Structure-function analyses of tyrosine phosphatase PTP69D in giant fiber synapse formation of Drosophila.

PTP69D is a receptor protein tyrosine phosphatase (RPTP) with two intracellular catalytic domains (Cat1 and Cat2) and has been shown to play a role in axon guidance of embryonic motoneurons as well as targeting of photoreceptor neurons in the visual system of Drosophila melanogaster. Here, we characterized the developmental role of PTP69D in the giant fiber (GF) neurons, two interneurons in the central nervous system (CNS) that control the escape response of the fly. Our studies revealed that PTP69D has a function in synaptic terminal growth in the CNS. We found that missense mutations in the first immunoglobulin (Ig) domain and in the Cat1 domain, present in Ptp69D10 and Ptp69D20 mutants, respectively, did not affect axon guidance or targeting but resulted in stunted terminal growth of the GFs. Cell autonomous rescue experiments demonstrated a function for the Cat1 and the first Ig domain of PTP69D in the GFs but not in its postsynaptic target neurons. In addition, complementation studies and structure-function analyses revealed that for GF terminal growth Cat1 function of PTP69D requires the immunoglobulin and the Cat2 domains, but not the fibronectin III or the membrane proximal region domains. In contrast, the fibronectin III but not the immunoglobulin domains were previously shown to be essential for axon targeting of photoreceptor neurons. Thus, our studies uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically distinct from its function in photoreceptor targeting.

CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila.

Aminoacyl-tRNA synthetases are ubiquitously expressed proteins that charge tRNAs with their cognate amino acids. By ensuring the fidelity of protein synthesis, these enzymes are essential for the viability of every cell. Yet, mutations in six tRNA synthetases specifically affect the peripheral nerves and cause Charcot-Marie-Tooth (CMT) disease. The CMT-causing mutations in tyrosyl- and glycyl-tRNA synthetases (YARS and GARS, respectively) alter the activity of the proteins in a range of ways (some mutations do not impact charging function, while others abrogate it), making a loss of function in tRNA charging unlikely to be the cause of disease pathology. It is currently unknown which cellular mechanisms are triggered by the mutant enzymes and how this leads to neurodegeneration. Here, by expressing two pathogenic mutations (G240R, P234KY) in Drosophila, we generated a model for GARS-associated neuropathy. We observed compromised viability, and behavioral, electrophysiological and morphological impairment in flies expressing the cytoplasmic isoform of mutant GARS. Their features recapitulated several hallmarks of CMT pathophysiology and were similar to the phenotypes identified in our previously described Drosophila model of YARS-associated neuropathy. Furthermore, CG8316 and CG15599 - genes identified in a retinal degeneration screen to modify mutant YARS, also modified the mutant GARS phenotypes. Our study presents genetic evidence for common mutant-specific interactions between two CMT-associated aminoacyl-tRNA synthetases, lending support for a shared mechanism responsible for the synthetase-induced peripheral neuropathies.

Nucleocapsid and Spike Protein-Based Anti-SARS-CoV-2 Assay Performance in the Minority and Rural Coronavirus Insights Study: Characteristics of Socioeconomically Disadvantaged Populations with Health Disparities.

BACKGROUND: COVID-19 has had a devastating impact on Black, Hispanic, and other underserved, disadvantaged populations. Here anti-SARS-CoV-2 tests are characterized in disadvantaged patients to examine equivalence in US populations. METHODS: Underserved participant adults (age > 18 years) were enrolled before the availability of SARS-CoV-2 vaccines in Federal Qualified Health Centers in California, Florida, Louisiana, Illinois, and Ohio and contributed samples to the Minority and Rural Coronavirus Insights Study (MRCIS). A subset coined the MRCIS SARS-CoV-2 Antibody Cohort of 2365 participants was tested with the Roche Anti-SARS-CoV-2 assay (Cobas e601). Five hundred ninety-five of these were also tested with the Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 IgG assay (VITROS-5600); 1770 were also tested with the Abbott ARCHITECT SARS-CoV-2 IgG assay (ARCHITECT-2000). Assay-specific cutoffs classified negative/positive results. RESULTS: Eight point four percent (199/2365) of the MRCIS SARS-CoV-2 Antibody Cohort was SARS-CoV-2 RNA positive at enrollment. Agreement between the Ortho/Roche and the Abbott/Roche antibody testing did not vary by enrollment RNA status. The Ortho (anti-spike protein) vs Roche (anti-nucleocapsid protein) comparison agreed substantially: kappa = 0.63 (95% CI: 0.57-0.69); overall agreement, 83%. However, agreement was even better for the Abbott vs Roche assays (both anti-nucleocapsid protein tests): kappa = 0.85 (95% CI: 0.81-0.87); overall agreement, 95%. Anti-SARS-CoV-2 comparisons stratified by demographic criteria demonstrated no significant variability in agreement by sex, race/ethnicity, or age. CONCLUSIONS: Analytical agreement is 96.4% for anti-spike-protein vs anti-nucleocapsid-protein comparisons. Physiologically, seroreversion of anti-nucleocapsid reactivity after infection occurred in the disadvantaged population similarly to general populations. No anti-SARS-CoV-2 assays included demonstrated a clinically significant difference due to the demographics of the disadvantaged MRCIS SARS-CoV-2 Antibody Cohort.

Demographic Determinants and Geographical Variability of COVID-19 Vaccine Hesitancy in Underserved Communities: Cross-sectional Study.

BACKGROUND: COVID-19 hospitalizations and deaths disproportionately affect underserved and minority populations, emphasizing that vaccine hesitancy can be an especially important public health risk factor in these populations. OBJECTIVE: This study aims to characterize COVID-19 vaccine hesitancy in underserved diverse populations. METHODS: The Minority and Rural Coronavirus Insights Study (MRCIS) recruited a convenience sample of adults (age≥18, N=3735) from federally qualified health centers (FQHCs) in California, the Midwest (Illinois/Ohio), Florida, and Louisiana and collected baseline data in November 2020-April 2021. Vaccine hesitancy status was defined as a response of "no" or "undecided" to the question "Would you get a coronavirus vaccine if it was available?" ("yes" categorized as not hesitant). Cross-sectional descriptive analyses and logistic regression models examined vaccine hesitancy prevalence by age, gender, race/ethnicity, and geography. The expected vaccine hesitancy estimates for the general population were calculated for the study counties using published county-level data. Crude associations with demographic characteristics within each region were assessed using the chi-square test. The main effect model included age, gender, race/ethnicity, and geographical region to estimate adjusted odds ratios (ORs) and 95% CIs. Interactions between geography and each demographic characteristic were evaluated in separate models. RESULTS: The strongest vaccine hesitancy variability was by geographic region: California, 27.8% (range 25.0%-30.6%); the Midwest, 31.4% (range 27.3%-35.4%); Louisiana, 59.1% (range 56.1%-62.1%); and Florida, 67.3% (range 64.3%-70.2%). The expected estimates for the general population were lower: 9.7% (California), 15.3% (Midwest), 18.2% (Florida), and 27.0% (Louisiana). The demographic patterns also varied by geography. An inverted U-shaped age pattern was found, with the highest prevalence among ages 25-34 years in Florida (n=88, 80.0%,) and Louisiana (n=54, 79.4%; P<.05). Females were more hesitant than males in the Midwest (n= 110, 36.4% vs n= 48, 23.5%), Florida (n=458, 71.6% vs n=195, 59.3%), and Louisiana (n= 425, 66.5% vs. n=172, 46.5%; P<.05). Racial/ethnic differences were found in California, with the highest prevalence among non-Hispanic Black participants (n=86, 45.5%), and in Florida, with the highest among Hispanic (n=567, 69.3%) participants (P<.05), but not in the Midwest and Louisiana. The main effect model confirmed the U-shaped association with age: strongest association with age 25-34 years (OR 2.29, 95% CI 1.74-3.01). Statistical interactions of gender and race/ethnicity with the region were significant, following the pattern found by the crude analysis. Compared to males in California, the associations with the female gender were strongest in Florida (OR=7.88, 95% CI 5.96-10.41) and Louisiana (OR=6.09, 95% CI 4.55-8.14). Compared to non-Hispanic White participants in California, the strongest associations were found with being Hispanic in Florida (OR=11.18, 95% CI 7.01-17.85) and Black in Louisiana (OR=8.94, 95% CI 5.53-14.47). However, the strongest race/ethnicity variability was observed within California and Florida: the ORs varied 4.6- and 2-fold between racial/ethnic groups in these regions, respectively. CONCLUSIONS: These findings highlight the role of local contextual factors in driving vaccine hesitancy and its demographic patterns.

The Minority and Rural Coronavirus Insights Study: Design and Baseline Characteristics of a Minority Cohort.

The Minority and Rural Coronavirus Insights Study (MRCIS) is an ongoing prospective cohort study examining health disparities associated with SARS-CoV-2 infection among medically underserved populations. This report describes procedures implemented to establish the MRCIS cohort and examines the factors associated with the molecular and serological assessment of SARS-CoV-2 infection status at participant enrollment. Participants were recruited from 5 geographically dispersed federally qualified health centers between November 2020 and April 2021. At baseline, participants completed a detailed demographic survey and biological samples were collected for testing. SARS-CoV-2 infection status was determined based on the combined molecular and serological test results. Chi-squared and logistic regression analyses were conducted to examine associations between sociodemographic factors, COVID-19 safety measures, existing comorbidities, and SARS-CoV-2 infection status. The final cohort included 3238 participants. The mean age of participants was 50.2 ± 15.8 years. Most participants identified as female (60.0%), heterosexual or straight (93.0%), White (47.6%), and Hispanic or Latino (49.1%). Approximately 26.1% of participants had at least one positive SARS-CoV-2 test result. The main effect model included age, sex, and race/ethnicity. Compared with adults ≥65 years, participants in all other age groups had ∼2 times increased odds of a positive SARS-CoV-2 test result. In addition, racial/ethnic minorities had ∼2 times increased odds of a positive SARS-CoV-2 infection status compared with non-Hispanic Whites. A unique cohort of a traditionally medically underserved minority population was established. Significant racial and ethnic disparities in SARS-CoV-2 infection status at baseline were discovered.

Tyrosyl-tRNA synthetase has a noncanonical function in actin bundling.

Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, we link YARS1 dysfunction to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, in this work, we show that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration.

A community-centered approach to sickle cell disease and clinical trial participation: an evaluation of perceptions, facilitators, and barriers.

Sickle cell disease (SCD) is the most common inherited red blood cell disorder in the United States, affecting 70 000 to 100 000 Americans and causing a range of serious medical complications. Although the cause of SCD was established decades ago, existing therapies have varied effectiveness and side effects, and development of novel therapies has been slow. The limitations of existing treatment options highlight the need for new therapies that are aligned with the desires of the community. To date, little has been done to systematically seek and report the opinions and experiences of people with SCD regarding clinical research. In 2019, the American Society of Hematology Research Collaborative conducted 8 community workshops across the United States engaging 472 people, including persons with SCD and caregivers of those living with the disease. The workshop goals included assessing understanding, awareness, and perceptions of clinical research; and identifying the most critical clinical trial considerations of this community. Participants were asked about their experiences living with SCD and their satisfaction with treatment options. Pain and fatigue were reported as symptoms requiring better therapies. Although few participants reported being asked to enroll in a clinical trial, they expressed conditional willingness to participate. A majority were willing to share personal health information to further research and improve health outcomes. To actively engage the SCD community and increase enrollment and retention in clinical trials, researchers should address the treatment priorities of this population and ensure they have access to trusted information about clinical research and opportunities for participation.

Reactions to the National Academies/Royal Society Report on Heritable Human Genome Editing.

In September 2020, a detailed report on Heritable Human Genome Editing was published. The report offers a translational pathway for the limited approval of germline editing under limited circumstances and assuming various criteria have been met. In this perspective, some three dozen experts from the fields of genome editing, medicine, bioethics, law, and related fields offer their candid reactions to the National Academies/Royal Society report, highlighting areas of support, omissions, disagreements, and priorities moving forward.

Reducing Health Care Disparities in Sickle Cell Disease: A Review.

Sickle cell disease (SCD) is an inherited blood disorder most common among African American and Hispanic American persons. The disease can cause substantial, long-term, and costly health problems, including infections, stroke, and kidney failure, many of which can reduce life expectancy. Disparities in receiving health care among African Americans and other racial/ethnic minority groups in the United States are well known and directly related to poor outcomes associated with SCD. As an orphan disease-one that affects <200 000 persons nationwide-SCD does not receive the research funding and pharmaceutical investment directed to other orphan diseases. For example, cystic fibrosis affects fewer than half the number of persons but receives 3.5 times the funding from the National Institutes of Health and 440 times the funding from national foundations. In this review, we discuss the health inequities affecting persons with SCD, describe programs intended to improve their care, and identify actions that could be taken to further reduce these inequities, improve care, control treatment costs, and ease the burden of disease.

Lissencephaly-1 dependent axonal retrograde transport of L1-type CAM Neuroglian in the adult drosophila central nervous system.

Here, we established the Drosophila Giant Fiber neurons (GF) as a novel model to study axonal trafficking of L1-type Cell Adhesion Molecules (CAM) Neuroglian (Nrg) in the adult CNS using live imaging. L1-type CAMs are well known for their importance in nervous system development and we previously demonstrated a role for Nrg in GF synapse formation. However, in the adult they have also been implicated in synaptic plasticity and regeneration. In addition, to its canonical role in organizing cytoskeletal elements at the plasma membrane, vertebrate L1CAM has also been shown to regulate transcription indirectly as well as directly via its import to the nucleus. Here, we intend to determine if the sole L1CAM homolog Nrg is retrogradley transported and thus has the potential to relay signals from the synapse to the soma. Live imaging of c-terminally tagged Nrg in the GF revealed that there are at least two populations of retrograde vesicles that differ in speed, and either move with consistent or varying velocity. To determine if endogenous Nrg is retrogradely transported, we inhibited two key regulators, Lissencephaly-1 (Lis1) and Dynactin, of the retrograde motor protein Dynein. Similar to previously described phenotypes for expression of poisonous subunits of Dynactin, we found that developmental knock down of Lis1 disrupted GF synaptic terminal growth and that Nrg vesicles accumulated inside the stunted terminals in both mutant backgrounds. Moreover, post mitotic Lis1 knock down in mature GFs by either RNAi or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) induced mutations, resulted in normal length terminals with fully functional GF synapses which also exhibited severe accumulation of endogenous Nrg vesicles. Thus, our data suggests that accumulation of Nrg vesicles is due to failure of retrograde transport rather than a failure of terminal development. Together with the finding that post mitotic knock down of Lis1 also disrupted retrograde transport of tagged Nrg vesicles in GF axons, it demonstrates that endogenous Nrg protein is transported from the synapse to the soma in the adult central nervous system in a Lis1-dependent manner.