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The CO2 receptor in mosquitoes is broadly tuned to detect many diverse odorants. The receptor consists of three subunits (Gr1, Gr2, and Gr3) in mosquitoes but only two subunits in Drosophila: Gr21a (Gr1 ortholog) and Gr63a (Gr3 ortholog). We demonstrate that Gr21a is required for CO2 responses in Drosophila, as has been shown for Gr63a. Next, we generate a Drosophila double mutant for Gr21a and Gr63a, and in this background, we functionally express combinations of Aedes Gr1, Gr2, and Gr3 genes in the CO2 empty neuron. Only two subunits, Gr2 and Gr3, suffice for response to CO2. Addition of Gr1 increases sensitivity to CO2, whereas it decreases the response to pyridine. The inhibitory effect of the antagonist isobutyric acid is observed upon addition of Gr1. Gr1 therefore increases the diversity of ligands of the receptor and modulates the response of the receptor complex.

AbstractThe CO2 receptor in mosquitoes is broadly tuned to detect many diverse odorants. The receptor consists of three 7-TM subunits (Gr1, Gr2, and Gr3) in mosquitoes but only two subunits in Drosophila: Gr21a (Gr1 ortholog) and Gr63a (Gr3 ortholog). We demonstrate that Gr21a is required for CO2 responses in Drosophila as has been shown for Gr63a. Next, we generate a Drosophila double mutant for Gr21a and Gr63a, and in this background we functionally express combinations of Aedes Gr1, 2, and 3 genes in the “CO2 empty neuron.” Only two subunits, Gr2 and Gr3, suffice for response to CO2. Addition of Gr1 increases sensitivity to CO2 while it decreases the response to pyridine. The inhibitory effect of the antagonist isobutyric acid is observed upon addition of Gr1. Gr1 therefore increases the diversity of ligands of the receptor, and also modulates the response of the receptor complex.

Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity. Author summary: We have developed a tissue-specific Cas9 toolkit that enables gene knockout specifically in motor neurons, glial cells, and muscle cells, the three principle cell types of the Drosophila peripheral motor system. Complementary to existing RNAi methods, this versatile tissue-specific knockout system offers unique advantages for dissecting gene functions at the neuromuscular junction (NMJ). Using these tools, we discovered that SNARE-mediated secretory pathways are required to maintain the integrity of the NMJ and that ESCRT components play critical yet differential roles in the biogenesis of extracellular vesicles, bouton growth, and membrane turnover at the NMJ. This CRISPR toolkit can be applied to study many biological questions in the neuromuscular system. [ABSTRACT FROM AUTHOR]

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease- contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway., Introduction Complex diseases of aging provide unique challenges for the identification of specific disease-causing genetic variants and associated cellular dysfunctions using traditional genetic approaches. Disease modeling using iPSCs is a [...]

Biomolecules exist and function in cellular microenvironments that control their spatial organization, local concentration, and biochemical reactivity. Due to the complexity of native cytoplasm, the development of artificial bioreactors and cellular mimics to compartmentalize, concentrate, and control the local physico-chemical properties is of great interest. Here, we employ self-assembling polypeptide coacervates to explore the partitioning of the ubiquitous cytoskeletal protein actin into liquid polymer-rich droplets. We find that actin spontaneously partitions into coacervate droplets and is enriched by up to ∼30-fold. Actin polymerizes into micrometer-long filaments and, in contrast to the globular protein BSA, these filaments localize predominately to the droplet periphery. We observe up to a 50-fold enhancement in the actin filament assembly rate inside coacervate droplets, consistent with the enrichment of actin within the coacervate phase. Together these results suggest that coacervates can serve as a versatile platform in which to localize and enrich biomolecules to study their reactivity in physiological environments.

Histone deacetylases (HDACs) have been extensively studied as drug targets in neurodegenerative diseases, but less is known about their role in healthy neurons. We tested zinc-dependent HDACs using RNAi in Drosophila melanogaster and found memory deficits with RPD3 and HDAC6. We demonstrate that HDAC6 is required in both the larval and adult stages for normal olfactory memory retention. Neuronal expression of HDAC6 rescued memory deficits, and we demonstrate that the N-terminal deacetylase (DAC) domain is required for this ability. This suggests that deacetylation of synaptic targets associated with the first DAC domain, such as the active-zone scaffold Bruchpilot, is required for memory retention. Finally, electrophysiological experiments at the neuromuscular junction reveal that HDAC6 mutants exhibit a partial block of homeostatic plasticity, suggesting that HDAC6 may be required for the stabilization of synaptic strength. The learning deficit we observe in HDAC6 mutants could be a behavioral consequence of these synaptic defects.

Purpose: Reflex cough is an essential airway protective mechanism that often occurs in the context of divided attention. The effect of divided attention on measures of reflexive cough airflow and sensitivity remains unknown. We present findings from a study testing the effects of divided attention (via a dual-task paradigm) on measures of reflex cough in healthy young adults. Method: Volunteers (N = 20, age = 20-40 years) underwent 4 blocks of capsaicin-induced cough challenges. Within each block, capsaicin ranging from 0 to 200 [micro]M was presented in a randomized order. Two blocks consisted of cough testing only (single task). During the other 2 blocks, participants counted tones while simultaneously undergoing cough testing (dual task). Measures of cough motor response, self-reported urge-to-cough, cough frequency, and cough airflow were collected. Results: Participants coughed more in the single-task condition compared to the dual-task condition (p = 0.001). Participants' urge-to-cough ratings were lower in the dual-task condition (x¯ = 2, "slight") compared to the single-task condition (x¯ = 3, "moderate"; p = 0.007). Participants' cough reflex sensitivity thresholds were significantly increased in the dual-task condition (p = 0.002). Cough peak expiratory flow rates did not change between the 2 conditions (p = 0.34). Conclusions: Somatosensation of tussive stimuli changes during dual tasking. Abnormal cortical resource allocation may be a mechanism involved in silent aspiration.

While religion and spirituality (R/S) have been broadly studied for their positive mental health impacts, instances of abuse within religious or spiritual contexts remain under-researched. This scoping review aims to elucidate how individuals experiencing such abuse navigate their trauma, find meaning, and foster posttraumatic growth (PTG). The research was conducted using a scoping review methodology as a guide, and 10 articles were selected based on predefined inclusion and exclusion criteria. Synthesizing these articles revealed the following three central themes: recognizing abuse, relaying one's story, and redefining spirituality. Survivors often face disbelief and stigma, hindering their ability to process their experiences. However, narrative sharing enables many to reclaim agency and healing through validation and the integration of the narrative into one's life story. Additionally, survivors often transform spirituality, shifting from rigid frameworks to more nuanced and flexible understandings of the Divine and self. These findings underscore the importance of trauma-informed, spiritually sensitive clinical approaches that validate survivors' experiences, facilitate narrative sharing, and support spiritual redefining. Future research must address knowledge gaps, including the development of improved assessment tools, exploration of effective treatment strategies, and the unifying of terms to better support survivors' healing journeys and promote meaning-making and PTG in the aftermath of R/S abuse. [ABSTRACT FROM AUTHOR]

Background:In December 2021, the Region 1 Disaster Health Response System, the state of Vermont, and the National Emergency Tele-Critical Care Network partnered to provide statewide access to disaster teleconsultations during COVID-19 surge conditions. In this case report, we describe how a disaster teleconsultation system was implemented in Vermont to provide access to temporary tele-critical care consultations during the Omicron COVID-19 surge. Methods:We measured the time from request of service to implementation and calculated descriptive statistics. Results:Seven of Vermont's 14 hospitals requested the service. Despite a technology solution capable of providing services within hours, mean time to service implementation was 27 days (interquartile range 20–41 days). Conclusions:Integration of disaster teleconsultation systems into state and local emergency management plans are needed to bring administrative start-up times in line with technical readiness. [ABSTRACT FROM AUTHOR]