Multiple distinct evolutionary mechanisms govern the dynamics of selfish mitochondrial genomes in Caenorhabditis elegans.

Cells possess multiple mitochondrial DNA (mtDNA) copies, which undergo semi-autonomous replication and stochastic inheritance. This enables mutant mtDNA variants to arise and selfishly compete with cooperative (wildtype) mtDNA. Selfish mitochondrial genomes are subject to selection at different levels: they compete against wildtype mtDNA directly within hosts and indirectly through organism-level selection. However, determining the relative contributions of selection at different levels has proven challenging. We overcome this challenge by combining mathematical modeling with experiments designed to isolate the levels of selection. Applying this approach to many selfish mitochondrial genotypes in Caenorhabditis elegans reveals an unexpected diversity of evolutionary mechanisms. Some mutant genomes persist at high frequency for many generations, despite a host fitness cost, by aggressively outcompeting cooperative genomes within hosts. Conversely, some mutant genomes persist by evading inter-organismal selection. Strikingly, the mutant genomes vary dramatically in their susceptibility to genetic drift. Although different mechanisms can cause high frequency of selfish mtDNA, we show how they give rise to characteristically different distributions of mutant frequency among individuals. Given that heteroplasmic frequency represents a key determinant of phenotypic severity, this work outlines an evolutionary theoretic framework for predicting the distribution of phenotypic consequences among individuals carrying a selfish mitochondrial genome.

Simple improvements in vector design afford substantial gains in AAV delivery of aggregation-slowing Aß variants.

Adeno-associated virus (AAV) gene therapy for neurological disease has gained traction due to stunning advances in capsid evolution for CNS targeting. With AAV brain delivery now in focus, conventional improvements in viral expression vectors offer a complementary route for optimizing gene delivery. We previously introduced a novel AAV gene therapy to slow amyloid aggregation in the brain based on neuronal release of an Aß sequence variant that inhibited fibrilization of wild-type Aß. Here we explore three coding elements of the virally delivered DNA plasmid in an effort to maximize the production of therapeutic peptide in the brain. We demonstrate that simply replacing the Gaussia luciferase signal peptide with the mouse immunoglobulin heavy chain signal peptide increased release of variant Aß by ∼5-fold. Sequence modifications within the expressed minigene further increased peptide release by promoting γ-secretase cleavage. Addition of a cytosolic fusion tag compatible with γ-secretase interaction allowed viral transduction to be tracked by immunostaining, independent from the variant Aß peptide. Collectively these construct modifications increased neuronal production of therapeutic peptide by 10-fold upon intracranial AAV injection of neonatal mice. These findings demonstrate that modest changes in expression vector design can yield substantial gains in AAV efficiency for therapeutic applications.

Human microbiome: Impact of newly approved treatments on C. difficile infection.

DISCLAIMER: In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. PURPOSE: The primary purposes of this review are to provide a brief overview of the microbiome, discuss the most relevant outcome data and key characteristics of each live microbiome agent, and pose questions for consideration going forward as these agents are integrated into clinical practice. SUMMARY: The management of Clostridiodes difficile infection (CDI) remains a difficult clinical conundrum, with recurrent CDI occurring in 15% to 35% of patients and causing significant morbidity and decreased quality of life. For patients with frequent CDI recurrences, fecal microbiota transplantation (FMT) has been demonstrated to have significant benefit but also significant risks, and FMT is not approved by the US Food and Drug Administration (FDA) for that indication. FDA has established a new therapeutic class for agents known as live biotherapeutic products (LBPs) that offer significant advantages over FMT, including standardized screening, testing, and manufacturing as well as known quantities of organisms contained within. Two new live microbiome products within this class were recently approved by FDA for prevention of CDI recurrences in adult patients following treatment for recurrent CDI with standard antimicrobial therapy. Both agents had demonstrated efficacy in registry trials in preventing CDI recurrence but differ significantly in a number of characteristics, such as route of administration. Cost as well as logistics are current obstacles to use of these therapies. CONCLUSION: Live microbiome therapy is a promising solution for patients with recurrent CDI. Future studies should provide further evidence within yet-to-be-evaluated populations not included in registry studies. This along with real-world evidence will inform future use and clinical guideline placement.

A new species of karst-dwelling bent-toed gecko of the Cyrtodactylusintermedius group (Squamata, Gekkonidae) from eastern Thailand and the phylogenetic placement of C.intermedius.

A new karst-dwelling bent-toed gecko of the Cyrtodactylusintermedius group is described from Khlong Hat District, Sa Kaeo Province, eastern Thailand, based on an integrative taxonomic analysis of genetic data and morphological characteristics. Phylogenetic analyses using the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene revealed that topotypes of C.intermedius were sister to a clade containing C.kulenensis from Cambodia, an unnamed lineage from Sakaerat Biosphere Reserve in Nakhon Ratchasima Province, Thailand, and the Khlong Hat lineage described here as Cyrtodactyluskhlonghatensis sp. nov. Multivariate analyses of morphometric and meristic characters showed that C.khlonghatensis sp. nov. is morphologically distinct from all other species in the group by having the combination of SVL 76.5-82.8 mm in adult males and 88.5 mm in an adult female; eight supralabial and nine infralabial scales; 30-32 paravertebral tubercles; 20 or 21 longitudinal rows of dorsal tubercles; 43 or 44 ventral scales; seven or eight expanded subdigital lamellae on the 4th toe; 12 unmodified subdigital lamellae on the 4th toe; 19 or 20 total subdigital lamellae on the 4th toe; 31 or 32 total number of enlarged femoral scales; enlarged femoral and precloacal scales continuous; 6-8 pore-bearing precloacal scales in males; three or four rows of enlarged post-precloacal scales; 1-3 postcloacal tubercles; proximal femoral scales less than one-half the size of distal femoral scales; absence of interdigital pocketing between digits of forefeet and hindfeet; and posterior border of the nuchal loop rounded. Uncorrected pairwise genetic divergences (p-distances) between the new species and other species of the intermedius group ranged from 4.73-22.55%. The discovery of this new species exclusively in isolated karst formations from the Thai-Cambodia border suggests that there may be further undiscovered Cyrtodactylus in unexplored karst landscapes along the border of eastern Thailand and western Cambodia.

Impact of waning immunity against SARS-CoV-2 severity exacerbated by vaccine hesitancy.

The SARS-CoV-2 pandemic has generated a considerable number of infections and associated morbidity and mortality across the world. Recovery from these infections, combined with the onset of large-scale vaccination, have led to rapidly-changing population-level immunological landscapes. In turn, these complexities have highlighted a number of important unknowns related to the breadth and strength of immunity following recovery or vaccination. Using simple mathematical models, we investigate the medium-term impacts of waning immunity against severe disease on immuno-epidemiological dynamics. We find that uncertainties in the duration of severity-blocking immunity (imparted by either infection or vaccination) can lead to a large range of medium-term population-level outcomes (i.e. infection characteristics and immune landscapes). Furthermore, we show that epidemiological dynamics are sensitive to the strength and duration of underlying host immune responses; this implies that determining infection levels from hospitalizations requires accurate estimates of these immune parameters. More durable vaccines both reduce these uncertainties and alleviate the burden of SARS-CoV-2 in pessimistic outcomes. However, heterogeneity in vaccine uptake drastically changes immune landscapes toward larger fractions of individuals with waned severity-blocking immunity. In particular, if hesitancy is substantial, more robust vaccines have almost no effects on population-level immuno-epidemiology, even if vaccination rates are compensatorily high among vaccine-adopters. This pessimistic scenario for vaccination heterogeneity arises because those few individuals that are vaccine-adopters are so readily re-vaccinated that the duration of vaccinal immunity has no appreciable consequences on their immune status. Furthermore, we find that this effect is heightened if vaccine-hesitants have increased transmissibility (e.g. due to riskier behavior). Overall, our results illustrate the necessity to characterize both transmission-blocking and severity-blocking immune time scales. Our findings also underline the importance of developing robust next-generation vaccines with equitable mass vaccine deployment.

Impacts of invasion on a freshwater cleaning symbiosis.

Organismal invasions have repeatedly been cited as a driving force behind the loss of biodiversity. Unlike many other impacts of invasion, the effect of invasion on native symbiont communities has received less attention. The introduction of invasive hosts presents a potential opportunity to native symbionts; invasive hosts could benefit native symbionts through providing a novel host environment that improves symbiont fitness relative to their fitness on native hosts. Alternatively, invasive hosts could noncompetent hosts for native symbionts, resulting in negative impacts on native symbiont abundance and diversity. Crayfish in the northern hemisphere host diverse assemblages of obligate annelid symbionts (P: Anellida, O: Branchiobdellida). Two invasive crayfish hosts in the genus Faxonius have been introduced and are interacting with the native crayfish hosts and their symbionts in three watersheds in western Virginia, USA. Previous studies suggest that the invasive host F. cristavarius is a less competent host for symbionts compared to native hosts in the genus Cambarus. We carried out an extensive survey in these watersheds to determine impacts of varying degrees of invasion on branchiobdellidan abundance and diversity. We also conducted a complementary host replacement experiment to investigate how increases in the relative abundance of invasive hosts contributes to observed patterns of symbiont abundance and diversity in the field. In our survey, as the proportion of invasive hosts at a site increased, branchiobdellidan abundance and diversity declined significantly. In the experiment, the worms dispersed onto both native and invasive hosts. As the percentage of noncompetent F. cristavarius hosts increased, the survival of branchiobdellidans declined. Both symbiont survival and opportunities for successful dispersal are reduced as this noncompetent invasive host progressively displaces native hosts, which imperils the integrity of native host-symbiont systems. Given that many native hosts accrue significant fitness benefits from their relationships with native symbionts, including hosts in our study system, losses of beneficial symbionts may produce a positive feedback loop that decreases invasion resistance of native species, exacerbates the effects of invasions, and presents a major conservation issue in invaded systems.

A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state.

Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein ßγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gßγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.

Significant Reduction of Radiation-Induced Death in Mice Treated with PrC-210 and G-CSF after Irradiation.

The search for single or combined radiation countermeasures that mitigate the development of Acute Radiation Syndrome (ARS) after radiation exposure remains a prominent goal of the U.S. government. This study was undertaken to determine whether PrC-210 and G-CSF, when administered 24-48 h postirradiation, would confer an additive or synergistic survival benefit and mitigate ARS in mice that had received an otherwise 96% lethal radiation dose. Our results show that optimum systemic doses of PrC-210 and G-CSF, when administered 24 h or later after a 96% lethal dose of whole-body irradiation, conferred: 1. strong individual survival benefits (PrC-210 44%, P = 0.003), (G-CSF 48%, P = 0.0002), 2. a profound combined 85% survival benefit (P < 0.0001) when administered together, and on day 14 postirradiation, 3. peripheral white blood cell/lymphocyte counts equal to unirradiated controls, 4. dense bone marrow cell density (>65% of unirradiated controls), 5. jejunal villi density that equaled 90% of unirradiated controls, and 6. spleen weights that equaled 93% of unirradiated controls. Our results show that PrC-210 and G-CSF given together 24 h after irradiation confer strong additive efficacy by protecting the immune system, and enabling recovery of the bone marrow, and they work synergistically to enable recovery of peripheral white blood cells in circulating blood.

Xylazine is an agonist at kappa opioid receptors and exhibits sex-specific responses to opioid antagonism.

Xylazine is in the unregulated drug supply at increasing rates, usually combined with fentanyl, necessitating understanding of its pharmacology. Despite commentary from politicians, and public health officials, it is unknown how xylazine impacts naloxone efficacy, and. few studies have examined it alone. Here, we examine the impact of xylazine alone and in combination with fentanyl on several behaviors in mice. Surprisingly, naloxone precipitates withdrawal from xylazine and fentanyl/xylazine coadministration, with enhanced sensitivity in females. Further, xylazine is a full agonist at kappa opioid receptors, a potential mechanism for its naloxone sensitivity. Finally, we demonstrate surprising effects of xylazine to kappa opioid antagonism, which are relevant for public health considerations. These data address an ongoing health crisis and will help inform critical policy and healthcare decisions.

Optimization of Tumor Dissection Procedures Leads to Measurable Improvement in the Quality of Molecular Testing.

Molecular tests have an inherent limit of detection (LOD) and, therefore, require samples with sufficiently high percentages of neoplastic cells. Many laboratories use tissue dissection; however, optimal procedures for dissection and quality assurance measures have not been established. In this study, several modifications to tissue dissection procedures and workflow were introduced over 4 years. Each modification resulted in a significant improvement in one or more quality assurance measures. The review of materials following dissection resulted in a 90% reduction in KRAS mutations below the stated LOD (P = 0.004). Mutation allele frequencies correlated best with estimated tumor percentages for pathologists with more experience in this process. The direct marking of unstained slides, use of a stereomicroscope, validation of extraction from diagnostic slides, and use of a robust, targeted next-generation sequencing platform all resulted in reduction of quantity not sufficient specimens from 20% to 25% to nearly 0%, without a significant increase in test failures or mutations below the LOD. These data indicate that post-dissection review of unstained slides and monitoring quantity not sufficient rate, test failure rate, and mutation allele frequencies are important tumor dissection quality assurance measures that should be considered by laboratories performing tissue dissections. The amendments to tissue dissection procedures enacted during this study resulted in a measurable improvement in the quality and reliability of this process based on these metrics.

Tournament Recovery Profiles and Physical Demands in a Collegiate Women's Tennis Team.

ABSTRACT: Tendero-Ortiz, E, Johnson, MJ, Horsfall, CM, Vondrasek, JD, Grosicki, GJ, Riemann, BL, and Flatt, AA. Tournament recovery profiles and physical demands in a collegiate women's tennis team. J Strength Cond Res XX(X): 000-000, 2024-We aimed to characterize recovery profiles and tournament physical demands in women's collegiate tennis players. A Division 1 team (n = 9) participated in the study. Markers of cardiac autonomic (resting heart rate [HR], HR variability), neuromuscular (isometric handgrip strength, seated single-arm shot-put test [SSAPT], hexagon agility, countermovement jump characteristics), and perceptual recovery were obtained before the tournament (baseline) and again 1 and 2 days posttournament. Cardiorespiratory (HR) and movement characteristics from matches were quantified with wearable devices. p values < 0.05 were statistically significant. No recovery markers differed from baseline (ps > 0.05), although small effect size reductions 1 day posttournament were noted for SSAPT, hexagon agility, and select countermovement jump characteristics. In addition, hexagon agility times and SSAPT were slower (p < 0.01) and shorter (p < 0.05), respectively, at 1 versus 2 days posttournament. Similarly, relative to 1 day posttournament, perceptual makers were improved 2 days posttournament (ps < 0.05). Mean and peak HR were higher for singles versus doubles matches  (ps < 0.05). Except for average speed, movement parameters were greater during singles versus doubles matches (ps < 0.05). Markers of recovery were minimally affected 1 day posttournament relative to baseline, but perceptual and select neuromuscular markers were most improved 2 days posttournament. Thus, passive rest or limited intensity training 1 day posttournament seems advisable. Competition HR and movement profiles inform practitioners of the cardiorespiratory and locomotor demands of women's collegiate tennis, which may be useful in designing preparatory conditioning programs to ensure that players attain match-specific physical capacities in training before competition.

Demonstration of Tunable Control over a Delayed-Release Vaccine Using Atomic Layer Deposition.

Many vaccines require multiple doses for full efficacy, posing a barrier for patient adherence and protection. One solution to achieve full vaccination may be attained with single-administration vaccines containing multiple controlled release doses. In this study, delayed-release vaccines were generated using atomic layer deposition (ALD) to coat antigen-containing powders with alumina. Using in vitro and in vivo methods, we show that increasing the coat thickness controls the kinetics of antigen release and antibody response, ranging from weeks to months. Our results establish an in vitro-in vivo correlation with a level of tunable control over the antigen release and antibody response times with the potential to impact future vaccine design.

A proximity proteomics pipeline with improved reproducibility and throughput.

Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.

Prevalence of and Risks for Bacterial Infections in Hospitalized Children With Bronchiolitis.

BACKGROUND AND OBJECTIVES: Viral bronchiolitis is a common pediatric illness. Treatment is supportive; however, some children have concurrent serious bacterial infections (cSBIs) requiring antibiotics. Identifying children with cSBI is challenging and may lead to unnecessary treatment. Improved understanding of the prevalence of and risk factors for cSBI are needed to guide treatment. We sought to determine the prevalence of cSBI and identify factors associated with cSBI in children hospitalized with bronchiolitis. METHODS: We performed a retrospective cohort study of children <2 years old hospitalized with bronchiolitis at a free-standing children's hospital from 2012 to 2019 identified by International Classification of Diseases codes. cSBI was defined as bacteremia, urinary tract infection, meningitis, or pneumonia. Risk factors for cSBI were identified using logistic regression. RESULTS: We identified 7871 admissions for bronchiolitis. At least 1 cSBI occurred in 4.2% of these admissions; with 3.5% meeting our bacterial pneumonia definition, 0.4% bacteremia, 0.3% urinary tract infection, and 0.02% meningitis. cSBI were more likely to occur in children with invasive mechanical ventilation (odds ratio [OR] 2.53, 95% confidence interval [CI] 1.78-3.63), a C-reactive protein ≥4 mg/dL (OR 2.20, 95% CI 1.47-3.32), a concurrent complex chronic condition (OR 1.67, 95% CI 1.22-2.25) or admission to the PICU (OR 1.46, 95% CI 1.02-2.07). CONCLUSIONS: cSBI is uncommon among children hospitalized with bronchiolitis, with pneumonia being the most common cSBI. Invasive mechanical ventilation, elevated C-reactive protein, presence of complex chronic conditions, and PICU admission were associated with an increased risk of cSBI.

The Role of Risk Tolerance in a Patient's Decision to Undergo Total Knee and Hip Arthroplasty.

BACKGROUND: A patient's decision-making process to undergo surgery is crucial for surgeons to understand for patient-counseling purposes. Total knee and hip arthroplasty, like any other major surgery, is associated with serious, sometimes life-threatening, complications. Using the results of discrete choice experiments (DCEs), we aimed to understand the relationship between a patient's risk tolerance and choosing to undergo surgery in real life. METHODS: This is a retrospective study of prospectively collected DCE results for 142 potential knee or hip arthroplasty clinic patients from October 2021 to March 2022. The DCE presented the patient with 2 scenarios, each of which was made up of different combinations of attributes and levels. A hierarchal Bayesian model was used to obtain a risk score that reflected the risk attributes chosen by each patient. Logistic regressions were then used to evaluate the association between a patient's willingness to incur risk and their decision to undergo a total joint arthroplasty. RESULTS: Of the 142 patients enrolled in the DCE, 89 (62.3%) underwent a total joint arthroplasty. Risk score (odds ratio [OR] = 2.6, 95% confidence interval [CI] 1.1 to 6.6, P = 0.04), men (OR = 2.5, 95% CI 1.1 to 5.9, P = 0.028), and patients who have hip osteoarthritis (OR = 2.4, 95% CI 1.1 to 5.5, P = 0.036) increased the odds of undergoing arthroplasty, whereas physical function of at least 75% at the initial visit (OR = 0.3, 95% CI 0.1 to 0.7, P = 0.004) decreased these odds. CONCLUSIONS: We found that a patient's willingness to incur risk, lower baseline physical function, and men were all independently associated with undergoing total knee arthroplasty. We believe that these findings prompt much-needed future studies that focus solely on the relationship between patients' inherent risk behavior and surgical and patient-reported outcomes.

Molecular glues as potential GPCR therapeutics.

"Molecular Glues" are defined as small molecules that can either be endogenous or synthetic which promote interactions between proteins at their interface. Allosteric modulators, specifically GPCR allosteric modulators, can promote both the association and the dissociation of a given receptor's transducer but accomplishes this "at a distance" from the interface. However, recent structures of GPCR G protein complexes in the presence of allosteric modulators indicate that some GPCR allosteric modulators can act as "molecular glues" interacting with both the receptor and the transducer at the interface biasing transducer signaling in both a positive and negative manner depending on the transducer. Given these phenomena we discuss the implications for this class of allosteric modulators to be used as molecular tools and for future drug development.

Impact of CYP3A5 Polymorphisms on Pediatric Asthma Outcomes.

Genetic variation among inhaled corticosteroid (ICS)-metabolizing enzymes may affect asthma control, but evidence is limited. This study tested the hypothesis that single-nucleotide polymorphisms (SNPs) in Cytochrome P450 3A5 (CYP3A5) would affect asthma outcomes. Patients aged 2-18 years with persistent asthma were recruited to use the electronic AsthmaTracker (e-AT), a self-monitoring tool that records weekly asthma control, medication use, and asthma outcomes. A subset of patients provided saliva samples for SNP analysis and participated in a pharmacokinetic study. Multivariable regression analysis adjusted for age, sex, race, and ethnicity was used to evaluate the impact of CYP3A5 SNPs on asthma outcomes, including asthma control (measured using the asthma symptom tracker, a modified version of the asthma control test or ACT), exacerbations, and hospital admissions. Plasma corticosteroid and cortisol concentrations post-ICS dosing were also assayed using liquid chromatography-tandem mass spectrometry. Of the 751 patients using the e-AT, 166 (22.1%) provided saliva samples and 16 completed the PK study. The e-AT cohort was 65.1% male, and 89.6% White, 6.0% Native Hawaiian, 1.2% Black, 1.2% Native American, 1.8% of unknown race, and 15.7% Hispanic/Latino; the median age was 8.35 (IQR: 5.51-11.3) years. CYP3A5*3/*3 frequency was 75.8% in White subjects, 50% in Native Hawaiians and 76.9% in Hispanic/Latino subjects. Compared with CYP3A5*3/*3, the CYP3A5*1/*x genotype was associated with reduced weekly asthma control (OR: 0.98; 95% CI: 0.97-0.98; p < 0.001), increased exacerbations (OR: 6.43; 95% CI: 4.56-9.07; p < 0.001), and increased asthma hospitalizations (OR: 1.66; 95% CI: 1.43-1.93; p < 0.001); analysis of 3/*3, *1/*1 and *1/*3 separately showed an allelic copy effect. Finally, PK analysis post-ICS dosing suggested muted changes in cortisol concentrations for patients with the CYP3A5*3/*3 genotype, as opposed to an effect on ICS PK. Detection of CYP3A5*3/3, CYPA35*1/*3, and CYP3A5*1/*1 could impact inhaled steroid treatment strategies for asthma in the future.

An active electronic bidirectional interface for high resolution interrogation of the spinal cord.

Epidural electrical stimulation (EES) has shown promise as both a clinical therapeutic tool and research aid in the study of nervous system function. However, available clinical paddles are limited to using a small number of contacts due to the burden of wires necessary to connect each contact to the therapeutic device. Here, we introduce for the first time the integration of a hermetic active electronic multiplexer onto the electrode paddle array itself, removing this interconnect limitation. We evaluated the chronic implantation of an active electronic 60-contact paddle (the HD64) on the lumbosacral spinal cord of two sheep. The HD64 was implanted for 13 months and 15 months, with no device-related malfunctions or adverse events. We identified increased selectivity in EES-evoked motor responses using dense stimulating bipoles. Further, we found that dense recording bipoles decreased the spatial correlation between channels during recordings. Finally, spatial electrode encoding enabled a neural network to accurately perform EES parameter inference for unseen stimulation electrodes, reducing training data requirements. A high-density EES paddle, containing active electronics safely integrated into neural interfaces, opens new avenues for the study of nervous system function and new therapies to treat neural injury and dysfunction.

MRGPRX4 mediates phospho-drug-associated pruritus in a humanized mouse model.

The phosphate modification of drugs is a common chemical strategy to increase solubility and allow for parenteral administration. Unfortunately, phosphate modifications often elicit treatment- or dose-limiting pruritus through an unknown mechanism. Using unbiased high-throughput drug screens, we identified the Mas-related G protein-coupled receptor X4 (MRGPRX4), a primate-specific, sensory neuron receptor previously implicated in itch, as a potential target for phosphate-modified compounds. Using both Gq-mediated calcium mobilization and G protein-independent GPCR assays, we found that phosphate-modified compounds potently activate MRGPRX4. Furthermore, a humanized mouse model expressing MRGPRX4 in sensory neurons exhibited robust phosphomonoester prodrug-evoked itch. To characterize and confirm this interaction, we further determined the structure of MRGPRX4 in complex with a phosphate-modified drug through single-particle cryo-electron microscopy (cryo-EM) and identified critical amino acid residues responsible for the binding of the phosphate group. Together, these findings explain how phosphorylated drugs can elicit treatment-limiting itch and identify MRGPRX4 as a potential therapeutic target to suppress itch and to guide future drug design.

The Cytochrome P450 2C8*3 Variant (rs11572080) Is Associated with Improved Asthma Symptom Control in Children and Altered Lipid Mediator Production and Inflammatory Response in Human Bronchial Epithelial Cells.

This study investigated an association between the cytochrome P450 (CYP) 2C8*3 polymorphism with asthma symptom control in children and changes in lipid metabolism and pro-inflammatory signaling by human bronchial epithelial cells (HBECs) treated with cigarette smoke condensate (CSC). CYP genes are inherently variable in sequence, and while such variations are known to produce clinically relevant effects on drug pharmacokinetics and pharmacodynamics, the effects on endogenous substrate metabolism and associated physiologic processes are less understood. In this study, CYP2C8*3 was associated with improved asthma symptom control among children: Mean asthma control scores were 3.68 (n = 207) for patients with one or more copies of the CYP2C8*3 allele versus 4.42 (n = 965) for CYP2C8*1/*1 (P = 0.0133). In vitro, CYP2C8*3 was associated with an increase in montelukast 36-hydroxylation and a decrease in linoleic acid metabolism despite lower mRNA and protein expression. Additionally, CYP2C8*3 was associated with reduced mRNA expression of interleukin-6 (IL-6) and C-X-C motif chemokine ligand 8 (CXCL-8) by HBECs in response to CSC, which was replicated using the soluble epoxide hydrolase inhibitor, 12-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]-dodecanoic acid. Interestingly, 9(10)- and 12(13)- dihydroxyoctadecenoic acid, the hydrolyzed metabolites of 9(10)- and 12(13)- epoxyoctadecenoic acid, increased the expression of IL-6 and CXCL-8 mRNA by HBECs. This study reveals previously undocumented effects of the CYP2C8*3 variant on the response of HBECs to exogenous stimuli. SIGNIFICANCE STATEMENT: These findings suggest a role for CYP2C8 in regulating the epoxyoctadecenoic acid:dihydroxyoctadecenoic acid ratio leading to a change in cellular inflammatory responses elicited by environmental stimuli that exacerbate asthma.