Rapid initiation of nasal saline irrigation to reduce severity in high-risk COVID+ outpatients.

OBJECTIVE: To determine whether initiating saline nasal irrigation after COVID-19 diagnosis reduces hospitalization and death in high-risk outpatients compared with observational controls, and if irrigant composition impacts severity. METHODS: Participants 55 and older were enrolled within 24 hours of a + PCR COVID-19 test between September 24 and December 21, 2020. Among 826 screened, 79 participants were enrolled and randomly assigned to add 2.5 mL povidone-iodine 10% or 2.5 mL sodium bicarbonate to 240 mL of isotonic nasal irrigation twice daily for 14 days. The primary outcome was hospitalization or death from COVID-19 within 28 days of enrollment by daily self-report confirmed with phone calls and hospital records, compared to the CDC Surveillance Dataset covering the same time. Secondary outcomes compared symptom resolution by irrigant additive. RESULTS: Seventy-nine high-risk participants were enrolled (mean [SD] age, 64 [8] years; 36 [46%] women; 71% Non-Hispanic White), with mean BMI 30.3. Analyzed by intention-to-treat, by day 28, COVID-19 symptoms resulted in one ED visit and no hospitalizations in 42 irrigating with alkalinization, one hospitalization of 37 in the povidone-iodine group, (1.27%) and no deaths. Of nearly three million CDC cases, 9.47% were known to be hospitalized, with an additional 1.5% mortality in those without hospitalization data. Age, sex, and percentage with pre-existing conditions did not significantly differ by exact binomial test from the CDC dataset, while reported race and hospitalization rate did. The total risk of hospitalization or death (11%) was 8.57 times that of enrolled nasal irrigation participants (SE = 2.74; P = .006). Sixty-two participants completed daily surveys (78%), averaging 1.8 irrigations/day. Eleven reported irrigation-related complaints and four discontinued use. Symptom resolution was more likely for those reporting twice daily irrigation (X2 = 8.728, P = .0031) regardless of additive. CONCLUSION: SARS-CoV-2+ participants initiating nasal irrigation were over 8 times less likely to be hospitalized than the national rate.

EGL-38/Pax coordinates development in the Caenhorhabditis elegans egg-laying system through EGF pathway dependent and independent functions.

Paired box (Pax) proteins function as regulators of coordinated development in organogenesis by controlling factors such as cell growth and differentiation necessary to organize multiple cell types into a single, cohesive organ. Previous work has suggested that Pax transcription factors may regulate diverse cell types through participation in inductive cell-to-cell signaling, which has not been well explored. Here we show that EGL-38, a Pax2/5/8 ortholog, coordinates differentiation of the C. elegans egg-laying system through separate autonomous and non-autonomous functions synchronized by the EGF pathway. We find that EGL-38 protein is expressed at the correct times to both participate in and respond to the EGF pathway specifying uterine ventral (uv1) cell fate, and that EGL-38 is required for uv1 expression of nlp-2 and nlp-7, which are both markers of and participants in uv1 identity. Additionally, we have separated uv1 cell placement and gene expression as distinct hallmarks of uv1 identity and specification, with different dependencies on EGL-38. The parallels between EGL-38 participation in cell signaling events and previous Pax studies argue that coordination of signaling and response to an inductive pathway may be a common feature of Pax protein function.

Muscle atrophy in mechanically-ventilated critically ill children.

IMPORTANCE: ICU-acquired muscle atrophy occurs commonly and worsens outcomes in adults. The incidence and severity of muscle atrophy in critically ill children are poorly characterized. OBJECTIVE: To determine incidence, severity and risk factors for muscle atrophy in critically ill children. DESIGN, SETTING AND PARTICIPANTS: A single-center, prospective cohort study of 34 children receiving invasive mechanical ventilation for ≥48 hours. Patients 1 week- 18 years old with respiratory failure and without preexisting neuromuscular disease or skeletal trauma were recruited from a tertiary Pediatric Intensive Care Unit (PICU) between June 2015 and May 2016. We used serial bedside ultrasound to assess thickness of the diaphragm, biceps brachii/brachialis, quadriceps femoris and tibialis anterior. Serial electrical impedance myography (EIM) was assessed in children >1 year old. Medical records were abstracted from an electronic database. EXPOSURES: Respiratory failure requiring endotracheal intubation for ≥48 hours. MAIN OUTCOME AND MEASURES: The primary outcome was percent change in muscle thickness. Secondary outcomes were changes in EIM-derived fat percentage and "quality". RESULTS: Of 34 enrolled patients, 30 completed ≥2 ultrasound assessments with a median interval of 6 (IQR 6-7) days. Mean age was 5.42 years, with 12 infants <1 year (40%) and 18 children >1 year old (60%). In the entire cohort, diaphragm thickness decreased 11.1% (95%CI, -19.7% to -2.52%) between the first two assessments or 2.2%/day. Quadriceps thickness decreased 8.62% (95%CI, -15.7% to -1.54%) or 1.5%/day. Biceps (-1.71%; 95%CI, -8.15% to 4.73%) and tibialis (0.52%; 95%CI, -5.81% to 3.40%) thicknesses did not change. Among the entire cohort, 47% (14/30) experienced diaphragm atrophy (defined a priori as ≥10% decrease in thickness). Eighty three percent of patients (25/30) experienced atrophy in ≥1 muscle group, and 47% (14/30)-in ≥2 muscle groups. On multivariate linear regression, increasing age and traumatic brain injury (TBI) were associated with greater muscle loss. EIM revealed increased fat percentage and decreased muscle "quality". CONCLUSIONS AND RELEVANCE: In children receiving invasive mechanical ventilation, diaphragm and other skeletal muscle atrophy is common and rapid. Increasing age and TBI may increase severity of limb muscle atrophy. Prospective studies are required to link muscle atrophy to functional outcomes in critically ill children.

Systemic Delivery of Dysferlin Overlap Vectors Provides Long-Term Gene Expression and Functional Improvement for Dysferlinopathy.

Dysferlinopathies comprise a family of disorders caused by mutations in the dysferlin (DYSF) gene, leading to a progressive dystrophy characterized by chronic muscle fiber loss, fat replacement, and fibrosis. To correct the underlying histopathology and function, expression of full-length DYSF is required. Dual adeno-associated virus vectors have been developed, defined by a region of homology, to serve as a substrate for reconstitution of the full 6.5 kb dysferlin cDNA. Previous work studied the efficacy of this treatment through intramuscular and regional delivery routes. To maximize clinical efficacy, dysferlin-deficient mice were treated systemically to target all muscles through the vasculature for efficacy and safety studies. Mice were evaluated at multiple time points between 4 and 13 months post treatment for dysferlin expression and functional improvement using magnetic resonance imaging and magnetic resonance spectroscopy and membrane repair. A systemic dose of 6 × 1012 vector genomes resulted in widespread gene expression in the muscles. Treated muscles showed a significant decrease in central nucleation, collagen deposition, and improvement of membrane repair to wild-type levels. Treated gluteus muscles were significantly improved compared to placebo-treated muscles and were equivalent to wild type in volume, intra- and extramyocellular lipid accumulation, and fat percentage using magnetic resonance imaging and magnetic resonance spectroscopy. Dual-vector treatment allows for production of full-length functional dysferlin with no toxicity. This confirms previous safety data and validates translation of systemic gene delivery for dysferlinopathy patients.

Defective membrane fusion and repair in Anoctamin5-deficient muscular dystrophy.

Limb-girdle muscular dystrophies are a genetically diverse group of diseases characterized by chronic muscle wasting and weakness. Recessive mutations in ANO5 (TMEM16E) have been directly linked to several clinical phenotypes including limb-girdle muscular dystrophy type 2L and Miyoshi myopathy type 3, although the pathogenic mechanism has remained elusive. ANO5 is a member of the Anoctamin/TMEM16 superfamily that encodes both ion channels and regulators of membrane phospholipid scrambling. The phenotypic overlap of ANO5 myopathies with dysferlin-associated muscular dystrophies has inspired the hypothesis that ANO5, like dysferlin, may be involved in the repair of muscle membranes following injury. Here we show that Ano5-deficient mice have reduced capacity to repair the sarcolemma following laser-induced damage, exhibit delayed regeneration after cardiotoxin injury and suffer from defective myoblast fusion necessary for the proper repair and regeneration of multinucleated myotubes. Together, these data suggest that ANO5 plays an important role in sarcolemmal membrane dynamics. Genbank Mouse Genome Informatics accession no. 3576659.

AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models.

OBJECTIVE: Dysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment. METHODS: Traditional gene therapy is restricted by the packaging capacity limit of adeno-associated virus (AAV), however, use of a dual vector strategy allows for delivery of over-sized genes, including dysferlin. The two vector system (AAV.DYSF.DV) packages the dysferlin cDNA utilizing AAV serotype rh.74 through the use of two discrete vectors defined by a 1 kb region of homology. Delivery of AAV.DYSF.DV via intramuscular and vascular delivery routes in dysferlin deficient mice and nonhuman primates was compared for efficiency and safety. RESULTS: Treated muscles were tested for dysferlin expression, overall muscle histology, and ability to repair following injury. High levels of dysferlin overexpression was shown for all muscle groups treated as well as restoration of functional outcome measures (membrane repair ability and diaphragm specific force) to wild-type levels. In primates, strong dysferlin expression was demonstrated with no safety concerns. INTERPRETATION: Treated muscles showed high levels of dysferlin expression with functional restoration with no evidence of toxicity or immune response providing proof of principle for translation to dysferlinopathy patients.

Abrogation of nontypeable Haemophilus influenzae protein D function reduces phosphorylcholine decoration, adherence to airway epithelial cells, and fitness in a chinchilla model of otitis media.

The pneumococcal polysaccharide conjugate vaccine which includes a nonacylated protein D carrier from Haemophilus influenzae has been recently licensed for use in many countries. While this vaccine is protective against nontypeable Haemophilus influenzae (NTHI)-induced acute otitis media (OM), the mechanism underlying this protective efficacy is not yet fully understood. Protein D/glycerophosphodiester phosphodiesterase (PD/GlpQ) is an outer membrane lipoprotein expressed by NTHI that has been ascribed several functions, including host cell adherence and phosphorylcholine (PCho) acquisition. We found that a pd/glpQ NTHI mutant exhibited reduced adherence to airway epithelial cells, diminished phosphorylcholine (PCho) decoration of biofilms, and compromised fitness during experimental acute OM compared to the parent strain. We also found that exposure of NTHI to antibodies directed against the vaccine formulation recapitulated the PCho decoration and NTHI adherence phenotypes exhibited by PD/GlpQ-deficient NTHI, providing at least two likely mechanisms by which the pneumococcal polysaccharide-PD/GlpQ conjugate vaccine induces protection from NTHI-induced OM.

The Caenorhabditis elegans heterochronic gene lin-14 coordinates temporal progression and maturation in the egg-laying system.

Heterochronic genes function to ensure the timing of stage-specific developmental events in C. elegans. Mutations in these genes cause certain developmental programs to be executed in a precocious or retarded manner. Canonical precocious (loss-of-function) and retarded (gain-of-function) mutations in the lin-14 gene lead to elimination or reiteration of larval stage-specific cellular events. Here, we describe a hypomorphic, missense allele of lin-14, sa485. lin-14(sa485) hermaphrodites pass through normal larval stages, but exhibit asynchrony between vulval and gonadal maturation in the L4 larval stage. We show that a subtly precocious morphogenetic event in the vulva disrupts tissue synchrony and is followed by retarded vulval eversion. Additionally, uterine uv1 cell differentiation is retarded in lin-14(sa485) animals that exhibit delayed vulval eversion. Together, these experiments outline a function for LIN-14 in coordinating the temporal progression of development, which is separable from its role in regulating stage-specific events during C. elegans postembryonic development.

Positive and negative regulatory inputs restrict pax-6/vab-3 transcription to sensory organ precursors in Caenorhabditis elegans.

The Pax-6 gene encodes a transcription factor essential for the development of eyes and other sensory organs in species ranging from planaria to mice. Because Pax-6 activity can be both necessary and sufficient for eye organogenesis, much work has focused on PAX-6 function and regulation of target genes. However, less is known about the genetic mechanisms that establish the Pax-6 expression pattern. We have utilized Caenorhabditis elegans as a relatively simple model system to characterize the regulation of Pax-6 transcription in sensory organ precursors. In C. elegans males, two sensory mating structures, the copulatory spicules and the post-cloacal sensilla, are formed from stereotyped divisions of the two post-embryonic blast cells, B.a and Y.p, respectively. A C. elegans pax-6 transcript, vab-3, is necessary for the development of these sensory structures. Using a green fluorescent protein (GFP)-based vab-3 transcriptional reporter, we show that expression is restricted to the sensory organ lineages of B.a and Y.p. Transcription of vab-3 in the tail region of the worm requires the Abdominal B homeobox gene, egl-5. Opposing this activation, a transcription factor cascade and a Wnt signaling pathway each act to restrict vab-3 expression to the appropriate cell lineages. Thus we have identified multiple genetic pathways that act to restrict pax-6/vab-3 gene expression to the sensory organ precursor cells.