ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD.

The hexanucleotide repeat expansion GGGGCC (G4C2)n in the C9orf72 gene is the most common genetic abnormality associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent findings suggest that dysfunction of nuclear-cytoplasmic trafficking could affect the transport of RNA binding proteins in C9orf72 ALS/FTD. Here, we provide evidence that the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is mislocalized in C9orf72 repeat expansion mediated ALS/FTD. ADAR2 is responsible for adenosine (A) to inosine (I) editing of double-stranded RNA, and its function has been shown to be essential for survival. Here we show the mislocalization of ADAR2 in human induced pluripotent stem cell-derived motor neurons (hiPSC-MNs) from C9orf72 patients, in mice expressing (G4C2)149, and in C9orf72 ALS/FTD patient postmortem tissue. As a consequence of this mislocalization we observe alterations in RNA editing in our model systems and across multiple brain regions. Analysis of editing at 408,580 known RNA editing sites indicates that there are vast RNA A to I editing aberrations in C9orf72-mediated ALS/FTD. These RNA editing aberrations are found in many cellular pathways, such as the ALS pathway and the crucial EIF2 signaling pathway. Our findings suggest that the mislocalization of ADAR2 in C9orf72 mediated ALS/FTD is responsible for the alteration of RNA processing events that may impact vast cellular functions, including the integrated stress response (ISR) and protein translation.

Correction to: ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD.

The original article was published erroneously without mentioning the support of the U.S.

Collagen VI deficiency reduces muscle pathology, but does not improve muscle function, in the γ-sarcoglycan-null mouse.

Muscular dystrophy is characterized by progressive skeletal muscle weakness and dystrophic muscle exhibits degeneration and regeneration of muscle cells, inflammation and fibrosis. Skeletal muscle fibrosis is an excessive deposition of components of the extracellular matrix including an accumulation of Collagen VI. We hypothesized that a reduction of Collagen VI in a muscular dystrophy model that presents with fibrosis would result in reduced muscle pathology and improved muscle function. To test this hypothesis, we crossed γ-sarcoglycan-null mice, a model of limb-girdle muscular dystrophy type 2C, with a Col6a2-deficient mouse model. We found that the resulting γ-sarcoglycan-null/Col6a2Δex5 mice indeed exhibit reduced muscle pathology compared with γ-sarcoglycan-null mice. Specifically, fewer muscle fibers are degenerating, fiber size varies less, Evans blue dye uptake is reduced and serum creatine kinase levels are lower. Surprisingly, in spite of this reduction in muscle pathology, muscle function is not significantly improved. In fact, grip strength and maximum isometric tetanic force are even lower in γ-sarcoglycan-null/Col6a2Δex5 mice than in γ-sarcoglycan-null mice. In conclusion, our results reveal that Collagen VI-mediated fibrosis contributes to skeletal muscle pathology in γ-sarcoglycan-null mice. Importantly, however, our data also demonstrate that a reduction in skeletal muscle pathology does not necessarily lead to an improvement of skeletal muscle function, and this should be considered in future translational studies.

Bringing Menu Labelling to Independent Restaurants: Findings from a Voluntary Pilot Project in Toronto.

Toronto Public Health conducted a pilot project to assess the feasibility of menu labelling by independent restaurants. The pilot project was informed by consultations with the industry and other jurisdictions that have implemented a similar initiative. Public Health Dietitians worked closely with these restaurants to help them work toward posting calories and sodium on their menus. This paper reports on the findings of a feasibility assessment that took a mixed-methods approach resulting in a comprehensive process evaluation. Results showed that having highly motivated restaurants and early adopters of menu labelling is a necessary starting point. However, this alone is not sufficient to make voluntary menu labelling successful. It may be feasible only for select independent restaurants who: (i) are highly motivated and ready to make a substantial time commitment; (ii) value offering healthy food choices; (iii) have fairly standardized recipes to begin with; (iv) receive extensive specialized, individualized support; and (v) receive incentives, cost offsetting, and recognition. Full-scale implementation of a menu labelling program with Toronto independent restaurants was not justified given the current level of interest and capacity.

Findings of Esophagography for 25 Patients After Peroral Endoscopic Myotomy for Achalasia.

OBJECTIVE: The purpose of this study is to better characterize the findings of esophagography after peroral endoscopic myotomy for achalasia. MATERIALS AND METHODS: We evaluated 25 patients who underwent peroral endoscopic myotomy for achalasia. The findings noted on pre- and postprocedural esophagrams were reviewed retrospectively and were correlated with clinical outcomes. RESULTS: None of the patients had esophageal perforation noted on esophagrams obtained after myotomy, and all but two patients had a hospital stay that lasted 1 day only. Esophagrams obtained on postoperative day 1 revealed endoscopic clips in 25 patients (100%), pneumoperitoneum in 18 (72%), retroperitoneal gas in 10 (40%), gastric pneumatosis in nine (36%), intramural dissections in seven (28%), and pneumomediastinum in four (16%). Repeat esophagrams obtained 3 weeks later for 22 of the patients revealed endoscopic clips in 16 patients (73%) and intramural dissections in five patients (23%), but the remaining findings had resolved. Eighteen patients (72%) had a successful myotomy and seven (28%) had suboptimal results on the basis of clinical outcomes. Observation of a distal esophageal width of 5 mm or less on postprocedural esophagrams was often associated with suboptimal results. CONCLUSION: Peroral endoscopic myotomy is a novel procedure that is less invasive than is laparoscopic Heller myotomy for the treatment of achalasia, with fewer complications and shorter recovery times. Radiologists should be aware of the findings expected on esophagography (including pneumoperitoneum, retroperitoneal gas, gastric pneumatosis, intramural dissections, and pneumomediastinum) and should also know that fluoroscopic studies may be helpful for predicting patient outcomes on the basis of the width of the distal esophagus after myotomy.

Closing the gaps in adolescent vaccinations: Rhode Island's Vaccinate Before You Graduate program as a model for other jurisdictions.

Objective: The northeastern state of Rhode Island (RI) has a Vaccinate Before You Graduate (VBYG) program that supplements the traditional primary care infrastructure by providing vaccines to adolescents while they are in school, with no out-of-pocket expenses. We analyzed data from RI's immunization registry to evaluate whether VBYG also reduces disparities in adolescent immunization rates. Methods: We identified adolescent and catch-up vaccines administered in RI to people who were aged 11-18 at any point during the 5-year study period of 2019-2023, and conducted bivariate and multivariate analyses of vaccine administration data by setting (VBYG clinics, community health centers [CHCs], all other primary care practices [oPCPs], other school-based clinics, and other sites) and adolescent demographics (racial and ethnic identity, insurance status, sex, and age at time of vaccine). Results: Of over 387,000 routine vaccines administered during the study period, 3.3 % were administered by a VBYG clinic despite significant declines during school closures associated with the early COVID-19 pandemic. VBYG-administered doses went to slightly older youth, and a higher proportion were catch-up doses (25.7 % versus 14.1 % for CHC doses and 6.5 % for oPCP). Youths received an average of 2.71 vaccines in VBYG clinics compared to 1.77 from oPCPs and 2.08 from CHCs. A higher proportion of vaccines administered by VBYG went to adolescents of color and those without private insurance than those administered by oPCPs. Conclusions: VBYG provides a model to other jurisdictions of a vaccine safety net for adolescents who may not otherwise receive recommended vaccines before exiting the school system.

Expert panel curation of 31 genes in relation to limb girdle muscular dystrophy.

OBJECTIVE: Limb girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous autosomal conditions with some degree of phenotypic homogeneity. LGMD is defined as having onset >2 years of age with progressive proximal weakness, elevated serum creatine kinase levels and dystrophic features on muscle biopsy. Advances in massively parallel sequencing have led to a surge in genes linked to LGMD. METHODS: The ClinGen Muscular Dystrophies and Myopathies gene curation expert panel (MDM GCEP, formerly Limb Girdle Muscular Dystrophy GCEP) convened to evaluate the strength of evidence supporting gene-disease relationships (GDR) using the ClinGen gene-disease clinical validity framework to evaluate 31 genes implicated in LGMD. RESULTS: The GDR was exclusively LGMD for 17 genes, whereas an additional 14 genes were related to a broader phenotype encompassing congenital weakness. Four genes (CAPN3, COL6A1, COL6A2, and COL6A3) were split into two separate disease entities, based on each displaying both dominant and recessive inheritance patterns, resulting in curation of 35 GDRs. Of these, 30 (86%) were classified as definitive, 4 (11%) as moderate, and 1 (3%) as limited. Two genes, POMGNT1 and DAG1, though definitively related to myopathy, currently have insufficient evidence to support a relationship specifically with LGMD. INTERPRETATION: The expert-reviewed assertions on the clinical validity of genes implicated in LGMDs form an invaluable resource for clinicians and molecular geneticists. We encourage the global neuromuscular community to publish case-level data that help clarify disputed or novel LGMD associations.

Expert Panel Curation of 31 Genes in Relation to Limb Girdle Muscular Dystrophy.

Introduction: Limb girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous autosomal conditions with some degree of phenotypic homogeneity. LGMD is defined as having onset >2 years of age with progressive proximal weakness, elevated serum creatine kinase levels and dystrophic features on muscle biopsy. Advances in massively parallel sequencing have led to a surge in genes linked to LGMD. Methods: The ClinGen Muscular Dystrophies and Myopathies gene curation expert panel (MDM GCEP, formerly Limb Girdle Muscular Dystrophy GCEP) convened to evaluate the strength of evidence supporting gene-disease relationships (GDR) using the ClinGen gene-disease clinical validity framework to evaluate 31 genes implicated in LGMD. Results: The GDR was exclusively LGMD for 17 genes, whereas an additional 14 genes were related to a broader phenotype encompassing congenital weakness. Four genes (CAPN3, COL6A1, COL6A2, COL6A3) were split into two separate disease entities, based on each displaying both dominant and recessive inheritance patterns, resulting in curation of 35 GDRs. Of these, 30 (86%) were classified as Definitive, 4 (11%) as Moderate and 1 (3%) as Limited. Two genes, POMGNT1 and DAG1, though definitively related to myopathy, currently have insufficient evidence to support a relationship specifically with LGMD. Conclusions: The expert-reviewed assertions on the clinical validity of genes implicated in LGMDs form an invaluable resource for clinicians and molecular geneticists. We encourage the global neuromuscular community to publish case-level data that help clarify disputed or novel LGMD associations.

Mechanism-free repurposing of drugs for C9orf72-related ALS/FTD using large-scale genomic data.

Repeat expansions in the C9orf72 gene are the most common genetic cause of (ALS) and frontotemporal dementia (FTD). Like other genetic forms of neurodegeneration, pinpointing the precise mechanism(s) by which this mutation leads to neuronal death remains elusive, and this lack of knowledge hampers the development of therapy for C9orf72-related disease. We used an agnostic approach based on genomic data (n = 41,273 ALS and healthy samples, and n = 1,516 C9orf72 carriers) to overcome these bottlenecks. Our drug-repurposing screen, based on gene- and expression-pattern matching and information about the genetic variants influencing onset age among C9orf72 carriers, identified acamprosate, a γ-aminobutyric acid analog, as a potentially repurposable treatment for patients carrying C9orf72 repeat expansions. We validated its neuroprotective effect in cell models and showed comparable efficacy to riluzole, the current standard of care. Our work highlights the potential value of genomics in repurposing drugs in situations where the underlying pathomechanisms are inherently complex. VIDEO ABSTRACT.

Moderate intrinsic phenotypic alterations in C9orf72 ALS/FTD iPSC-microglia despite the presence of C9orf72 pathological features.

While motor and cortical neurons are affected in C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), it remains largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We differentiated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G4C2 repeat RNA foci, exhibit reduced C9orf72 protein levels, and generate dipeptide repeat proteins. Healthy control and C9orf72 ALS/FTD iPSC-MG equally express microglial specific genes and perform microglial functions, including inflammatory cytokine release and phagocytosis of extracellular cargos, such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. RNA sequencing analysis revealed select transcriptional changes of genes associated with neuroinflammation or neurodegeneration in diseased microglia yet no significant differentially expressed microglial-enriched genes. Moderate molecular and functional differences were observed in C9orf72 iPSC-MG mono-cultures despite the presence of C9orf72 pathological features suggesting that a diseased microenvironment may be required to induce phenotypic changes in microglial cells and the associated neuronal dysfunction seen in C9orf72 ALS/FTD neurodegeneration.

Special delivery: dynamic targeting via cortical capture of microtubules.

Gap junction formation depends on the proper transport of connexin hemichannels to sites of cell-cell contact. Recently in Cell, Shaw et al. implicate microtubule tip tracking proteins in the trafficking of connexin43 to adherens junctions (Shaw et al., 2007). This finding suggests a mechanism for targeted delivery of membrane proteins by microtubule capture at the cortex.

A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation.

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

Enhancing cultural competence for improved access to quality care.

The Hospital for Sick Children (SickKids) is situated in one of the most diverse cities in the world. This is reflected in the patient population it serves. In 2009, the hospital embarked on a quality improvement initiative to address the existing evidence base on health disparities and to enhance health equity through cultural competence programming. The goal was to achieve optimal health outcomes for all patients and families, with a particular focus on new immigrant and other vulnerable populations. Evaluation results indicate changes in clinical practice as a result of this initiative and increased patient satisfaction with regard to staff members' level of cultural sensitivity. This article provides an overview of this hospital-wide initiative, as well as the evaluation methods and outcomes. Based on a needs assessment, we developed an institutionally meaningful curriculum with SickKids' values of family-centred care, patient safety and service excellence embedded in the program. Educational sessions were delivered to clinical and non-clinical hospital staff, focusing on health disparities, the case for culturally competent care and practical tools for healthcare practitioners. Organizational change strategies, including the use of champions as change agents and role models, were used to embed cultural competence as integral to family-centred care at SickKids.

Focal reactive periostitis ossificans in a long bone: radiologic and pathologic findings.

Florid reactive periostitis ossificans (FRPO) is a benign juxta-cortical lesion of unknown etiology which most commonly occurs in the hands and feet. We report the radiographic, CT, and MR features of a pathologically confirmed FRPO in the distal femur, a location in which only a handful of cases has been reported. A 26-year-old male who presented with distal thigh pain initially underwent radiograph and CT, which illustrated a well-circumscribed, ossified lesion associated with the cortex of the femur without contiguity with the medullary canal. A subsequent MRI demonstrated heterogeneous signal intensity corresponding to the ossified portion of the lesion with a T2 hyperintense cartilaginous cap and surrounding edema. The lesion was surgically excised and pathologic diagnosis of FRPO, a mixture of osteoid, mature bone, cartilage and fibrous tissue, with associated inflammatory cells, was confirmed. Follow up four months after surgery revealed significant improvement in the patient's pain.

Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: implications for cell proliferation control.

Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

Insulin metabolism in human adipocytes from subcutaneous and visceral depots.

Subjects with the metabolic syndrome (insulin resistance, glucose intolerance, dyslipidemia, hypertension, etc.) have a relative increase in abdominal fat tissue compared to normal individuals and obesity has also been shown to be associated with a decrease in insulin clearance. The majority of the clearance of insulin is due to the action of insulin-degrading enzyme (IDE) and IDE is present throughout all tissues. Since abdominal fat is increased in obesity we hypothesized that IDE may be altered in the different fat depots. Adipocytes were isolated from fat samples obtained from subjects during elective abdominal surgery. Fat samples were taken from subcutaneous (SQ) and visceral (VIS) sites. Insulin metabolism was compared in adipocytes isolated from SQ and VIS fat tissue. Adipocytes from the VIS site degraded more insulin that those from SQ fat tissue. Inhibitors of cathepsins B and D has no effect on the degradation of insulin, while bacitracin, an inhibitor of IDE, inhibited degradation by approx. 33% in both SQ and VIS adipocytes. These data show that insulin metabolism is relatively greater in VIS than in SQ fat tissue and potentially due to IDE.

Evaluating the Impact of a Call Triage Assistant on Resident Efficiency, Errors, and Stress.

OBJECTIVE: The aim of this study was to evaluate whether a call triage assistant, who answered telephone calls to the main reading room during the busiest hours of weekend call, would impact resident workflow efficiency, diagnostic errors, and stress level. METHODS: The call triage assistant answered all telephone calls to the main reading room from 12 pm to 7 pm on 6 weekend days over a 3-month period. We compared report turnaround times and resident discrepancy rates on these days with control days, when the same residents were on call without the assistant. We also surveyed residents to determine whether the assistants relieved anxiety associated with the call shift. RESULTS: We recorded 168 telephone calls over the study period. We found the majority of telephone calls could be handled by the assistant without disturbing the on-call resident, resulting in a 71% reduction in interruptions. The mean turnaround time for studies read on the days the assistant was on duty was 44.3 min, compared with 75.2 min on the control days (P < .01). Resident major discrepancy rates (0.4% on the intervention days compared to 0.2% on the control days) were similar (P = .58), as were minor discrepancy rates (7.5% on the intervention days compared with 6.7% on the control days; P = .61). Residents reported fewer distractions, improved workflow efficiency, and decreased call-related stress when the assistant was on duty. CONCLUSIONS: A call triage assistant effectively improved workflow efficiency and reduced resident stress on call. Resident error rates were unaffected by the presence of the assistant.

Women's expectations of treatment and care after an antenatal HIV diagnosis in Lilongwe, Malawi.

Women in sub-Saharan Africa are increasingly learning their HIV status in prevention of mother-to-child transmission of HIV (PMTCT) programmes in the context of antenatal care. This paper examines women's decisions about HIV testing and their experience of PMTCT and HIV-related care in one clinic in Lilongwe, Malawi. It is based on qualitative, ethnographic research conducted in 2004 and 2005, including interviews and focus group discussions with 55 HIV-positive women participating in a PMTCT programme, and 21 interviews with key informants from the programme and the health system. Women's expectations from testing were consistent with the benefits for their own health and their infants' health, as communicated by nurses. However, the PMTCT programme only poorly met their expectations. Reasons for this disjuncture included the construction of women as still healthy even when they needed treatment, a focus only on infant health, health system weaknesses, lack of integrated care and timely referral, and defining HIV exclusively as a medical issue, while ignoring the social determinants of health. Women's own health was particularly marginalised within the PMTCT programme, yet good models exist for comprehensive care for women, infants and their families that should be implemented as testing is scaled up.

A Census of Midsize to Large Supermarkets in Toronto: A Cross-Sectional Analysis of the Consumer Nutrition Environment.

OBJECTIVE: Assess the consumer nutrition environment in midsize to large supermarkets by supermarket type and area-level socioeconomic variables. DESIGN: Cross-sectional census of 257 supermarkets using the Toronto Nutrition Environment Measures Survey in Stores. SETTING: Toronto, Canada. VARIABLES MEASURED: Availability; price and linear shelf space of fruits and vegetables vs energy-dense snack foods by supermarket type; after-tax, low-income measure; and neighborhood improvement area. ANALYSIS: Multivariate linear regression. RESULTS: There was a high availability of fruits (7.7 of 8) and vegetables (9.5 of 11). There was similar linear shelf space for fruits and vegetables vs energy-dense snack foods (ratio, 1.1 m). Adjusted fruit prices were lowest in quintiles 1 (ß = -$1.30; P = .008), 2 (ß = -$1.41; P = .005), and 3 (ß = -$1.89; P < .001) vs quintile 5 (lowest percentage of people living with low income) and in ethnic (ß = -$3.47; P < .001) and discount stores (ß = -$5.64; P < .001) vs conventional. Adjusted vegetable prices were lowest in quintiles 2 (ß = -$1.87; P = .04), 3 (ß = -$1.78; P = .03), and 4 (ß = -$2.65; P = .001) vs quintile 5 and in ethnic (ß = -$7.10; P < .001) and discount (ß = -$5.49; P < .001) stores. They were highest in other (ß = + $3.08; P = .003) vs conventional stores. Adjusted soda and chips prices were lower in discount (ß = -$1.16; P < .001) and higher in other stores (ß = + $0.67; P < .001) vs conventional. CONCLUSIONS AND IMPLICATIONS: Findings do not indicate inequities in shelf space, availability, or price across diverse neighborhoods. Practitioners can use findings to help consumers navigate supermarkets to make healthy choices.

Stenosis of gastric sleeve after laparoscopic sleeve gastrectomy: clinical, radiographic and endoscopic findings.

OBJECTIVE: To determine the clinical, radiographic, and endoscopic findings of sleeve stenosis after sleeve gastrectomy and to correlate treatment with outcomes. METHODS: We identified 43 patients who underwent barium studies to evaluate upper GI symptoms after laparoscopic sleeve gastrectomy. The clinical, radiographic, and endoscopic findings were reviewed and correlated with treatment and outcomes. RESULTS: 26 patients (60%) had sleeve stenoses. All stenoses appeared as short segments of smooth, tapered narrowing, with a mean length of 8.0 mm and mean width of 7.5 mm, and 24 (92%) were located in the proximal or distal third of the sleeve. 23 patients (88%) had upstream dilation, and 1 (4%) had retained food proximal to the stenosis. 23 (70%) of 33 patients with obstructive symptoms and 3 (30%) of 10 without obstructive symptoms had sleeve stenoses. Endoscopy revealed sleeve stenosis in 8 (67%) of 12 patients with radiographic stenosis. Endoscopic dilation resulted in improvement/resolution of symptoms in seven (88%) of 8 patients. CONCLUSION: Sleeve stenosis after sleeve gastrectomy was characterized radiographically by a short segment of smooth, tapered narrowing, typically in the proximal or distal third of the sleeve. Approximately, 70% of patients with obstructive symptoms and 30% with non-obstructive symptoms had sleeve stenosis. One-third of radiographically diagnosed stenoses were not seen at endoscopy. The barium study, therefore, is a useful test for sleeve stenosis in patients with obstructive or nonobstructive symptoms after sleeve gastrectomy. Advances in knowledge: This article describes the appearance and location of sleeve stenoses after laparoscopic sleeve gastrectomy and the clinical presentation and treatment options for these patients.