Unmasking the Impact of Oxygenator Induced Hypocapnia on Blood Lactate in Veno-Arterial Extracorporeal Membrane Oxygenation.

Extracorporeal membrane oxygenation (ECMO) is often associated with disturbances in acid/base status that can be triggered by the underlying pathology or the ECMO circuit itself. Extracorporeal membrane oxygenation is known to cause hypocapnia, but the impact of reduced partial pressure of carbon dioxide (pCO2) on biomarkers of tissue perfusion during veno-arterial (VA)-ECMO has not been evaluated. To study the impact of low pCO2 on perfusion indices in VA-ECMO, we placed Sprague-Dawley rats on an established VA-ECMO circuit using either an oxygen/carbon dioxide mixture (O2 95%, CO2 5%) or 100% O2 delivered through the oxygenator (n = 5 per cohort). Animals receiving 100% O2 developed a significant VA CO2 difference (pCO2 gap) and rising blood lactate levels that were inversely proportional to the decrease in pCO2 values. In contrast, pCO2 gap and lactate levels remained similar to pre-ECMO baseline levels in animals receiving the O2/CO2 mixture. More importantly, there was no significant difference in venous oxygen saturation (SvO2) between the two groups, suggesting that elevated blood lactate levels observed in the rats receiving 100% O2 were a response to oxygenator induced hypocapnia and alkaline pH rather than reduced perfusion or underlying tissue hypoxia. These findings have implications in clinical and experimental extracorporeal support contexts.

A holistic approach to integrating patient, family, and lived experience voices in the development of the BrainHealth Databank: a digital learning health system to enable artificial intelligence in the clinic.

Artificial intelligence, machine learning, and digital health innovations have tremendous potential to advance patient-centred, data-driven mental healthcare. To enable the clinical application of such innovations, the Krembil Centre for Neuroinformatics at the Centre for Addiction and Mental Health, Canada's largest mental health hospital, embarked on a journey to co-create a digital learning health system called the BrainHealth Databank (BHDB). Working with clinicians, scientists, and administrators alongside patients, families, and persons with lived experience (PFLE), this hospital-wide team has adopted a systems approach that integrates clinical and research data and practices to improve care and accelerate research. PFLE engagement was intentional and initiated at the conception stage of the BHDB to help ensure the initiative would achieve its goal of understanding the community's needs while improving patient care and experience. The BHDB team implemented an evolving, dynamic strategy to support continuous and active PFLE engagement in all aspects of the BHDB that has and will continue to impact patients and families directly. We describe PFLE consultation, co-design, and partnership in various BHDB activities and projects. In all three examples, we discuss the factors contributing to successful PFLE engagement, share lessons learned, and highlight areas for growth and improvement. By sharing how the BHDB navigated and fostered PFLE engagement, we hope to motivate and inspire the health informatics community to collectively chart their paths in PFLE engagement to support advancements in digital health and artificial intelligence.

Innate and adaptive AAV-mediated immune responses in a mouse model of Duchenne muscular dystrophy.

High systemic doses of adeno-associated viruses (AAVs) have been associated with immune-related serious adverse events (SAEs). Although AAV was well tolerated in preclinical models, SAEs were observed in clinical trials, indicating the need for improved preclinical models to understand AAV-induced immune responses. Here, we show that mice dual-dosed with AAV9 at 4-week intervals better recapitulate aspects of human immunity to AAV. In the model, anti-AAV9 immunoglobulin G (IgGs) increased in a linear fashion between the first and second AAV administrations. Complement activation was only observed in the presence of high levels of both AAV and anti-AAV IgG. Myeloid-derived pro-inflammatory cytokines were significantly induced in the same pattern as complement activation, suggesting that myeloid cell activation to AAV may rely on the presence of both AAV and anti-AAV IgG complexes. Single-cell RNA sequencing of peripheral blood mononuclear cells confirmed that activated monocytes were a primary source of pro-inflammatory cytokines and chemokines, which were significantly increased after a second AAV9 exposure. The same activated monocyte clusters expressed both Fcγ and complement receptors, suggesting that anti-AAV-mediated activation of myeloid cells through Fcγ receptors and/or complement receptors is one mechanism by which anti-AAV antigen complexes may prime antigen-presenting cells and amplify downstream immunity.

Nanoparticles systemically biodistribute to regenerating skeletal muscle in DMD.

Skeletal muscle disease severity can often progress asymmetrically across muscle groups and heterogeneously within tissues. An example is Duchenne Muscular Dystrophy (DMD) in which lack of dystrophin results in devastating skeletal muscle wasting in some muscles whereas others are spared or undergo hypertrophy. An efficient, non-invasive approach to identify sites of asymmetry and degenerative lesions could enable better patient monitoring and therapeutic targeting of disease. In this study, we utilized a versatile intravenously injectable mesoporous silica nanoparticle (MSNP) based nanocarrier system to explore mechanisms of biodistribution in skeletal muscle of mdx mouse models of DMD including wildtype, dystrophic, and severely dystrophic mice. Moreover, MSNPs could be imaged in live mice and whole muscle tissues enabling investigation of how biodistribution is altered by different types of muscle pathology such as inflammation or fibrosis. We found MSNPs were tenfold more likely to aggregate within select mdx muscles relative to wild type, such as gastrocnemius and quadriceps. This was accompanied by decreased biodistribution in off-target organs. We found the greatest factor affecting preferential delivery was the regenerative state of the dystrophic skeletal muscle with the highest MSNP abundance coinciding with the regions showing the highest level of embryonic myosin staining and intramuscular macrophage uptake. To demonstrate, muscle regeneration regulated MSNP distribution, we experimentally induced regeneration using barium chloride which resulted in a threefold increase of intravenously injected MSNPs to sites of regeneration 7 days after injury. These discoveries provide the first evidence that nanoparticles have selective biodistribution to skeletal muscle in DMD to areas of active regeneration and that nanoparticles could enable diagnostic and selective drug delivery in DMD skeletal muscle.

A Mixed Methods Study Examining Citizenship Among Youth With Mental Health Challenges.

Introduction: Multiple stakeholders have recently called for greater research on the barriers to citizenship and community belonging faced by people with mental health challenges. Citizenship has been defined as a person's access to the rights, roles, responsibilities, resources and relationships that help people feel a sense of belonging. Factors that may impact citizenship include financial precarity; intersecting forms of marginalization and oppression (e.g., racism); and the mental health care people receive. Research has yet to examine experiences of citizenship among youth with mental health challenges. To address this gap, this study will examine how youth experience citizenship; predictors of citizenship; how citizenship shapes recovery; and the degree to which youth are receiving citizenship-oriented care. Methods: The research objectives will be evaluated using a multiphase mixed methods research design. Quantitative data will be collected cross-sectionally using validated self-report questionnaires. Qualitative data will be collected using a hermeneutic phenomenological method using semi-structured interviews and focus groups. Analyses: Multiple stepwise regression analyses will be used to determine predictors of citizenship and if of citizenship predict recovery. Pearson correlations will be computed to determine the relationship between participants' perceived desire for, and receipt of citizenship-oriented care. Phenomenological analysis will be used to analyze qualitative data. Findings will then be mixed using a weaving method in the final paper discussion section. Conclusion: Findings from this study may support the development of citizenship-oriented healthcare in Canada.

Integrative Multi-OMICs Identifies Therapeutic Response Biomarkers and Confirms Fidelity of Clinically Annotated, Serially Passaged Patient-Derived Xenografts Established from Primary and Metastatic Pediatric and AYA Solid Tumors.

Establishment of clinically annotated, molecularly characterized, patient-derived xenografts (PDXs) from treatment-naïve and pretreated patients provides a platform to test precision genomics-guided therapies. An integrated multi-OMICS pipeline was developed to identify cancer-associated pathways and evaluate stability of molecular signatures in a panel of pediatric and AYA PDXs following serial passaging in mice. Original solid tumor samples and their corresponding PDXs were evaluated by whole-genome sequencing, RNA-seq, immunoblotting, pathway enrichment analyses, and the drug−gene interaction database to identify as well as cross-validate actionable targets in patients with sarcomas or Wilms tumors. While some divergence between original tumor and the respective PDX was evident, majority of alterations were not functionally impactful, and oncogenic pathway activation was maintained following serial passaging. CDK4/6 and BETs were prioritized as biomarkers of therapeutic response in osteosarcoma PDXs with pertinent molecular signatures. Inhibition of CDK4/6 or BETs decreased osteosarcoma PDX growth (two-way ANOVA, p < 0.05) confirming mechanistic involvement in growth. Linking patient treatment history with molecular and efficacy data in PDX will provide a strong rationale for targeted therapy and improve our understanding of which therapy is most beneficial in patients at diagnosis and in those already exposed to therapy.

Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis from Human Pluripotent Stem Cells.

This protocol describes the use of CRISPR/Cas9-mediated homology-directed recombination to construct a PAX7-GFP reporter in human pluripotent stem cells (hPSCs). PAX7 is a key transcription factor and regulator of skeletal muscle stem/progenitor cells. We obtained heterozygous knockin reporter cells and validated their PAX7 expression using both artificial activation by the CRISPR/dCas9-VPR system and physiological activation during hPSC myogenic differentiation. These cells can serve as tools for better understanding of in vitro hPSC myogenesis and enriching myogenic cells for downstream analysis. For complete details on the use and execution of this protocol, please refer to Xi et al. (2017) and Xi et al. (2020).

Using Photovoice to Improve Healthy Eating for Children Participating in an Obesity Prevention Program.

Background: Children have benefited from participation in obesity prevention programs. Aims: The objectives of this study were to evaluate the impact of a healthy eating intervention for children in after-school programs and to use photovoice to document change in eating behavior. Methods: Forty-two children in three after-school programs participated. Children participated in lessons from an existing program to learn about healthy eating. A mixed methods study was conducted, using surveys to assess parent and child perceptions, and photovoice to capture children's perceptions of how they and their family changed eating habits. Member-checking was used to verify themes in the data. Twenty parents completed surveys evaluating the program. Results: Findings indicated that children learned program information, were interested in eating healthier (more fruits and vegetables), and quantitative data revealed there was a pre-post trend for eating more fruits at home. They reported that using the photovoice method helped them to monitor their behavior and helped them improve personal and family eating habits. Member checking confirmed themes. A majority of parents were satisfied with the program and reported that their children were discussing what they learned at home. Conclusions: Findings suggested that the photovoice methodology helped children to change in a positive way, increasing their agency in improving their own health and that of their family. Assessing longitudinal change in attitudes about healthy eating and eating behaviors will provide information about whether children maintain gains in knowledge and healthy eating over time.

Systems Biology Approach Identifies Prognostic Signatures of Poor Overall Survival and Guides the Prioritization of Novel BET-CHK1 Combination Therapy for Osteosarcoma.

Osteosarcoma (OS) patients exhibit poor overall survival, partly due to copy number variations (CNVs) resulting in dysregulated gene expression and therapeutic resistance. To identify actionable prognostic signatures of poor overall survival, we employed a systems biology approach using public databases to integrate CNVs, gene expression, and survival outcomes in pediatric, adolescent, and young adult OS patients. Chromosome 8 was a hotspot for poor prognostic signatures. The MYC-RAD21 copy number gain (8q24) correlated with increased gene expression and poor overall survival in 90% of the patients (n = 85). MYC and RAD21 play a role in replication-stress, which is a therapeutically actionable network. We prioritized replication-stress regulators, bromodomain and extra-terminal proteins (BETs), and CHK1, in order to test the hypothesis that the inhibition of BET + CHK1 in MYC-RAD21+ pediatric OS models would be efficacious and safe. We demonstrate that MYC-RAD21+ pediatric OS cell lines were sensitive to the inhibition of BET (BETi) and CHK1 (CHK1i) at clinically achievable concentrations. While the potentiation of CHK1i-mediated effects by BETi was BET-BRD4-dependent, MYC expression was BET-BRD4-independent. In MYC-RAD21+ pediatric OS xenografts, BETi + CHK1i significantly decreased tumor growth, increased survival, and was well tolerated. Therefore, targeting replication stress is a promising strategy to pursue as a therapeutic option for this devastating disease.

A Human Skeletal Muscle Atlas Identifies the Trajectories of Stem and Progenitor Cells across Development and from Human Pluripotent Stem Cells.

The developmental trajectory of human skeletal myogenesis and the transition between progenitor and stem cell states are unclear. We used single-cell RNA sequencing to profile human skeletal muscle tissues from embryonic, fetal, and postnatal stages. In silico, we identified myogenic as well as other cell types and constructed a "roadmap" of human skeletal muscle ontogeny across development. In a similar fashion, we also profiled the heterogeneous cell cultures generated from multiple human pluripotent stem cell (hPSC) myogenic differentiation protocols and mapped hPSC-derived myogenic progenitors to an embryonic-to-fetal transition period. We found differentially enriched biological processes and discovered co-regulated gene networks and transcription factors present at distinct myogenic stages. This work serves as a resource for advancing our knowledge of human myogenesis. It also provides a tool for a better understanding of hPSC-derived myogenic progenitors for translational applications in skeletal muscle-based regenerative medicine.

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond.

This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic strategies for multiple NMDs. Last, therapeutic considerations, including delivery and off-target effects, are also discussed.

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies.

Quality of Cardiac Care in Canada: Recommendations for Building a Sustainable Future.

Cardiovascular (CV) disease continues to present a significant disease and economic burden in Canada. To improve the quality of care and ensure sustainability of services, a national quality improvement initiative is required. The purpose of this analysis was to review the evidence for public reporting (PR) and external benchmarking (EB) to improve patient outcomes, and to recommend a strategy to improve CV care in Canada. To incorporate recent literature, the Canadian Cardiovascular Society (CCS) commissioned the Institute of Health Economics to provide a rapid update on the literature of PR and EB. The review showed that EB is more likely to promote positive effects, such as improved mortality, morbidity, and evidence-based clinical practice, and to limit negative effects, such as access restrictions or unintended provider behaviour associated with some forms of "top-down" PR. On the basis of these findings, this we recommend the following: (1) secure funding for the provincial collection of CV quality indicators and the creation of annual National CV Quality Reports; (2) enhance the culture of using CV quality indicator data for continuous quality improvement and opportunities for national or regional EB and sharing best practices; and (3) implement ongoing evaluation and revision of CCS clinical practice guidelines incorporating key quality indicators. This is already under way to a limited extent by the CCS with its Quality Project, but intentional, sustained support needs to be secured to enhance this ongoing effort and improve the quality of CV care for all Canadians.

ERBB3 and NGFR mark a distinct skeletal muscle progenitor cell in human development and hPSCs.

Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. We observed that hPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Using RNA sequencing, we found that the cell surface receptors ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of transforming growth factor-ß signalling during differentiation improved fusion efficiency, ultrastructural organization and the expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibres after engraftment of CRISPR-Cas9-corrected Duchenne muscular dystrophy human induced pluripotent stem cell-SMPCs. The work provides an in-depth characterization of human myogenesis, and identifies candidates that improve the in vivo myogenic potential of hPSC-SMPCs to levels that are equal to directly isolated human fetal muscle cells.

Recovery of metal values from copper slag and reuse of residual secondary slag.

Resource and environmental factors have become major forces in mining and metallurgy sectors driving research for sustainability purposes. The concept of zero-waste processing has been gaining ground readily. The scant availability of high quality raw materials has forced the researchers to shift their focus to recycling while the exceedingly stringent environmental regulations have forced researchers to explore new frontiers of minimizing/eliminating waste generation. The present work is aimed at addressing both aspects by employing recycling to generate wealth from copper slag and producing utilizable materials at the same time thus restoring the ecosystem. Copper slag was characterized and processed. The pyro-metallurgical processing prospects to generate utilizable materials were arrived at through rigorous thermodynamic analysis. Carbothermal reduction at elevated temperature (near 1440°C) helped recover a majority of the metal values (e.g., Fe, Cu and Mo) into the iron-rich alloy product which can be a feed material for steel making. On the other hand, the non-metallic residue, the secondary slag, can be used in the glass and ceramic industries. Reduction time and temperature and carbon content were shown to be the most important process variables for the reaction which were optimized to identify the most favored operating regime that maximizes the metal recovery and simultaneously maximizes the hardness of the secondary slag and minimizes its density, the two major criteria for the secondary slag product to be utilizable. The flux addition level was shown to have relatively less impact on the process performance if these are maintained at an adequate level. The work established that the copper slag, a waste material, can be successfully processed to generate reusable products through pyrometallurgical processing.

Creation of a Novel Humanized Dystrophic Mouse Model of Duchenne Muscular Dystrophy and Application of a CRISPR/Cas9 Gene Editing Therapy.

Duchenne muscular dystrophy is caused by mutations in DMD which disrupt the reading frame. Therapeutic strategies that restore DMD's reading frame, such as exon skipping and CRISPR/Cas9, need to be tested in the context of the human DMD sequence in vivo. We have developed a novel dystrophic mouse model by using CRISPR/Cas9 to delete exon 45 in the human DMD gene in hDMD mice, which places DMD out-of-frame. We have utilized this model to demonstrate that our clinically-relevant CRISPR/Cas9 platform, which targets deletion of human DMD exons 45-55, can be directly applied in vivo to restore dystrophin.

Why do caterpillars whistle at birds? Insect defence sounds startle avian predators.

Many insects produce sounds when attacked by a predator, yet the functions of these signals are poorly understood. It is debated whether such sounds function as startle, warning or alarm signals, or merely serve to augment other defences. Direct evidence is limited owing to difficulties in disentangling the effects of sounds from other defences that often occur simultaneously in live insects. We conducted an experiment to test whether an insect sound can function as a deimatic (i.e. startle) display. Variations of a whistle of the walnut sphinx caterpillar (Amorpha juglandis) were presented to a predator, red-winged blackbirds (Agelaius phoeniceus), when birds activated a sensor while feeding on mealworms (Tenebrio molitor). Birds exposed to whistles played back at natural sound levels exhibited significantly higher startle scores (by flying away, flinching, and hopping) and took longer to return to the feeding dish than during control conditions where no sounds were played. Birds habituated to sounds during a one-hour session, but after two days the startling effects were restored. Our results provide empirical evidence that an insect sound alone can function as a deimatic display against an avian predator. We discuss how whistles might be particularly effective 'acoustic eye spots' on avian predators.

Exon Skipping Therapy.

Exondys 51 is the first therapy for Duchenne muscular dystrophy (DMD) to have been granted accelerated approval by the FDA. Approval was granted based on using dystrophin expression as a surrogate marker. Exondys 51 targets DMD exon 51 for skipping to restore the reading frame for 13% of Duchenne patients.

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells.

Targeted genome editing technology can correct the sickle cell disease mutation of the ß-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the ß-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.