Age and donor site do not affect cell growth and biologic activity in Autologous Tendon-cell Implantation (OrthoATI™) for treatment of patellar tendon and palmaris longus tendon.

OBJECTIVES: Autologous tendon cell implantation (OrthoATI™) therapy has demonstrated efficacy in treating patients with tendinopathy at various anatomical sites. This study evaluates the effect of patient age, gender and tendon biopsy site on morphology, growth and gene expression of autologous tendon cells used to treat chronic tendinopathy. METHODS: Patients undergoing OrthoATI™ for tendinopathies between 2020 and 2022 were initially treated by biopsies taken from patella tendon (PT) or palmaris longus tendon (PL). Autologous tenocytes were treated at a Good Manufacturing Practice (GMP) cell laboratory where they were isolated, cultured and expanded for four to six weeks. Cell morphology was assessed using phase contrast microscopy. Droplet digital PCR (ddPCR) was utilised for gene expression analysis. Dichotomous results were compared between groups using x2 or Fisher's exact tests with no adjustment for multiple comparisons. The non-parametric Mann-Whitney U and Kruskal-Wallis tests were utilised for the sex and age (<35y, 35-44y, 45-54y, >55y) analyses respectively. All analyses were performed using IBM SPSS v27, and a two-tailed P-value of <0.05 was considered statistically significant. RESULTS: 149 patients were included in the analysis. The PT was biopsied in 63 patients, and PL in 86 patients. There were no observer effects for age and gender between PT and PL groups. There was no statistical significance between the PT and PL tendons for cell morphology, average cell population doubling time (PDT) (PT 83.9 vs PL 82.7 hours, p=0.482), cellular yield (PT 16.2 vs PL 15.2×106 , p=0.099), and cell viability (PT 98.7 vs PL 99.0%, p=0.277). Additionally, ddPCR analyses showed no statistical significance found in tenogenic gene expression including collagen type I (COL1, p=0.86), tenomodulin (TNMD, p=0.837) and scleraxis (SCX, p=0.331) between PT- and PL-derived tendon cells. An age stratification analysis found no effect on growth and gene expression. COL1 was found to be higher in males when compared to females (P<0.001), but otherwise no difference was seen in growth and gene expression in the gender analysis. No post-biopsy clinical complications were reported for either group. CONCLUSION: This study has shown that the growth and bioactivities of tendon cells from tendon biopsies for OrthoATI™ are not affected by tendon donor site and age. LEVEL OF EVIDENCE: IV.

Self-Assembled STING-Activating Coordination Nanoparticles for Cancer Immunotherapy and Vaccine Applications.

The cGAS-STING pathway plays a crucial role in innate immune activation against cancer and infections, and STING agonists based on cyclic dinucleotides (CDN) have garnered attention for their potential use in cancer immunotherapy and vaccines. However, the limited drug-like properties of CDN necessitate an efficient delivery system to the immune system. To address these challenges, we developed an immunostimulatory delivery system for STING agonists. Here, we have examined aqueous coordination interactions between CDN and metal ions and report that CDN mixed with Zn2+ and Mn2+ formed distinctive crystal structures. Further pharmaceutical engineering led to the development of a functional coordination nanoparticle, termed the Zinc-Mn-CDN Particle (ZMCP), produced by a simple aqueous one-pot synthesis. Local or systemic administration of ZMCP exerted robust antitumor efficacy in mice. Importantly, recombinant protein antigens from SARS-CoV-2 can be simply loaded during the aqueous one-pot synthesis. The resulting ZMCP antigens elicited strong cellular and humoral immune responses that neutralized SARS-CoV-2, highlighting ZMCP as a self-adjuvant vaccine platform against COVID-19 and other infectious pathogens. Overall, this work establishes a paradigm for developing translational coordination nanomedicine based on drug-metal ion coordination and broadens the applicability of coordination medicine for the delivery of proteins and other biologics.

Eligibility and Potential Benefit of Transcatheter Edge-to-Edge Repair in a Contemporary Cohort With Heart Failure: Evidence From a Large Integrated Health Care Delivery System.

Background: The eligibility and potential benefit of transcatheter edge-to-edge repair (TEER) in addition to guideline-directed medical therapy to treat moderate-severe or severe secondary mitral regurgitation (MR) has not been reported in a contemporary heart failure (HF) population. Methods: Eligibility for TEER based on Food and Drug Administration (FDA) labeling: (1) HF symptoms, (2) moderate-severe or severe MR, (3) left ventricular ejection fraction (LVEF) 20% to 50%, (4) left ventricular end-systolic dimension 7.0 cm, and (5) receiving GDMT (blocker + angiotensin-converting enzyme inhibitor/angiotensin receptor blocker). The proportion (%) of patients eligible for TEER. The hypothetical number needed to treat to prevent or postpone adverse outcomes was estimated using relative risk reductions from published hazard ratios in the registration trial and the observed event rates. Results: We identified 50,841 adults with HF and known LVEF. After applying FDA criteria, 2461 patients (4.8%) were considered eligible for transcatheter mitral valve replacement (FDA+), with the vast majority of patients excluded (FDA-) based on a lack of clinically significant MR (N = 47,279). FDA+ patients had higher natriuretic peptide levels and were more likely to have a prior HF hospitalization compared to FDA- patients. Although FDA+ patients had a more dilated left ventricle and lower LVEF, median (25th-75th) left ventricular end-systolic dimension (cm) was low at 4.4 (3.7-5.1) and only 30.8% had severely reduced LVEF. FDA+ patients were at higher risk of HF-related morbidity and mortality. The estimated number needed to treat to potentially prevent or postpone all-cause hospitalization was 4.4, 8.8 for HF hospitalization, and 5.3 for all-cause death at 24 months in FDA+ patients. Conclusions: There is a low prevalence of TEER eligibility based on FDA criteria primarily due to absence of moderate-severe or severe MR. FDA+ patients are a high acuity population and may potentially derive a robust clinical benefit from TEER based on pivotal studies. Additional research is necessary to validate the scope of eligibility and comparative effectiveness of TEER in real-world populations.

Mechanical overload-induced release of extracellular mitochondrial particles from tendon cells leads to inflammation in tendinopathy.

Tendinopathy is one of the most common musculoskeletal diseases, and mechanical overload is considered its primary cause. However, the underlying mechanism through which mechanical overload induces tendinopathy has not been determined. In this study, we identified for the first time that tendon cells can release extracellular mitochondria (ExtraMito) particles, a subtype of medium extracellular particles (mEPs), into the environment through a process regulated by mechanical loading. RNA sequencing systematically revealed that oxygen-related reactions, extracellular particles, and inflammation were present in diseased human tendons, suggesting that these factors play a role in the pathogenesis of tendinopathy. We simulated the disease condition by imposing a 9% strain overload on three-dimensional mouse tendon constructs in our cyclic uniaxial stretching bioreactor. The three-dimensional mouse tendon constructs under normal loading with 6% strain exhibited an extended mitochondrial network, as observed through live-cell confocal laser scanning microscopy. In contrast, mechanical overload led to a fragmented mitochondrial network. Our microscopic and immunoblot results demonstrated that mechanical loading induced tendon cells to release ExtraMito particles. Furthermore, we showed that mEPs released from tendon cells overloaded with a 9% strain (mEP9%) induced macrophage chemotaxis and increased the production of proinflammatory cytokines, including IL-6, CXCL1, and IL-18, from macrophages compared to mEP0%, mEP3%, and mEP6%. Partial depletion of the ExtraMito particles from mEP9% by magnetic-activated cell sorting significantly reduced macrophage chemotaxis. N-acetyl-L-cysteine treatment preserved the mitochondrial network in overloaded tendon cells, diminishing overload-induced macrophage chemotaxis toward mEP9%. These findings revealed a novel mechanism of tendinopathy; in an overloaded environment, ExtraMito particles convey mechanical response signals from tendon cells to the immune microenvironment, culminating in tendinopathy.

Pilot study of paediatric regional lung function assessment via X-ray velocimetry (XV) imaging in children with normal lungs and in children with cystic fibrosis.

INTRODUCTION: Cystic fibrosis (CF) is a life-limiting autosomal recessive genetic condition. It is caused by mutations in the gene that encodes for a chloride and bicarbonate conducting transmembrane channel. X-ray velocimetry (XV) is a novel form of X-ray imaging that can generate lung ventilation data through the breathing cycle. XV technology has been validated in multiple animal models, including the ß-ENaC mouse model of CF lung disease. It has since been assessed in early-phase clinical trials in adult human subjects; however, there is a paucity of data in the paediatric cohort, including in CF. The aim of this pilot study was to investigate the feasibility of performing a single-centre cohort study in paediatric patients with CF and in those with normal lungs to demonstrate the appropriateness of proceeding with further studies of XV in these cohorts. METHODS AND ANALYSIS: This is a cross-sectional, single-centre, pilot study. It will recruit children aged 3-18 years to have XV lung imaging performed, as well as paired pulmonary function testing. The study will aim to recruit 20 children without CF with normal lungs and 20 children with CF. The primary outcome will be the feasibility of recruiting children and performing XV testing. Secondary outcomes will include comparisons between XV and current assessments of pulmonary function and structure. ETHICS AND DISSEMINATION: This project has ethical approval granted by The Women's and Children's Hospital Human Research Ethics Committee (HREC ID 2021/HRE00396). Findings will be disseminated through peer-reviewed publication and conferences. TRIAL REGISTRATION NUMBER: ACTRN12623000109606.

Type I interferon signaling induces a delayed antiproliferative response in respiratory epithelial cells during SARS-CoV-2 infection.

ABSTRACT: Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain unclear. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top four genes identified in our screen encode components of the same type I interferon (IFN-I) signaling complex­IFNAR1, IFNAR2, JAK1, and TYK2. The fifth gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response in both Calu-3 cells and iPSC-derived type 2 alveolar epithelial cells. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

Developing Clinical Risk Prediction Models for Worsening Heart Failure Events and Death by Left Ventricular Ejection Fraction.

Background There is a need to develop electronic health record-based predictive models for worsening heart failure (WHF) events across clinical settings and across the spectrum of left ventricular ejection fraction (LVEF). Methods and Results We studied adults with heart failure (HF) from 2011 to 2019 within an integrated health care delivery system. WHF encounters were ascertained using natural language processing and structured data. We conducted boosted decision tree ensemble models to predict 1-year hospitalizations, emergency department visits/observation stays, and outpatient encounters for WHF and all-cause death within each LVEF category: HF with reduced ejection fraction (EF) (LVEF <40%), HF with mildly reduced EF (LVEF 40%-49%), and HF with preserved EF (LVEF ≥50%). Model discrimination was evaluated using area under the curve and calibration using mean squared error. We identified 338 426 adults with HF: 61 045 (18.0%) had HF with reduced EF, 49 618 (14.7%) had HF with mildly reduced EF, and 227 763 (67.3%) had HF with preserved EF. The 1-year risks of any WHF event and death were, respectively, 22.3% and 13.0% for HF with reduced EF, 17.0% and 10.1% for HF with mildly reduced EF, and 16.3% and 10.3% for HF with preserved EF. The WHF model displayed an area under the curve of 0.76 and mean squared error of 0.13, whereas the model for death displayed an area under the curve of 0.83 and mean squared error of 0.076. Performance and predictors were similar across WHF encounter types and LVEF categories. Conclusions We developed risk prediction models for 1-year WHF events and death across the LVEF spectrum using structured and unstructured electronic health record data and observed no substantial differences in model performance or predictors except for death, despite differences in underlying HF cause.

Elevated plasma sclerostin is associated with high brain amyloid-ß load in cognitively normal older adults.

Osteoporosis and Alzheimer's disease (AD) mainly affect older individuals, and the possibility of an underlying link contributing to their shared epidemiological features has rarely been investigated. In the current study, we investigated the association between levels of plasma sclerostin (SOST), a protein primarily produced by bone, and brain amyloid-beta (Aß) load, a pathological hallmark of AD. The study enrolled participants meeting a set of screening inclusion and exclusion criteria and were stratified into Aß- (n = 65) and Aß+ (n = 35) according to their brain Aß load assessed using Aß-PET (positron emission tomography) imaging. Plasma SOST levels, apolipoprotein E gene (APOE) genotype and several putative AD blood-biomarkers including Aß40, Aß42, Aß42/Aß40, neurofilament light (NFL), glial fibrillary acidic protein (GFAP), total tau (t-tau) and phosphorylated tau (p-tau181 and p-tau231) were detected and compared. It was found that plasma SOST levels were significantly higher in the Aß+ group (71.49 ± 25.00 pmol/L) compared with the Aß- group (56.51 ± 22.14 pmol/L) (P < 0.01). Moreover, Spearman's correlation analysis showed that plasma SOST concentrations were positively correlated with brain Aß load (ρ = 0.321, P = 0.001). Importantly, plasma SOST combined with Aß42/Aß40 ratio significantly increased the area under the curve (AUC) when compared with using Aß42/Aß40 ratio alone (AUC = 0.768 vs 0.669, P = 0.027). In conclusion, plasma SOST levels are elevated in cognitively unimpaired older adults at high risk of AD and SOST could complement existing plasma biomarkers to assist in the detection of preclinical AD.

Examining barriers and facilitators in asthma inhaler technique education and technology-enhanced educational tools in children: A mixed methods evaluation using the theoretical domains framework.

INTRODUCTION: Educational interventions for asthma inhaler technique have been identified as successful in improving technique in children, yet inhaler technique has not improved over time. New approaches should be considered, including the use of technology-based interventions such as smartphone and tablet applications. Adoption and implementation of such technology in healthcare has been historically slow. This mixed-methods study aimed to identify the barriers and facilitators of delivering and receiving asthma inhaler education for children in a hospital setting, including technology-based interventions. METHODS: Children with asthma, their caregivers, and healthcare professionals who regularly provide asthma education, were invited to participate in a qualitative interview and brief questionnaire to describe their experiences, knowledge, beliefs, and recommendations about asthma education delivery. The Theoretical Domains Framework was used to develop questions for the semistructured moderator guide, questionnaire, and provide the rigorous evaluation framework for deductive thematic analysis. RESULTS: Sixteen interviews and questionnaires were conducted with participants. Overall, healthcare professionals perceived more barriers in asthma inhaler education delivery than asthmatic children and their caregivers to receiving the education. Healthcare professionals and caregivers identified time-pressures within a hospital setting as a barrier for providing sufficient education. However, all participants felt they had adequate knowledge in their asthma management skills and inhaler technique. Technology-based innovations were viewed positively by all participant groups to improve asthma education. CONCLUSIONS: Several barriers and facilitators to current hospital-based asthma education delivery were reported by target end-users. Future programs should consider these findings when developing asthma inhaler educational interventions, particularly those using technology-enhanced information delivery.

Co-design of an Augmented Reality Asthma Inhaler Educational Intervention for Children: Development and Usability Study.

BACKGROUND: Smartphone and tablet apps that deliver health care education have been identified as effective in improving patient knowledge and treatment adherence in asthma populations. Despite asthma being the most common chronic disease in pediatrics, there are few apps that are targeted specifically for children. Only half of children with asthma have acceptable control of their symptoms, and 40%-98% do not use their inhalers correctly. With children being increasingly connected to technology, there is an opportunity to improve asthma inhaler technique education by delivery via smartphone or tablet apps. Augmented reality (AR) technology was used in this study to capitalize on growing technological innovations. Digital health interventions that use a co-design process for development have the highest likelihood of successful uptake and effectiveness on their intended outcomes. Perceived usability also has been shown to improve the effectiveness of education as well as the acceptance of the intervention. OBJECTIVE: The aims of this study were to describe the co-design process, development, and design outcomes of a smartphone or tablet app that incorporates AR technology to deliver asthma inhaler technique education to children with asthma. This study also aimed to provide a usability evaluation, using the System Usability Scale to inform our work and future research, and recommendations for others performing similar work. METHODS: The development of the AR asthma inhaler technique education app was based on an iterative co-design process with likely end users (children with asthma, their caregivers, and health care professionals). This involved multiple stages: recruitment of end users for qualitative interviews and usability testing with a previously designed educational intervention, which used an AR-embedded smartphone or tablet app; ideation of content for a specific asthma inhaler technique education intervention with end users; development of the specific asthma inhaler intervention; and 2 further rounds of interviews and usability testing with the redesign of the initial prototype. RESULTS: We included 16 participants aged 9-45 years. Using the co-design process, the AR asthma inhaler technique education app was designed, incorporating the preferences of end users. After iteration 1, animation was included based on the feedback provided. Iteration 2 feedback resulted in increased AR experiences and the removal of the requirement of a paper-based resource to trigger AR in the third iteration. Throughout all rounds, the ease of use of the app and the novel nature of the intervention were frequently described. The usability of the intervention overall was perceived to be excellent, and the mean System Usability Scale score of the intervention was found to be highest in the final round of evaluation (90.14). CONCLUSIONS: The results from this co-design process and usability evaluation will be used to develop a final AR asthma inhaler technique educational intervention, which will be evaluated in the clinical setting. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1177/16094069211042229.

Safety and efficacy of AK0529 in respiratory syncytial virus-infected infant patients: A phase 2 proof-of-concept trial.

Background: Respiratory syncytial virus (RSV) infection is a cause of substantial morbidity and mortality in young children. There is currently no effective therapy available. Methods: This was a Phase 2 study of the oral RSV fusion protein inhibitor AK0529 in infants aged 1-24 months, hospitalized with RSV infection. In Part 1, patients (n = 24) were randomized 2:1 to receive a single dose of AK0529 up to 4 mg/kg or placebo. In Part 2, patients (n = 48) were randomized 2:1 to receive AK0529 at 0.5, 1, or 2 mg/kg bid or placebo for 5 days. Sparse pharmacokinetic samples were assessed using population pharmacokinetics modelling. Safety, tolerability, viral load, and respiratory signs and symptoms were assessed daily during treatment. Results: No safety or tolerability signals were detected for AK0529: grade ≥3 treatment-emergent adverse events occurring in 4.1% of patients in AK0529 and 4.2% in placebo groups, respectively, and none led to death or withdrawal from the study. In Part 2, targeted drug exposure was reached with 2 mg/kg bid. A numerically greater reduction in median viral load with 2 mg/kg bid AK0529 than with placebo at 96 h was observed. A -4.0 (95% CI: -4.51, -2.03) median reduction in Wang Respiratory Score from baseline to 96 h was observed in the 2 mg/kg group compared with -2.0 (95% CI: -3.42, -1.82) in the placebo group. Conclusions: AK0529 was well tolerated in hospitalized RSV-infected infant patients. Treatment with AK0529 2 mg/kg bid was observed to reduce viral load and Wang Respiratory Score. Clinical Trials Registration: NCT02654171.

Mixed Reality Technology to Deliver Psychological Interventions to Adolescents With Asthma: Qualitative Study Using the Theoretical Framework of Acceptability.

BACKGROUND: Interactive, mixed reality technologies such as augmented reality, virtual reality, and holographic technology may provide a novel solution to fast-track the translation of evidence into practice. They may also help overcome barriers to both mental health and asthma management service uptake, such as cost, availability of appointments, fear of judgment, and quality of care. OBJECTIVE: This study aimed to investigate if mixed reality technology is an acceptable mechanism for the delivery of a component of cognitive and behavioral therapies for the management of elevated psychological distress among young people with asthma. METHODS: To explore the perceived acceptability of these technologies, mixed reality tools were evaluated via qualitative, 1-on-1 interviews with young people with asthma and symptoms of psychological distress, parents/caregivers of young people with asthma and symptoms of psychological distress, and relevant health professionals. The Theoretical Framework of Acceptability was used for the deductive coding of the recorded interview transcripts. RESULTS: This study enrolled the following participants: (1) 3 adolescents with asthma and symptoms of psychological distress with a mean age of 14 (SD 1.7) years; (2) 4 parents/caregivers of adolescents with asthma with a mean age of 55 (SD 14.6) years; and (3) 6 health professionals with a mean age of 40.8 (SD 4.3) years. A total of 4 constructs-experienced affective attitude, experienced effectiveness, self-efficacy, and intervention coherence-were coded in all participant transcripts. The most frequently coded constructs were experienced affective attitude and intervention coherence, which were reported a total of 96 times. The least frequently coded construct was anticipated opportunity cost, which was reported a total of 5 times. Participants were mostly positive about the mixed reality resources. However, some concerns were raised regarding ethicality, particularly regarding privacy, accessibility, and messaging. Participants noted the need for technology to be used in conjunction with face-to-face engagement with health professionals and that some patients would respond to this type of delivery mechanism better than others. CONCLUSIONS: These results suggest that mixed reality technology to deliver psychological interventions may be an acceptable addition to current health care practices for young people with asthma and symptoms of psychological distress. TRIAL REGISTRATION: Australia and New Zealand Clinical Trials Registry ACTRN12620001109998; https://anzctr.org.au/Trial/Registration/TrialReview.aspx?id=380427.

Systematic evaluation of three porcine-derived collagen membranes for guided bone regeneration.

Guided bone regeneration is one of the most common surgical treatment modalities performed when an additional alveolar bone is required to stabilize dental implants in partially and fully edentulous patients. The addition of a barrier membrane prevents non-osteogenic tissue invasion into the bone cavity, which is key to the success of guided bone regeneration. Barrier membranes can be broadly classified as non-resorbable or resorbable. In contrast to non-resorbable membranes, resorbable barrier membranes do not require a second surgical procedure for membrane removal. Commercially available resorbable barrier membranes are either synthetically manufactured or derived from xenogeneic collagen. Although collagen barrier membranes have become increasingly popular amongst clinicians, largely due to their superior handling qualities compared to other commercially available barrier membranes, there have been no studies to date that have compared commercially available porcine-derived collagen membranes with respect to surface topography, collagen fibril structure, physical barrier property, and immunogenic composition. This study evaluated three commercially available non-crosslinked porcine-derived collagen membranes (Striate+TM, Bio-Gide® and CreosTM Xenoprotect). Scanning electron microscopy revealed similar collagen fibril distribution on both the rough and smooth sides of the membranes as well as the similar diameters of collagen fibrils. However, D-periodicity of the fibrillar collagen is significantly different among the membranes, with Striate+TM membrane having the closest D-periodicity to native collagen I. This suggests that there is less deformation of collagen during manufacturing process. All collagen membranes showed superior barrier property evidenced by blocking 0.2-16.4 µm beads passing through the membranes. To examine the immunogenic agents in these membranes, we examined the membranes for the presence of DNA and alpha-gal by immunohistochemistry. No alpha-gal or DNA was detected in any membranes. However, using a more sensitive detection method (real-time polymerase chain reaction), a relatively strong DNA signal was detected in Bio-Gide® membrane, but not Striate+TM and CreosTM Xenoprotect membranes. Our study concluded that these membranes are similar but not identical, probably due to the different ages and sources of porcine tissues, as well as different manufacturing processes. We recommend further studies to understand the clinical implications of these findings.

Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.

Type I interferon signaling induces a delayed antiproliferative response in Calu-3 cells during SARS-CoV-2 infection.

Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain poorly understood. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top 4 genes identified in our screen encode components of the same type I interferon signaling complex - IFNAR1, IFNAR2, JAK1, and TYK2. The 5th gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

STING agonist-loaded mesoporous manganese-silica nanoparticles for vaccine applications.

Cyclic dinucleotides (CDNs), as one type of Stimulator of Interferon Genes (STING) pathway agonist, have shown promising results for eliciting immune responses against cancer and viral infection. However, the suboptimal drug-like properties of conventional CDNs, including their short in vivo half-life and poor cellular permeability, compromise their therapeutic efficacy. In this study, we have developed a manganese-silica nanoplatform (MnOx@HMSN) that enhances the adjuvant effects of CDN by achieving synergy with Mn2+ for vaccination against cancer and SARS-CoV-2. MnOx@HMSN with large mesopores were efficiently co-loaded with CDN and peptide/protein antigens. MnOx@HMSN(CDA) amplified the activation of the STING pathway and enhanced the production of type-I interferons and other proinflammatory cytokines from dendritic cells. MnOx@HMSN(CDA) carrying cancer neoantigens elicited robust antitumor T-cell immunity with therapeutic efficacy in two different murine tumor models. Furthermore, MnOx@HMSN(CDA) loaded with SARS-CoV-2 antigen achieved strong and durable (up to one year) humoral immune responses with neutralizing capability. These results demonstrate that MnOx@HMSN(CDA) is a versatile nanoplatform for vaccine applications.

Short Message Service Reminder Nudge for Parents and Influenza Vaccination Uptake in Children and Adolescents With Special Risk Medical Conditions: The Flutext-4U Randomized Clinical Trial.

Importance: Children with chronic medical conditions are at increased risk of severe influenza. Uptake of influenza vaccination in children and adolescents with these identified special risk medical conditions (SRMCs) is suboptimal. Objective: To assess the effectiveness of Flutext-4U, a parent short message service (SMS) reminder nudge intervention, in increasing influenza immunization in children and adolescents with SRMCs. Design, Setting, and Participants: This randomized clinical trial was conducted at a tertiary pediatric hospital in Adelaide, South Australia, from April 15 to September 30, 2021. Children and adolescents aged 6 months to younger than 18 years with SRMCs and a subspecialist outpatient appointment over a 5-month period during the Australian seasonal influenza vaccination season (April-August 2021) were eligible to participate. Follow-up was until September 30, 2021. Interventions: Participants were randomly assigned (1:1 ratio) to control: clinician nudges (hospital vaccine availability, ease of access, and recommendation from hospital subspecialists) or SMS intervention (control conditions plus an additional SMS reminder nudge to parents), with randomization stratified by age group (<5 years, 5-14 years, or >14 to <18 years). Main Outcomes and Measures: The primary outcome was influenza vaccination, as confirmed by the Australian Immunisation Register. Results: A total of 600 participants (intervention group: 298 [49.7%]; mean [SD] age, 11.5 [4.6] years; 162 female participants [54.4%]; control group: 302 [50.3%]; mean [SD] age, 11.4 [4.7] years; 155 female participants [51.3%]) were included. Influenza vaccination was 38.6% (113 of 293) in the SMS intervention group compared with 26.2% (79 of 302) in the control group (adjusted odds ratio [aOR], 1.79; 95% CI, 1.27-2.55; P = .001). Time to vaccine receipt was significantly lower among SMS participants (adjusted hazard ratio, 1.67; 95% CI, 1.25-2.22; P < .001). For participants randomly assigned by June 15, a significantly greater proportion receiving the SMS intervention were vaccinated during the optimal delivery period April to June 30 (SMS group: 40.0% [76 of 190] vs 25.4% [50 of 197]; aOR, 1.97; 95% CI, 1.28-3.06; P = .002). Conclusions and Relevance: Results of this randomized clinical trial suggest that an additional SMS reminder nudge for parents delivered in the tertiary care hospital setting to children and adolescents with SMRCs resulted in higher influenza vaccine uptake compared with clinician nudges alone. Trial Registration: ANZCTR Identifier: ACTRN12621000463875.

The Acceptability of Using Augmented Reality as a Mechanism to Engage Children in Asthma Inhaler Technique Training: Qualitative Interview Study With Deductive Thematic Analysis.

BACKGROUND: Inhaled medications or inhalers provide first-line pharmacotherapeutic treatment for patients with asthma for both acute symptomatic relief and long-term management to keep symptoms under control. A good technique requires only basic instruction and training; however, a recent study identified that 92% of children do not follow all correct steps when using inhalers. There is a growing interest in technology-enhanced asthma education, with evidence demonstrating improvements in knowledge and treatment adherence. Subsequently, there are calls to explore the role of technology-based solutions in improving asthma management and disease outcomes from public health experts, health professionals, and patients with asthma. Augmented reality (AR) technology is an information delivery mechanism with proven efficacy in educational settings. AR displays digital content in a real-world environment using the camera on a smartphone or tablet device to create an immersive learning experience. OBJECTIVE: The study aimed to evaluate the acceptability of AR as a mechanism for delivering asthma inhaler technique education from the perspective of children with asthma and their parents and health professionals, examined through the theoretical framework of acceptability (TFA). METHODS: An asthma education resource enhanced with AR technology was created to provide inhaler technique education to children. An iterative co-design process was undertaken with target end users for a qualitative evaluation. The participants were 8 to 12 years old with asthma, their caregivers, and health professionals who had experience in managing asthma. Qualitative data were obtained through semistructured one-on-one interviews. Deductive thematic analysis using TFA was undertaken using NVivo software 2020 to assess the acceptability of AR as a delivery modality for asthma inhaler technique education. RESULTS: Overall, 6 health care professionals, 5 asthmatic children, and 5 caregivers of children with asthma totaled a sample of 16. The use of AR in the asthma inhaler resource was found to be acceptable when responses were examined in accordance with TFA. Each of the 7 component constructs of TFA was coded throughout the 16 interviews, with perceived effectiveness (157 times) and affective attitude (63 times) coded most frequently. Positive responses included the intervention being accessible, easy to use, interesting, and fitting within the users' value systems. Negative responses included the need to maintain an interest in children and concerns about the loss of face-to-face interaction with health professionals. CONCLUSIONS: AR appears to be an acceptable modality for delivering asthma education to children when explored using TFA constructs. Although some challenges were identified with the use of AR, the results were predominantly positive. Future designs of asthma education interventions involving AR should consider the results of this study, and further research should focus on the feasibility, usability, and barriers and facilitators of behavior change to ensure the successful implementation and uptake of AR into clinical settings. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.1177/16094069211042229.

PINK1-mediated mitophagy contributes to glucocorticoid-induced cathepsin K production in osteocytes.

Background: Glucocorticoid (GC) is one of frequently used anti-inflammatory agents, but its administration is unfortunately accompanied with bone loss. Although sporadic studies indicated that osteocytes are subject to a series of pathological changes under GC stress, including overexpression of cathepsin K, the definite role of osteocytes in GC-induced bone loss remains largely unclear. Methods: Gene expression of Ctsk and protein levels of cathepsin K were assessed in MLO-Y4 cell lines exposed to dexamethasone (Dex) of different time (0, 12, 24 hours) and dose (0, 10-8 and 10-6 M) courses by RT-qPCR and western blotting, respectively. Confocal imaging and immunostaining were then performed to evaluate the effects of osteocyte-derived cathepsin K on type I collagen in a primary osteocyte ex vivo culture system. MitoTracker Red was used to stain mitochondria for mitochondria morphology assessment and JC-1 assay was employed to evaluate the mitochondria membrane potential in MLO-Y4 cells following Dex treatment. Activation of PINK1-mediated mitophagy was evaluated by immunostaining of the PINK1 protein and CytoID assay. Mdivi-1 was used to inhibit mitophagy and siRNAs were used for the inhibition of Pink1 and Atg5. Results: GC triggered osteocytes to produce excessive cathepsin K which in turn led to the degradation of type I collagen in the extracellular matrix in a primary osteocyte ex vivo culture system. Meanwhile, GC administration increased mitochondrial fission and membrane depolarization in osteocytes. Further, the activation of PINK1-mediated mitophagy was demonstrated to be responsible for the diminishment of dysfunctional mitochondria in osteocytes. Examination of relationship between mitophagy and cathepsin K production revealed that inhibition of mitophagy via knocking down Pink1 gene abolished the GC-triggered cathepsin K production. Interestingly, GC's activation effect towards cathepsin K via mitophagy was found to be independent on the canonical autophagy as this effect was not impeded when inhibiting the canonical autophagy via Atg5 suppression. Conclusion: GC-induced PINK1-mediated mitophagy substantially modulates the production of cathepsin K in osteocytes, which could be an underlying mechanism by which osteocytes contribute to the extracellular matrix degradation during bone loss. The Translational potential of this article: Findings of the current study indicate a possible role of osteocyte mitophagy in GC-induced bone loss, which provides a potential therapeutic approach to alleviate GC-induced osteoporosis by targeting PINK1-mediated osteocytic mitophagy.