Effects of gene replacement therapy with resamirigene bilparvovec (AT132) on skeletal muscle pathology in X-linked myotubular myopathy: results from a substudy of the ASPIRO open-label clinical trial.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a rare, life-threatening congenital muscle disease caused by mutations in the MTM1 gene that result in profound muscle weakness, significant respiratory insufficiency, and high infant mortality. There is no approved disease-modifying therapy for XLMTM. Resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) is an investigational adeno-associated virus (AAV8)-mediated gene replacement therapy designed to deliver MTM1 to skeletal muscle cells and achieve long-term correction of XLMTM-related muscle pathology. The clinical trial ASPIRO (NCT03199469) investigating resamirigene bilparvovec in XLMTM is currently paused while the risk:benefit balance associated with this gene therapy is further investigated. METHODS: Muscle biopsies were taken before treatment and 24 and 48 weeks after treatment from ten boys with XLMTM in a clinical trial of resamirigene bilparvovec (ASPIRO; NCT03199469). Comprehensive histopathological analysis was performed. FINDINGS: Baseline biopsies uniformly showed findings characteristic of XLMTM, including small myofibres, increased internal or central nucleation, and central aggregates of organelles. Biopsies taken at 24 weeks post-treatment showed marked improvement of organelle localisation, without apparent increases in myofibre size in most participants. Biopsies taken at 48 weeks, however, did show statistically significant increases in myofibre size in all nine biopsies evaluated at this timepoint. Histopathological endpoints that did not demonstrate statistically significant changes with treatment included the degree of internal/central nucleation, numbers of triad structures, fibre type distributions, and numbers of satellite cells. Limited (predominantly mild) treatment-associated inflammatory changes were seen in biopsy specimens from five participants. INTERPRETATION: Muscle biopsies from individuals with XLMTM treated with resamirigene bilparvovec display statistically significant improvement in organelle localisation and myofibre size during a period of substantial improvements in muscle strength and respiratory function. This study identifies valuable histological endpoints for tracking treatment-related gains with resamirigene bilparvovec, as well as endpoints that did not show strong correlation with clinical improvement in this human study. FUNDING: Astellas Gene Therapies (formerly Audentes Therapeutics, Inc.).

Experimental phase diagram and its temporal evolution for submicron 2-methylglutaric acid and ammonium sulfate aerosol particles.

Liquid-liquid phase separation (LLPS) in aerosol particles is important for the climate system due to its potential to impact heterogeneous chemistry, cloud condensation nuclei, and new particle growth. Our group and others have shown a lower separation relative humidity for submicron particles, but whether the suppression is due to thermodynamics or kinetics is unclear. Herein, we characterize the experimental LLPS phase diagram of submicron 2-methylglutaric acid and ammonium sulfate aerosol particles and compare it to that of supermicron-sized particles. Surprisingly, as the equilibration time of submicron-sized aerosol particles was increased from 20 min to 60 min, the experimental phase diagram converges with the results for supermicron-sized particles. Our findings indicate that nucleation kinetics are responsible for the observed lower separation relative humidities in submicron aerosol particles. Therefore, experiments and models that investigate atmospheric processes of organic aerosol particles may need to consider the temporal evolution of aerosol LLPS.

Aberrations in Energetic Metabolism and Stress-Related Pathways Contribute to Pathophysiology in the Neb Conditional Knockout Mouse Model of Nemaline Myopathy.

Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed on the basis of the presence of nemaline rods on skeletal muscle biopsy. Although NM has typically been classified by causative genes, disease severity or prognosis cannot be predicted. The common pathologic end point of nemaline rods (despite diverse genetic causes) and an unexplained range of muscle weakness suggest that shared secondary processes contribute to the pathogenesis of NM. We speculated that these processes could be identified through a proteome-wide interrogation using a mouse model of severe NM in combination with pathway validation and structural/functional analyses. A proteomic analysis was performed using skeletal muscle tissue from the Neb conditional knockout mouse model compared with its wild-type counterpart to identify pathophysiologically relevant biological processes that might impact disease severity or provide new treatment targets. A differential expression analysis and Ingenuity Pathway Core Analysis predicted perturbations in several cellular processes, including mitochondrial dysfunction and changes in energetic metabolism and stress-related pathways. Subsequent structural and functional studies demonstrated abnormal mitochondrial distribution, decreased mitochondrial respiratory function, an increase in mitochondrial transmembrane potential, and extremely low ATP content in Neb conditional knockout muscles relative to wild type. Overall, the findings of these studies support a role for severe mitochondrial dysfunction as a novel contributor to muscle weakness in NM.

Different Mouse Models of Nemaline Myopathy Harboring Acta1 Mutations Display Differing Abnormalities Related to Mitochondrial Biology.

ACTA1 encodes skeletal muscle-specific α-actin, which polymerizes to form the thin filament of the sarcomere. Mutations in ACTA1 are responsible for approximately 30% of nemaline myopathy (NM) cases. Previous studies of weakness in NM have focused on muscle structure and contractility, but genetic issues alone do not explain the phenotypic heterogeneity observed in patients with NM or NM mouse models. To identify additional biological processes related to NM phenotypic severity, proteomic analysis was performed using muscle protein isolates from wild-type mice in comparison to moderately affected knock-in (KI) Acta1H40Y and the minimally affected transgenic (Tg) ACTA1D286G NM mice. This analysis revealed abnormalities in mitochondrial function and stress-related pathways in both mouse models, supporting an in-depth assessment of mitochondrial biology. Interestingly, evaluating each model in comparison to its wild-type counterpart identified different degrees of mitochondrial abnormality that correlated well with the phenotypic severity of the mouse model. Muscle histology, mitochondrial respiration, electron transport chain function, and mitochondrial transmembrane potential were all normal or minimally affected in the TgACTA1D286G mouse model. In contrast, the more severely affected KI.Acta1H40Y mice displayed significant abnormalities in relation to muscle histology, mitochondrial respirometry, ATP, ADP, and phosphate content, and mitochondrial transmembrane potential. These findings suggest that abnormal energy metabolism is related to symptomatic severity in NM and may constitute a contributor to phenotypic variability and a novel treatment target.

Barriers to PrEP uptake among Black female adolescents and emerging adults.

HIV/AIDS disproportionately impacts Black cisgender female adolescents and emerging adults. Pre-Exposure Prophylaxis (PrEP) reduces the risk of HIV infection; however, structural barriers may exacerbate resistance to PrEP in this population. The purpose of this paper is to understand the characteristics of age, race, gender, history, and medical mistrust as barriers to PrEP uptake among Black female adolescents and emerging adults (N = 100 respondents) between the ages of 13-24 years in Chicago. Between January and June of 2019, participants completed the survey. We used directed content analysis to examine reported barriers to PrEP uptake. The most commonly identified barriers to PrEP uptake were side effects (N = 39), financial concerns (N = 15), and medical mistrust (N = 12). Less frequently reported barriers included lack of PrEP knowledge and misconceptions (N = 9), stigma (N = 2), privacy concerns (N = 4). We describe innovative multi-level strategies to provide culturally safe care to improve PrEP acceptability among Black female adolescents and emerging adults in Chicago. These recommendations may help mitigate the effect of medical mistrust, stigma, and misconceptions of PrEP within Black communities.

Knowledgeable, aware / interested: Young black women's perceptions of pre-exposure prophylaxis.

Purpose: HIV in the United States disproportionately affects young Black women. Pre-exposure prophylaxis (PrEP) is an effective HIV prevention option that has the potential to reduce HIV incidence among HIV-vulnerable populations. However, data regarding women's awareness, interest in starting, and feelings of acceptability or stigma about PrEP remains limited, particularly among adolescent and young Black women. Materials and methods: A cross-sectional survey was conducted with 100 sexually active young Black women ages 13-24 years attending women's health clinics in Chicago, IL. Descriptive statistics were used to characterize the sample and determine more about what the PrEP needs and barriers are in this community. Associations were modeled using logistic regression and 95% confidence intervals for both bivariate and multivariable models. Results: In our survey (N = 100), half of study participants (50%) expressed interest in starting PrEP in the next three months and a majority (80%) of young women were confident they could obtain PrEP. Pregnant young women were significantly more interested in starting PrEP than non-pregnant women [OR 2.3 95% CI (1.0, 5.4)], p = 0.05), however, this association did not remain significant in adjusted models. Conclusions: This study provides a more complete understanding of awareness, interest in, and acceptability of PrEP among adolescent and young Black women attending women's health clinics. Findings indicate sustained interest in starting PrEP, reduced stigma, and increased awareness of PrEP among young Black women. These findings suggest that integrating PrEP into women's health clinics is a promising strategy to increase awareness and utilization of PrEP and decrease HIV transmission among youth at highest risk.

Sexual Activity Screening and Referral to Gynecology Services among Adolescent Women with Chronic Disease in the Pediatric Subspecialty Setting.

STUDY OBJECTIVE: Medically complex adolescent girls have significantly increased risk of disease exacerbation from pregnancy, and their use of teratogenic medications poses a risk to a developing fetus. Pediatric subspecialists have an opportunity to screen for sexual activity and refer to gynecology if indicated. METHODS: Subspecialist pediatricians (n = 39) completed a survey of their screening and referral practices. A retrospective chart review of a proportion of visits by these providers in the previous 6 months (n = 222) was conducted to compare documented sexual activity, contraception counseling, and referral practices with self-report data. RESULTS: Less than half of providers reported routinely asking about sexual activity (46%), whereas 69% reported routinely referring sexually active adolescents to gynecology. Documentation indicated 11 instances of sexual activity screening (4.9%) and 7 referrals (2.7%). CONCLUSION: Despite frequent contact with the medical field and use of teratogenic medications, medically complex adolescent girls are inconsistently screened for sexual activity and are rarely referred to gynecology.

An mHealth Intervention to Improve Pre-Exposure Prophylaxis Knowledge Among Young Black Women in Family Planning Clinics: Development and Usability Study.

BACKGROUND: Young Black women between the ages of 18 and 24 years are disproportionately impacted by HIV, yet they have a low self-perception of HIV risk and limited exposure to prevention strategies. Pre-exposure prophylaxis (PrEP) is a safe and effective biomedical HIV prevention strategy for those at risk for HIV infection, but uptake has been slow among cisgender women. Family planning clinics are a primary source of health care access for young women, providing an ideal opportunity to integrate PrEP information and care into existing clinic practices. OBJECTIVE: The aim of this study was to use a multistage, community-engaged process to develop a mobile health app and to evaluate the feasibility and acceptability of the app. METHODS: Using user-centered design, the In the Loop app was developed in collaboration with a community advisory board of young Black women. This study employed a multistage design, which included community-engaged app development, user testing, and evaluation of the app's feasibility and acceptability. A pre- and postdesign was used to assess the impact of the app on PrEP knowledge immediately after app use. Descriptive statistics (eg, mean, SD, and percentage values) were used to describe the sample, and Wilcoxon matched-pairs signed-ranks test was used to detect changes in PrEP knowledge before and immediately after using the app. RESULTS: A total of 50 sexually active, young Black women, aged 18-24 (mean 21, SD 1.9) years, were enrolled in this study. Analysis comparing scores before and immediately after use of the app revealed a significant increase in PrEP content knowledge scores on a 7-item true or false scale (z=-6.04, P<.001). Overall, participants considered the In the Loop app feasible and acceptable to use while waiting for a family planning visit. The majority of participants (n=46, 92%) agreed that they would recommend In the Loop to friends to learn more about PrEP. Participants rated the overall quality of the app 4.3 on a 1-5 scale (1=very poor and 5=very good). Of 50 participants, 40 (80%) agreed that the app was easy to use, and 48 (96%) agreed that they found the information in the app easy to understand. Finally, 40 (80%) agreed that they had enjoyed using the app while waiting for their family planning visit. CONCLUSIONS: Our findings suggest that young Black women waiting for family planning visits found the In the Loop app to be feasible and acceptable. This study demonstrates the value of engaging young Black women in the app design process. As family planning clinics are a primary source of health care access for young women, they provide an ideal setting to integrate PrEP information and care into existing clinic practices. Next steps in the development of the In the Loop app include implementing user-suggested improvements and conducting efficacy testing in a randomized controlled trial to determine the app's impact on PrEP uptake.

The Effect of Immunomodulatory Treatments on Anti-Dystrophin Immune Response After AAV Gene Therapy in Dystrophin Deficient mdx Mice.

BACKGROUND: AAV-based gene therapy is an attractive approach to treat Duchenne muscular dystrophy (DMD) patients. Although the long-term consequences of a gene therapy approach for DMD are unknown, there is evidence in both DMD patients and animal models that dystrophin replacement by gene therapy leads to an anti-dystrophin immune response that is likely to limit the long-term use of these therapeutic strategies. OBJECTIVE: Our objective is to test whether the anti-dystrophin immune response is affected by immunomodulatory drugs in mdx mice after rAAV gene therapy. METHODS: mdx mice were treated with rAAV microdystrophin alone or in combination with immunomodulatory drugs. Dystrophin expression in skeletal muscle was assessed by mass spectrometry. Immune responses were assessed by immunophenotyping, western blot for anti-dystrophin antibodies and flow cytometry assays for antigen-specific T-cell cytokine expression. The impact on muscle was measured by grip strength assessment, in vivo torque, optical imaging for inflammation and H&E staining of sections to assess muscle damage. RESULTS: We found that AAV-9-microdystrophin gene therapy induced expression of microdystrophin, anti-dystrophin antibodies, and T-cell cytokine responses. Immunomodulatory treatments, rituximab and VBP6 completely abrogated the anti-dystrophin antibody response. Prednisolone, CTLA4-Ig, and eplerenone showed variable efficacy in blocking the anti-dystrophin immune response. In contrast, none of the drugs completely abrogated the antigen specific IFN-γ response. AAV-microdystrophin treatment significantly reduced inflammation in both forelimbs and hindlimbs, and the addition of prednisolone and VBP6 further reduced muscle inflammation. Treatment with immunomodulatory drugs, except eplerenone, enhanced the beneficial effects of AAV-microdystrophin therapy in terms of force generation. CONCLUSIONS: Our data suggest that AAV-microdystrophin treatment results in anti-dystrophin antibody and T-cell responses, and immunomodulatory treatments have variable efficacy on these responses.

Use of Transmission Electron Microscopy for Analysis of Aerosol Particles and Strategies for Imaging Fragile Particles.

For over 25 years, transmission electron microscopy (TEM) has provided a method for the study of aerosol particles with sizes from below the optical diffraction limit to several microns, resolving the particles as well as smaller features. The wide use of this technique to study aerosol particles has contributed important insights about environmental aerosol particle samples and model atmospheric systems. TEM produces an image that is a 2D projection of aerosol particles that have been impacted onto grids and, through associated techniques and spectroscopies, can contribute additional information such as the determination of elemental composition, crystal structure, and 3D particle structures. Soot, mineral dust, and organic/inorganic particles have all been analyzed using TEM and spectroscopic techniques. TEM, however, has limitations that are important to understand when interpreting data including the ability of the electron beam to damage and thereby change the structure and shape of particles, especially in the case of particles composed of organic compounds and salts. In this paper, we concentrate on the breadth of studies that have used TEM as the primary analysis technique. Another focus is on common issues with TEM and cryogenic-TEM. Insights for new users on best practices for fragile particles, that is, particles that are easily susceptible to damage from the electron beam, with this technique are discussed. Tips for readers on interpreting and evaluating the quality and accuracy of TEM data in the literature are also provided and explained.

Dynamics of Liquid-Liquid Phase Separation in Submicrometer Aerosol.

Nanoscale materials, when compared to their bulk components, possess unique properties. In particular, shifts in phase transitions can occur for submicrometer particles. For instance, small particles do not undergo the process of liquid-liquid phase separation (LLPS). LLPS has applications in emulsions such as Janus particles, controllable morphology to create drug-rich phases during drug delivery, and is often observed in atmospheric aqueous aerosol particles. In atmospheric particles, LLPS is tracked as a function of particle water activity, which is equivalent to the relative humidity (RH) at equilibrium. We probed three organic/inorganic aerosol systems in the range of RH over which phase separation occurs (SRH). Our findings indicate that SRH for submicrometer aerosol particles is lower than for micrometer-sized droplets. These findings show that it may be necessary to update the representation of phase transitions in aerosol particles in climate models. The vast majority of organic/inorganic aerosol particles have submicrometer diameters, and a decrease in SRH for submicrometer particles indicates that the current estimation of phase-separated aerosols may be overestimated. Furthermore, understanding the properties of LLPS at the nanoscale can provide key parameters to describe these systems and may lead to better control of phase separation in submicrometer particles.

Inhibition of Phase Separation in Aerosolized Water-Soluble Polymer-Polymer Nanoparticles at Small Sizes and the Effects of Molecular Weight.

The effects of confinement on the phase separation behavior of polymer-polymer mixtures have been frequently studied in morphologies such as thin films and rods, but little research exists with respect to the nanoscale droplet size regime. This paper addresses the phase separation of water-soluble polymers in submicron aerosol droplets. Atomized aerosol particles were prepared from aqueous solutions and dried using diffusion dryers. For poly(ethylene) glycol/dextran and poly(vinyl alcohol)/poly(4-styrene sulfonic acid) systems, small particles remain homogeneous, while larger particles undergo phase separation within a single particle. As the molecular weight of the polymers increases while a constant ratio between monomers of polymers A and B is maintained, phase separation occurs in smaller diameter particles. These trends are modeled using a combination of equations describing the nucleation of a new phase and the Flory-Huggins theory and provide qualitative agreement. These results provide insight into the phase separation of aqueous nanoscale polymer-polymer systems. Potential exists to make new polymer materials with unique properties due to the mixing of polymer combinations that normally undergo phase separation.

Flash Freeze Flow Tube to Vitrify Aerosol Particles at Fixed Relative Humidity Values.

Development of methods to measure the phase transitions and physical properties of submicron atmospheric aerosol particles is needed to better model these systems. In this paper, we present a method to flash freeze submicron particles to measure phase transitions as a function of relative humidity (RH). Particles are equilibrated at a fixed RH, vitrified in a temperature-controlled flow tube, and imaged with cryogenic transmission electron microscopy (cryo-TEM). We demonstrate the use of the technique for measuring the efflorescence relative humidity (ERH) of potassium sulfate and potassium chloride aerosol as well as the separation RH (SRH) for a multicomponent organic/inorganic system that undergoes liquid-liquid phase separation (LLPS). The location of phase transitions can shift between the micrometer and nanometer size regimes, and particles in a given population may have a range of RH over which a phase transition occurs. This technique addresses these requirements by allowing for characterization of the phase transitions for individual particles in a population on the submicron scale.

Awareness and Intent to Use Pre-exposure Prophylaxis (PrEP) Among African American Women in a Family Planning Clinic.

Due to the gap between cisgender women eligible for and those accessing pre-exposure prophylaxis (PrEP) for HIV prevention, it is critical to understand knowledge of and attitudes toward PrEP among HIV-vulnerable women. PrEP utilization is particularly low among African American women in the USA. Family planning clinics provide key access points to reach HIV-vulnerable African American women as well as to translate research findings into clinical practice. Our study aimed to (1) describe the awareness of and interest in PrEP among African American cisgender women attending a family planning clinic and (2) document the barriers and facilitators to PrEP uptake among these women. A cross-sectional survey was conducted with sexually active African American women of reproductive age attending a family planning clinic. Descriptive statistics were used to characterize the sample, and bivariate analysis was used to detect difference between categorical and outcome variables. In our survey (N = 109), over 80% of participants listed not knowing PrEP was available as the primary reason for not currently taking PrEP. Seventy percent reported they would probably or definitely like to take PrEP - demonstrating that barriers to uptake might stem from knowledge deficits rather than attitudes toward prevention. Study findings have the potential to inform strategies to increase awareness of PrEP as an HIV prevention option as well as to equip women with greater self-efficacy to access PrEP in family planning settings.

Physical activity and screen-viewing policies in Canadian childcare centers.

BACKGROUND: Physical activity (PA) offers numerous health benefits for young children; however, many children enrolled in childcare engage in low levels of PA and high levels of sedentary time. This study aimed to describe the prevalence and content of written PA and screen-viewing (SV) policies in Canadian childcare centers. METHODS: Using a modified version of the Environment and Policy Assessment and Observation Self-Report (EPAO-SR) tool, an online survey was distributed to all directors/administrators of center-based childcare facilities across Canada. Reminder emails were sent to encourage survey completion and a strong response rate. Descriptive statistics were used to explore demographic characteristics and frequencies were run to examine the number of centers that implemented a PA or SV policy. Deductive content analysis was completed to identify common themes in participants' open-ended responses. RESULTS: A total of 1158 childcare representatives participated in the study; 514 provided complete data. Of these, 295 (44%) centers indicated having a written PA policy (with the majority regulated at the provincial/territorial-level; n = 227; 42%). Content of these policies included amount of time: spent outdoors (n = 395; 63%); in teacher-led active play (n = 101; 16%); and PA education for children (n = 91; 16%). Additionally, 178 (29%) respondents reported a written policy regarding SV (with majority regulated at the center-level; n = 173; 34%) and primarily focusing on amount of time children watch television. CONCLUSIONS: PA regulations are more common than SV policies in Canadian childcare centers; however, less than half implement a PA policy and only a third adopt SV regulations. An opportunity exists to advance practice by adopting proactive approaches to encouraging young children to be more active and less sedentary in childcare (i.e., through written policies). Supplementing policy with accessible resources, as well as consistent provision of early childhood educator (staff) training, represent important steps for putting said policies into action.

Role of pH in Aerosol Processes and Measurement Challenges.

pH is one of the most basic chemical properties of aqueous solution, but its measurement in nanoscale aerosol particles presents many challenges. The pH of aerosol particles is of growing interest in the atmospheric chemistry community because of its demonstrated effects on heterogeneous chemistry and human health, as well as potential effects on climate. The authors have shown that phase transitions of aerosol particles are sensitive to pH, focusing on systems that undergo liquid-liquid phase separation. Currently, aerosol pH is calculated indirectly from knowledge of species present in the gas and aerosol phases through the use of thermodynamic models. From these models, ambient aerosol is expected to be highly acidic (pH ∼ 0-3). Direct measurements have focused on model systems due to the difficulty of this measurement. This area is one in which physical chemists should be encouraged to contribute because of the potential consequences for aerosol processes in the environment.

Effects of High Acidity on Phase Transitions of an Organic Aerosol.

Aerosol particle morphology influences the effect of particles on climate. Recent studies have documented the high acidity found in many ambient aerosol particles. The effect of this acidity on the phase transitions of mixed organic/inorganic aerosol particles has not been addressed. To investigate this effect, six organic compounds and ammonium sulfate were investigated individually with varying amounts of sulfuric acid to determine the role of low pH in the separation, efflorescence, and deliquescence transitions. All phase transitions were affected by the addition of sulfuric acid. This effect was attributed primarily to the change in the identity of the inorganic component as the ammonium/sulfate ratio (ASR) was changed from 2 to 1.5 to 1. The separation relative humidity (SRH) decreased with increasing amounts of sulfuric acid for each system studied, with the largest effect seen in compounds that have a lower SRH when mixed with ammonium sulfate. Control studies without an inorganic salt revealed that for some systems, phase separation occurs for mixtures of sulfuric acid and an organic acid. Overall, it was found that for aerosol particles at low pH (≤0.35) composed of organic acids and inorganic salts, phase separation can be impeded in some cases.

Balancing the Ecological Function of Residential Stormwater Ponds with Homeowner Landscaping Practices.

Stormwater ponds are installed in urban developments to provide the ecosystem services of flood control and water treatment. In coastal areas, these ponds are connected to watersheds that can drain directly into protected estuaries, making their design, function, and maintenance critical to environmental protection. However, stormwater ponds in residential areas are increasingly managed as aesthetic amenities that add value to real estate rather than as engineered devices with special maintenance requirements. To help extend the life of neighborhood stormwater systems and improve ecosystem services, homeowners should follow best management practices for nutrient management and add shoreline plantings and non-invasive, beneficial aquatic plants to their ponds. This study used focus group and survey research to document the knowledge, behaviors, and attitudes of homeowners living near stormwater ponds in a master-planned community in Florida. The study was designed to use a social marketing research approach to promote Extension best practices. Findings indicate that many residents were aware of the functional components of stormwater systems and respondents' receptivity to best management practices was mediated by age, their attitudes about water quality and whether their home was adjacent to a pond. These findings can be used to target Extension audiences and improve adoption of stormwater pond best management practices for increased protection of water quality.