion homeostasis, the maintenance of contraction-relaxation cycles and fibre elasticity, and the stabilisation of supramolecular complexes of the muscle periphery and cytoskeletal networks of contractile fibres. These findings will be helpful for the future functional systems analysis of giant muscle proteins.

Proteomic analysis of the sarcolemma-enriched fraction from dystrophic mdx-4cv skeletal muscle.

The highly progressive neuromuscular disorder dystrophinopathy is triggered by primary abnormalities in the Dmd gene, which causes cytoskeletal instability and loss of sarcolemmal integrity. Comparative organellar proteomics was employed to identify sarcolemma-associated proteins with an altered concentration in dystrophic muscle tissue from the mdx-4cv mouse model of dystrophinopathy. A lectin agglutination method was used to prepare a sarcolemma-enriched fraction and resulted in the identification of 190 significantly changed protein species. Proteomics established differential expression patterns for key components of the muscle plasma membrane, cytoskeletal network, extracellular matrix, metabolic pathways, cellular stress response, protein synthesis, immune response and neuromuscular junction. The deficiency in dystrophin and drastic reduction in dystrophin-associated proteins appears to trigger (i) enhanced membrane repair involving myoferlin, dysferlin and annexins, (ii) increased protein synthesis and the compensatory up-regulation of cytoskeletal proteins, (iii) the decrease in the scaffolding protein periaxin and myelin PO involved in myelination of motor neurons, (iv) complex changes in bioenergetic pathways, (v) elevated levels of molecular chaperones to prevent proteotoxic effects, (vi) increased collagen deposition causing reactive myofibrosis, (vii) disturbed ion homeostasis at the sarcolemma and associated membrane systems, and (viii) a robust inflammatory response by the innate immune system in response to chronic muscle damage. SIGNIFICANCE: Duchenne muscular dystrophy is a devastating muscle wasting disease and represents the most frequently inherited neuromuscular disorder in humans. Genetic abnormalities in the Dmd gene cause a loss of sarcolemmal integrity and highly progressive muscle fibre degeneration. Changes in the neuromuscular system are associated with necrosis, fibrosis and inflammation. In order to evaluate secondary changes in the sarcolemma membrane system due to the lack of the membrane cytoskeletal protein dystrophin, comparative organellar proteomics was used to study the mdx-4cv mouse model of dystrophinopathy. Mass spectrometric analyses identified a variety of altered components of the extracellular matrix-sarcolemma-cytoskeleton axis in dystrophic muscles. This included proteins involved in membrane repair, cytoskeletal restoration, calcium homeostasis, cellular signalling, stress response, neuromuscular transmission and reactive myofibrosis, as well as immune cell infiltration. These pathobiochemical alterations agree with the idea of highly complex secondary changes in X-linked muscular dystrophy and support the concept that micro-rupturing of the dystrophin-deficient plasma membrane is at the core of muscle wasting pathology.

High concordance of BRAF mutational status in matched primary and metastatic melanoma.

BACKGROUND: Techniques for the accurate identification of activating mutations of BRAF in metastatic melanoma are of great clinical importance, due to the availability of targeted therapies for these tumors. There is uncertainty regarding the frequency with which BRAF status differs between primary and metastatic sites. METHODS: Between 2011 and 2016, 219 melanoma cases underwent BRAF testing in our institution. In 53 of these cases, paired primary and metastatic specimens were available for polymerase chain reaction (PCR) and immunohistochemical evaluation. RESULTS: Fifty-two out of 53 cases (98%) showed concordant BRAF status between primary and metastatic site by immunohistochemistry (IHC). In one case, a metastasis and its matched primary were positive by IHC, but the metastasis was negative on PCR. On further investigation, PCR was positive in the primary, and repeat PCR in the metastasis was positive, following macrodissection. CONCLUSIONS: Our results suggest that discordance of BRAF mutational status between primaries and metastases is a rare occurrence. In one case, IHC provided strong evidence that initial PCR testing had provided a false-negative result due to low tumor volume. Thus, in cases where tissue is difficult to obtain from a metastasis or unavailable, the primary tumor can be used with confidence.