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Abstract The role of the cerebral cortex in self-initiated versus sensory-driven movements is central to understanding volitional action. Whether the differences in these two movement classes are due to specific cortical areas versus more cortex-wide engagement is debated. Using wide-field Ca2+ imaging, we compared neural dynamics during spontaneous and motorized treadmill locomotion, determining the similarities and differences in cortex-wide activation and functional connectivity (FC). During motorized locomotion, the cortex exhibits greater activation globally prior to and during locomotion starting compared to spontaneous and less during steady-state walking, during stopping, and after termination. Both conditions are characterized by FC increases in anterior secondary motor cortex (M2) nodes and decreases in all other regions. There are also cortex-wide differences; most notably, M2 decreases in FC with all other nodes during motorized stopping and after termination. Therefore, both internally- and externally-generated movements widely engage the cortex, with differences represented in cortex-wide activation and FC patterns.

Japanese climbing fern (Lygodium japonicum) is a vine native to the open forests of eastern Asia that has become an invasive species in the U.S. Herbarium records first noted this species in the U.S. in 1903 (Georgia), with spread to eight states by the end of the 1930s and current establishment in 10 states of the southeastern U.S. We aimed to ask three questions regarding the introduction of L. japonicum: (1) Was there a single Japanese climbing fern introduction or were there multiple introductions? (2) What is the distribution of genotypes in the U.S.? and (3) What are the source population(s) from the native range in Asia? We sequenced the chloroplast genome from 74 L. japonicum herbarium specimens representing 24 native and 50 invasive range populations. Seventeen haplotypes were found in the native range compared to three in the invasive range. Our results indicate L. japonicum has low genotypic diversity in the invasive range relative to the native range. Even with low genotypic diversity, these data suggest at least three introductions of L. japonicum. However, we were unable to define the native source population(s) of invasive L. japonicum.

Sarah Bentley,1 Elizabeth Exall,1 Lucy Morgan,1 Nicolas Roche,2 Kamlesh Khunti,3 Rebecca Rossom,4 James Piercy,5 Rob Arbuckle,1 Victoria S Higgins5 1Patient-Centered Outcomes, Adelphi Values, Bollington, UK; 2Respiratory Medicine, Cochin Hospital, APHP Centre University of Paris, Institut Cochin (UMR1016), Paris, France; 3Diabetes Research Centre, Leicester University, Leicester, UK; 4HealthPartners Institute, University of Minnesota Medical School, Minneapolis, MN, USA; 5Adelphi Real World, Bollington, UKCorrespondence: Rob Arbuckle, Adelphi Values, Adelphi Mill, Grimshaw Lane, Bollington, SK10 5JB, UK, Tel +44 1625 576815, Fax +44 1625 577328, Email rob.arbuckle@adelphivalues.comPurpose: Medication adherence is crucial for achieving clinical goals. Medication adherence drivers and behaviors were explored across multiple conditions, countries, and medication schedules/modalities to develop a conceptual model of medication adherence, which could later be used to support development of a patient-reported outcome (PRO) measure of adherence.Patients and Methods: Targeted review of qualitative literature identified important medication adherence concepts. Fifty-seven qualitative concept elicitation interviews were conducted (USA n=21, Spain n=18, Germany n=18). Participants were prescribed medication for: hypertension (n=9), asthma (n=8), multiple myeloma (n=8), psoriasis (n=8), diabetes (n=7), depression (n=7), multiple sclerosis (n=7), and/or schizophrenia (n=6). Thematic analysis of verbatim transcripts was performed. Expert clinicians (n=3) provided input throughout.Results: Nine qualitative articles were selected for review from 2168 screened abstracts. Forty-two medication adherence concepts were reported and grouped into 10 domains. Eight forms of medication adherence were reported during interviews, along with 27 drivers of non-adherence, all of which were incorporated into a conceptual model. Participants reported skipping medication doses (n=36/57; 63.2%) or taking medication later in the day than prescribed (n=29/57; 50.9%). Common drivers of non-adherence included forgetfulness (n=35/57; 61.4%), being out of the usual routine (n=31/57; 54.4%) and being busy (n=22/57; 38.6%). US participants were more likely to report non-adherence due to low perceived efficacy (n=6/21, 28.6%) and cost (n=5/21, 23.8%) than German (n=1/18, 5.6%; n=0/18, 0.0%) or Spanish (n=2/18, 11.1%; n=1/18, 5.6%) participants.Conclusion: Findings highlight the diverse forms and drivers of medication non-adherence, informing the development of a comprehensive conceptual model of medication adherence. The conceptual model builds on and advances previous models of medication adherence and can be used by healthcare professionals to understand and interpret barriers to medication adherence and how best to support patients in taking their medication as intended.Plain Language Summary: Medication adherence is the extent to which a patient takes their medication as prescribed. This paper describes a literature review and concept elicitation interviews to identify forms and drivers of medication adherence across a diverse sample of participants. Forms of non-adherence identified included: deviating from the prescription, skipping a dose, taking a different amount, and taking medication at a different time. Behaviours and drivers can vary by condition, treatment modality, and dosing schedule.This research highlights the variation in the prevalence of medication non-adherence, and the different forms and drivers of non-adherence, based on individuals’ demographic and clinical characteristics. The conceptual model developed advances previous models of medication adherence and may support healthcare professionals in the management of patients and how they can be supported to take medication as intended. The research ultimately informed the development of the Adelphi Adherence Questionnaire (ADAQ©), a novel generic patient-reported outcome measure.Keywords: qualitative research, interviews, literature review, medication adherence

Abstract Background The glycolytic enzyme alpha-enolase is a known biomarker of many cancers and involved in tumorigenic functions unrelated to its key role in glycolysis. Here, we show that expression of alpha-enolase correlates with subcellular localisation and tumorigenic status in the MCF10 triple negative breast cancer isogenic tumour progression model, where non-tumour cells show diffuse nucleocytoplasmic localisation of alpha-enolase, whereas tumorigenic cells show a predominantly cytoplasmic localisation. Alpha-enolase nucleocytoplasmic localisation may be regulated by tumour cell-specific phosphorylation at S419, previously reported in pancreatic cancer. Results Here we show ENO1 phosphorylation can also be observed in triple negative breast cancer patient samples and MCF10 tumour progression cell models. Furthermore, prevention of alpha-enolase-S419 phosphorylation by point mutation or a casein kinase-1 specific inhibitor D4476, induced tumour-specific nuclear accumulation of alpha-enolase, implicating S419 phosphorylation and casein kinase-1 in regulating subcellular localisation in tumour cell-specific fashion. Strikingly, alpha-enolase nuclear accumulation was induced in tumour cells by treatment with the specific exportin-1-mediated nuclear export inhibitor Leptomycin B. This suggests that S419 phosphorylation in tumour cells regulates alpha-enolase subcellular localisation by inducing its exportin-1-mediated nuclear export. Finally, as a first step to analyse the functional consequences of increased cytoplasmic alpha-enolase in tumour cells, we determined the alpha-enolase interactome in the absence/presence of D4476 treatment, with results suggesting clear differences with respect to interaction with cytoskeleton regulating proteins. Conclusions The results suggest for the first time that tumour-specific S419 phosphorylation may contribute integrally to alpha-enolase cytoplasmic localisation, to facilitate alpha-enolase’s role in modulating cytoskeletal organisation in triple negative breast cancer. This new information may be used for development of triple negative breast cancer specific therapeutics that target alpha-enolase.

The holobiont theory of evolution explains how individuals are deeply symbiotic with their gut microbes, such that microbes are adapted to influence host metabolism, immunity and behaviour, as signalled from the gut to the brain. For eusocial taxa like the Western honey bee (Apis mellifera), this brain-gut axis may scale up from the individual to affect entire colonies. Here, we examine how microbial supplementation of honey bee feeds could manipulate the brain-gut axis to affect hygienic and other social behaviours relevant to beekeeping, such as foraging, recruitment (dance language) and defence. To illustrate this concept, we focus on various lactic acid-producing bacteria that can synthesize neurotransmitters such as octopamine, dopamine, serotonin and γ-aminobutyric acid, which can influence an individual bee’s behavioural cycles and responsiveness to environmental cues. If the behaviour of a worker bee can be deliberately manipulated, and this effect multiplied across many workers, microbial neurotherapeutics could conceivably render colonies more behaviourally responsive to symptoms of disease, or more motivated to forage or possibly less aggressive towards beekeepers. Drawing from the scientific literature, we infer how microbial supplements, such as neurostimulatory or neurosuppressive probiotics, could be applied or even engineered to co-opt the brain-gut axis to bolster colony health or improve performance. The mechanistic link between the gut microbiota and the collective social behaviour of single colonies remains an understudied aspect of honey bee social biology with relevance to apiculture.

Abstract Pathogenic loss-of-function variants in BGN, an X-linked gene encoding biglycan, are associated with Meester-Loeys syndrome (MRLS), a thoracic aortic aneurysm/dissection syndrome. Since the initial publication of five probands in 2017, we have considerably expanded our MRLS cohort to a total of 18 probands (16 males and 2 females). Segregation analyses identified 36 additional BGN variant-harboring family members (9 males and 27 females). The identified BGN variants were shown to lead to loss-of-function by cDNA and Western Blot analyses of skin fibroblasts or were strongly predicted to lead to loss-of-function based on the nature of the variant. No (likely) pathogenic missense variants without additional (predicted) splice effects were identified. Interestingly, a male proband with a deletion spanning the coding sequence of BGN and the 5’ untranslated region of the downstream gene (ATP2B3) presented with a more severe skeletal phenotype. This may possibly be explained by expressional activation of the downstream ATPase ATP2B3 (normally repressed in skin fibroblasts) driven by the remnant BGN promotor. This study highlights that aneurysms and dissections in MRLS extend beyond the thoracic aorta, affecting the entire arterial tree, and cardiovascular symptoms may coincide with non-specific connective tissue features. Furthermore, the clinical presentation is more severe and penetrant in males compared to females. Extensive analysis at RNA, cDNA, and/or protein level is recommended to prove a loss-of-function effect before determining the pathogenicity of identified BGN missense and non-canonical splice variants. In conclusion, distinct mechanisms may underlie the wide phenotypic spectrum of MRLS patients carrying loss-of-function variants in BGN.

Hematogenous metastasis involves cancer cell migration to different locations from the primary tumor through the blood circulation. Von Willebrand factor (VWF) has been shown to play an important role in tumor cell adhesion to and extravasation from the endothelial cell lining of blood vessel walls during cancer metastasis. VWF may contribute to this process by interacting with tumor cells, endothelial cells, and platelets through various cell membrane receptors, such as platelet glycoprotein (GP)Ibα, P-selectin, ανβ3 and αIIbβ3 integrins, and glycocalyx. Blood flow can mechanically extend and activate VWF to bind platelets and associate intermolecularly with other VWF molecules in plasma or on the surface of endothelial cells, cancer cells, or platelets. This suggests a mechanoregulatory role of VWF in mediating the interactions between VWF and these cells to promote cancer cell adhesion to blood vessels. In this review, we will summarize the current knowledge of VWF function and the role of hydrodynamic forces in hematogenous cancer metastasis.

The Mount Meager Volcanic Complex (Q̓welq̓welústen) is an active glacier-capped volcanic massif in the Garibaldi Volcanic Belt (British Columbia) and the only known glaciovolcanic cave system in North America steadily releasing sulfur-rich gases. In September 2022, leveraging specialized cave explorer expertise, the fumarole-carved ice cave at the Job Glacier on Mt. Meager was surveyed. Direct measurements of fumarolic gas concentrations were taken at the source, with H2S >200 ppm, SO2 >100 ppm, CO2 ∼5,200 ppm, and CO ∼230 ppm. Snowpack and fumarole-associated sediments were characterized for microbial diversity, functional potential, and biogeochemistry including measurements of nutrients, major ions, dissolved organic and inorganic carbon concentrations as well as the stable isotope compositions of carbon, sulfur, hydrogen and oxygen. Green algae (Chlorophyta) dominated the snowpack, consistent with other Pacific Northwest glaciers. Representatives of Firmicutes were the most abundant bacterial sequences detected in our samples, contrasting with other glacier and snowpack samples which harbor abundant Sphingobacteria, Betaproteobacteria, and Alphaproteobacteria. Sediments and water collected inside the cave were mostly high in SO42- (5.3–185.2 mg/L) and acidic (pH = 3.6–6.0), while most other major anions and cations were below detection of the method used. Snow at the cave entrance had more SO42- (0.08 mg/L) and lower pH (5.9) than snow collected at a distance (SO42- undetectable, pH 7.6), suggesting influence by fumarole exhalations. Negative δ13C values of organic matter (−29.0‰ to −26.1‰, respectively) in sediments suggest in-situ microbial carbon transformations, findings that are supported by the presence of genes encoding complete heterotrophic and autotrophic carbon transformation pathways. The δ34S value of H2S was ∼0‰, suggesting a deep magmatic origin; however, both sulfur-oxidizing and sulfate-reducing microbial phyla were present in the sediment samples as were genes encoding both dissimilatory sulfur-oxidizing and sulfate-reducing pathways. Metagenomic data suggest diverse chemosynthetic lifestyles in the cave microbial community. This study provides insight on the microbiomes associated with a sulfidic glaciovolcanic system and identifies unique analog features for icy celestial bodies like Saturn’s moon Enceladus, where cryovolcanic activity may carry biomarkers from the subsurface and deposit them on surface ice.

Background: Despite limited evidence from intervention trials, replacing animal-source protein-rich foods with plant alternatives continues to be recommended as part of a healthy dietary pattern. Objectives: The objective of this study was to examine whether a diet containing fresh, lean beef elicits greater satiety, reduces ad libitum food intake, and is more acceptable compared with a diet containing plant alternatives in women with overweight. Methods: Seventeen women with overweight (mean ± SEM, age: 33 ± 1 y; BMI: 27.8 ± 0.1 kg/m2) completed an acute, tightly controlled, crossover design study. Participants were provided with eucaloric, isonitrogenous diets (15% of daily intake as protein) containing either 2 servings/d of fresh lean beef (BEEF) or plant equivalents (PLANT) for 7 d/pattern. During day 6 of each pattern, the participants completed a 10-h controlled-feeding, clinical testing day, which included repeated appetite and satiety questionnaires and blood sampling to assess pre- and postprandial plasma peptide YY (PYY) and GLP-1 across the day. On day 7, the participants completed a free-living testing day in which they consumed their respective protein foods and were provided with additional carbohydrate- and fat-rich foods to consume, ad libitum, during each eating occasion. Energy and macronutrient composition were assessed. A 2- to 3-wk washout period occurred between patterns. Results: No differences in daily satiety were detected between patterns. During the ad libitum testing day, 24-h food intake was not different between patterns (BEEF: 2714 ± 219 compared with PLANT: 2859 ± 147 kcals/d), BEEF led to fewer carbohydrates consumed compared with PLANT (338 ± 34 compared with 370 ± 22 g/d, P < 0.05), especially as sugar (169 ± 73 g compared with 186 ± 57 g, P = 0.05). Furthermore, BEEF was more well liked (i.e., higher flavor, texture, and acceptability) compared with PLANT (all, P < 0.05). Conclusions: Although satiety was similar between patterns, the consumption of animal-source protein-rich foods, such as fresh and lean beef, was more well liked and resulted in voluntary reductions in total carbohydrate and sugar intake in middle-aged women with overweight during a single ad libitum testing day.This study was registered at clinicaltrials.gov as NCT02614729.

ABSTRACT Despite the first-line recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are pressing barriers for optimizing its use for the treatment of non-Escherichia coli Enterobacterales UTI. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for minimal inhibitory concentration (MIC) determination is largely impractical. Using 160 clinical Klebsiella pneumoniae isolates, we sought to understand rates of skipped wells and MIC imprecision in broth microdilution (BMD) and how that compares to rates of error using AD. Though the Clinical and Laboratory Standards Institute refers to the skipped well phenomena in their recommendation against the use of BMD, there is a paucity of data on its frequency. While AD and BMD produced similar MIC50/90 values (32/256 µg/mL for AD and 64/256 µg/mL for BMD), essential agreement was poor. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells at a given concentration, as the most frequent scientific error. Growth in concentrations above the measured MIC occurred in up to 3.3% of wells and was seen within three dilutions of the MIC for BMD. Observation of single colonies either at or beyond the measured MIC for AD was also common and occurred up to 8.3% and 2.5% of the time, respectively. The frequent scientific error in both testing methods should prompt re-evaluation of AD guidelines and expansion of MIC testing methods for fosfomycin susceptibility testing, as poor agreement with another method prone to scientific error should not be the main detractor from BMD use.IMPORTANCEDespite the recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are barriers for optimizing its use. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for MIC determination is largely impractical. The use of broth microdilution (BMD) for fosfomycin testing is not recommended by the Clinical and Laboratory Standards Institute due to unsatisfactory precision and skipped wells—occurrence of no-growth in a single well before the minimal inhibitory concentration (MIC)—and trailing endpoints. We sought to understand rates of skipped wells and growth at concentrations above measured MICs in BMD and how that compares to scientific error using AD. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells for BMD and single colonies at or beyond measured MICs for AD were also common. Frequent scientific error in both methods should prompt re-evaluation of both AD and BMD for fosfomycin susceptibility testing.

ABSTRACT Over 160,000 people worldwide suffer from cystic fibrosis (CF), a genetic condition that causes mucus to accumulate in internal organs. Lung decline is a significant health burden for people with CF (pwCF), and chronic bacterial pulmonary infections are a major cause of death. Stenotrophomonas maltophilia complex (Smc) is an emerging, multidrug-resistant CF pathogen that can cause pulmonary exacerbations and result in higher mortality. However, little is known about the antagonistic interactions that occur between Smc isolates from pwCF and competitor bacteria. We obtained 13 Smc isolates from adult and pediatric pwCF located in the United States or Australia. We co-cultured these isolates with Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. We also performed whole-genome sequencing of these Smc isolates and compared their genomes using average nucleotide identity analyses. We observed that some Smc CF isolates can engage in antagonistic interactions with P. aeruginosa and S. aureus but recovered a substantial number of P. aeruginosa and S. aureus cells following co-cultures with all tested Smc isolates. By contrast, we discovered that most Smc CF isolates display strong antibacterial properties against E. coli cells and reduce recovery below detectable limits. Finally, we demonstrate that Smc CF strains from this study belong to diverse phylogenetic lineages.IMPORTANCEAntagonism toward competitor bacteria may be important for the survival of Stenotrophomonas maltophilia complex (Smc) in external environments, for the elimination of commensal species and colonization of upper respiratory tracts to enable early infections, and for competition against other pathogens after establishing chronic infections. These intermicrobial interactions could facilitate the acquisition of Smc by people with cystic fibrosis from environmental or nosocomial sources. Elucidating the mechanisms used by Smc to eliminate other bacteria could lead to new insights into the development of novel treatments.

Dust is a sink for many semi-volatile compounds including flame retardants of the organophosphate ester (OPE) and brominated flame-retardant (BFR) classes. Given the large amount of time that we spend indoors, our exposure to these compounds via dust is of significant interest. Here, we present a novel microextraction approach to determine quantitative levels of selected OPEs and BFRs sampled from residential air filters from HVAC systems using a small volume of solvent. Dust samples (25 mg) is extracted with 1 mL of hexane/acetone (50/50, v/v). Upon solvent extraction of these HVAC dust samples, the analytes (TCPP, TDCPP, TPHP, T24DtBPP, TBBPA, and TriBBPA) were quantified via gas chromatography-mass spectrometry (GC/MS) or liquid chromatography-mass spectrometry (LC/MS). The methods for extracting these compounds from HVAC dust samples are detailed here with extensive method validation data to demonstrate accuracy and precision of these methods. • Dust is a sink for many semi-volatile compounds, including novel or emerging indoor pollutants like the organophosphate ester flame retardant T24DtBPP. • Here, a small amount of dust (25 mg) is extracted with a small volume of solvent (1 mL hexane and acetone) prior to analysis via chromatographic separation and mass spectrometric detection.

Abstract Peptides or proteins containing small biomolecular aggregates, such as micelles, bicelles, droplets and nanodiscs, are pivotal in many fields ranging from structural biology to pharmaceutics. Monitoring dynamics of such systems has been limited by the lack of experimental methods that could directly detect their fast (picosecond to nanosecond) timescale dynamics. Spin relaxation times from NMR experiments are sensitive to such motions, but their interpretation for biomolecular aggregates is not straightforward. Here we show that the dynamic landscape of peptide-containing molecular assemblies can be determined by a synergistic combination of solution state NMR experiments and molecular dynamics (MD) simulations. Solution state NMR experiments are straightforward to implement without an excessive amount of sample, while direct combination of spin relaxation data to MD simulations enables interpretation of dynamic landscapes of peptides and other aggregated molecules. To demonstrate this, we interpret NMR data from transmembrane, peripheral, and tail anchored peptides embedded in micelles. Our results indicate that peptides and detergent molecules do not rotate together as a rigid body, but peptides rotate in a viscous medium composed of detergent micelle. Spin relaxation times also provide indirect information on peptide conformational ensembles. This work gives new perspectives on peptide dynamics in complex biomolecular assemblies.

Abstract Group A streptococcal strains potentially acquire new M protein gene types through genetic recombination (emm switching). To detect such variants, we screened 12,596 invasive GAS genomes for strains of differing emm types that shared the same multilocus sequence type (ST). Through this screening we detected a variant consisting of 16 serum opacity factor (SOF)-positive, emm pattern E, emm82 isolates that were ST36, previously only associated with SOF-negative, emm pattern A, emm12. The 16 emm82/ST36 isolates were closely interrelated (pairwise SNP distance of 0–43), and shared the same emm82-containing recombinational fragment. emm82/ST36 isolates carried the sof12 structural gene, however the sof12 indel characteristic of emm12 strains was corrected to confer the SOF-positive phenotype. Five independent emm82/ST36 invasive case isolates comprised two sets of genetically indistinguishable strains. The emm82/ST36 isolates were primarily macrolide resistant (12/16 isolates), displayed at least 4 different core genomic arrangements, and carried 11 different combinations of virulence and resistance determinants. Phylogenetic analysis revealed that emm82/ST36 was within a minor (non-clade 1) portion of ST36 that featured almost all ST36 antibiotic resistance. This work documents emergence of a rapidly diversifying variant that is the first confirmed example of an emm pattern A strain switched to a pattern E strain.

Abstract Oncogenic lesions in pancreatic ductal adenocarcinoma (PDAC) hijack the epigenetic machinery in stromal components to establish a desmoplastic and therapeutic resistant tumor microenvironment (TME). Here we identify Class I histone deacetylases (HDACs) as key epigenetic factors facilitating the induction of pro-desmoplastic and pro-tumorigenic transcriptional programs in pancreatic stromal fibroblasts. Mechanistically, HDAC-mediated changes in chromatin architecture enable the activation of pro-desmoplastic programs directed by serum response factor (SRF) and forkhead box M1 (FOXM1). HDACs also coordinate fibroblast pro-inflammatory programs inducing leukemia inhibitory factor (LIF) expression, supporting paracrine pro-tumorigenic crosstalk. HDAC depletion in cancer-associated fibroblasts (CAFs) and treatment with the HDAC inhibitor entinostat (Ent) in PDAC mouse models reduce stromal activation and curb tumor progression. Notably, HDAC inhibition (HDACi) enriches a lipogenic fibroblast subpopulation, a potential precursor for myofibroblasts in the PDAC stroma. Overall, our study reveals the stromal targeting potential of HDACi, highlighting the utility of this epigenetic modulating approach in PDAC therapeutics.

Anglers are a diverse population whose behaviours and perspectives are influenced by a myriad of factors including knowledge, expertise, management actions, and regulations. We examine similarities and differences in behaviours and perspectives amongst freshwater anglers of rainbow trout and steelhead (Oncorhynchus mykiss) in British Columbia, Canada, using an online survey. Findings from the survey suggest that subgroups or “types” of anglers are identifiable by differences in their behaviours and perspectives according to geographic area, gear type, fishery, and frequency of fishing activities. Our results indicate that angler types share many of the same motivations for engaging in fishing behaviours and similar concerns regarding threats to their preferred fishery; however, differences were evident across types of issues related to angler behaviour, as well as views on fisheries management. Overall, we argue that understanding fishery-scale angler heterogeneity can benefit fisheries management by highlighting areas of agreement and disagreement and encouraging tailored communications and relationship-building with important angler subgroups.

Killer whales (Orcinus orca) are currently recognized as a single ecologically and morphologically diverse, globally distributed species. Multiple morphotypes or ecotypes have been described, often associated with feeding specialization, and several studies have suggested taxonomic revision to include multiple subspecies or species in the genus. We review the ecological, morphological and genetic data for the well-studied ‘resident’ and Bigg’s (aka ‘transient’) ecotypes in the eastern North Pacific and use quantitative taxonomic guidelines and standards to determine whether the taxonomic status of these killer whale ecotypes should be revised. Our review and new analyses indicate that species-level status is justified in both cases, and we conclude that eastern North Pacific Bigg’s killer whales should be recognized as Orcinus rectipinnus (Cope in Scammon, 1869) and resident killer whales should be recognized as Orcinus ater (Cope in Scammon, 1869).

Objective: Patients undergoing congenital cardiac surgery require induction of anesthesia. Our objective was to identify the median anesthesia ready time and the predictors of this time. Methods: By using the Society of Thoracic Surgeons Congenital Heart Surgery Database, we identified patients who underwent cardiopulmonary bypass procedures from 2017 to 2021. Univariate and multivariable regression modeling to predict the anesthesia ready time was performed using mixed-effects linear regression. Results: After exclusion of outliers, 44,418 cases were analyzed. The median anesthesia ready time was 51 minutes (interquartile range, 38-66). On multivariable analysis, independent predictors of a longer anesthesia ready time included decreasing weight (0.3 min/10 kg, 95% CI, 0.1-0.6; P = .011), prematurity (1.5 minutes, 95% CI, 0.8-2.2; P

Abstract Despite complex pathways of drug disposition, clinical pharmacogenetic predictors currently rely on only a few high effect variants. Quantification of the polygenic contribution to variability in drug disposition is necessary to prioritize target drugs for pharmacogenomic approaches and guide analytic methods. Dexmedetomidine and fentanyl, often used in postoperative care of pediatric patients, have high rates of inter‐individual variability in dosing requirements. Analyzing previously generated population pharmacokinetic parameters, we used Bayesian hierarchical mixed modeling to measure narrow‐sense (additive) heritability (hSNP2) of dexmedetomidine and fentanyl clearance in children and identify relative contributions of small, moderate, and large effect‐size variants to hSNP2. We used genome‐wide association studies (GWAS) to identify variants contributing to variation in dexmedetomidine and fentanyl clearance, followed by functional analyses to identify associated pathways. For dexmedetomidine, median clearance was 33.0 L/h (interquartile range [IQR] 23.8–47.9 L/h) and hSNP2 was estimated to be 0.35 (90% credible interval 0.00–0.90), with 45% of hSNP2 attributed to large‐, 32% to moderate‐, and 23% to small‐effect variants. The fentanyl cohort had median clearance of 8.2 L/h (IQR 4.7–16.7 L/h), with estimated hSNP2 of 0.30 (90% credible interval 0.00–0.84). Large‐effect variants accounted for 30% of hSNP2, whereas moderate‐ and small‐effect variants accounted for 37% and 33%, respectively. As expected, given small sample sizes, no individual variants or pathways were significantly associated with dexmedetomidine or fentanyl clearance by GWAS. We conclude that clearance of both drugs is highly polygenic, motivating the future use of polygenic risk scores to guide appropriate dosing of dexmedetomidine and fentanyl.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy in need of new therapeutic options. Using unbiased analyses of super-enhancers (SEs) as sentinels of core genes involved in cell-specific function, here we uncover a druggable SE-mediated RNA-binding protein (RBP) cascade that supports PDAC growth through enhanced mRNA translation. This cascade is driven by a SE associated with the RBP heterogeneous nuclear ribonucleoprotein F, which stabilizes protein arginine methyltransferase 1 (PRMT1) to, in turn, control the translational mediator ubiquitin-associated protein 2-like. All three of these genes and the regulatory SE are essential for PDAC growth and coordinately regulated by the Myc oncogene. In line with this, modulation of the RBP network by PRMT1 inhibition reveals a unique vulnerability in Myc-high PDAC patient organoids and markedly reduces tumor growth in male mice. Our study highlights a functional link between epigenetic regulation and mRNA translation and identifies components that comprise unexpected therapeutic targets for PDAC.

Polyphenol-rich Aronia fruits have great potential as a functional food with anti-inflammatory, hypolipidemic, and hypoglycemic biologic activities. However, clinical intervention trials investigating the impact of Aronia fruit consumption on human health are limited. A randomized, controlled, double-blinded, parallel intervention trial was conducted using 14 human subjects who ingested either 0 mL or 100 mL of Aronia juice daily for 30 days. Anthropometric measurements, fasting, and postprandial measures of glucose and lipid metabolism and inflammation, 16S rRNA fecal microbial composition data, and mass spectrometry-acquired serum and fecal metabolomic data were collected before and after the intervention period. Data were analyzed using general linear models, ANOVA, and t-tests. Daily consumption of Aronia prevented a rise in cholesterol levels (β = −0.50, p = 0.03) and reduced postprandial glucose (β = −3.03, p < 0.01). No difference in microbial community composition by condition was identified at any taxonomic level, but a decrease (β = −18.2, p = 0.04) in microbial richness with Aronia was detected. Serum and fecal metabolomic profiles indicated shifts associated with central carbon and lipid metabolism and decreases in pro-inflammatory metabolites. Our study further informs the development of polyphenol-based dietary strategies to lower metabolic disease risk.

Abstract Transmission and secretion of signals via the choroid plexus (ChP) brain barrier can modulate brain states via regulation of cerebrospinal fluid (CSF) composition. Here, we developed a platform to analyze diurnal variations in male mouse ChP and CSF. Ribosome profiling of ChP epithelial cells revealed diurnal translatome differences in metabolic machinery, secreted proteins, and barrier components. Using ChP and CSF metabolomics and blood-CSF barrier analyses, we observed diurnal changes in metabolites and cellular junctions. We then focused on transthyretin (TTR), a diurnally regulated thyroid hormone chaperone secreted by the ChP. Diurnal variation in ChP TTR depended on Bmal1 clock gene expression. We achieved real-time tracking of CSF-TTR in awake Ttr mNeonGreen mice via multi-day intracerebroventricular fiber photometry. Diurnal changes in ChP and CSF TTR levels correlated with CSF thyroid hormone levels. These datasets highlight an integrated platform for investigating diurnal control of brain states by the ChP and CSF.

This study aimed to develop poly(lactic acid) (PLA) biodegradable film containing sodium nitrite and α-tocopherol, and to examine its effect on dark-cutting beef color attributes. Using a twin-screw extruder, PLA pellets were manufactured as a low-concentration (LC) PLA pellet containing 0.12% sodium nitrite and 0.5% α-tocopherol or a high-concentration (HC) PLA pellet containing 0.6% sodium nitrite and 2.5% α-tocopherol. Extruded PLA pellets were oven-dried before being compressed and molded into film sheets using a hot press. In this study, 7 normal-pH and 7 dark-cutting strip loins were selected from a commercial processor. Loins were sliced into 2.54-cm thick steaks and randomly assigned to the respective loin-type treatments. The packaging treatments include normal-pH polyvinyl chloride (PVC), dark-cutting PVC, dark-cutting vacuum, LC-PLA dark-cutting in vacuum, and HC-PLA dark-cutting in vacuum. Normal-pH steaks were packaged with PVC overwrap, while dark-cutting control steaks were packaged in PVC overwrap and vacuum-sealed. The respective PLA films (LC or HC) were placed directly on the surface of the dark-cutting steak surface before vacuum packaging, resulting in LC-PLA and HC-PLA treatments. All steaks were placed in a simulated retail dis-play maintained at 2±1°C for 6 d, during which their surface color was evaluated every 24 h using a HunterLab spectrophotometer and a trained panel (n=6). Half of the steaks were removed from display on d 5 and cooked to 71°C on a George Foreman grill to evaluate the cooked color. The remaining steaks were evaluated on d 6 for microbial growth and lipid oxidation. A significant increase (P0.05) compared to dark-cutting steaks in control vacuum packages (without nitrite). There were no differences (P>0.05) in aerobic plate count and lipid oxidation between nitrite-embedded and control vacuum dark-cutting treatments. Results indicate that using lower concentrations of sodium nitrite (0.12%) andα-tocopherol (0.5%) in PLA films can help improve the surface color of raw dark-cutting steaks while minimizing unpleasant cooked color associated with nitrite-embedded film.

Sexual bonds are central to the social lives of many species, including humans, and monogamous prairie voles have become the predominant model for investigating such attachments. We developed an automated whole-brain mapping pipeline to identify brain circuits underlying pair-bonding behavior. We identified bonding-related c-Fos induction in 68 brain regions clustered in seven major brain-wide neuronal circuits. These circuits include known regulators of bonding, such as the bed nucleus of the stria terminalis, paraventricular hypothalamus, ventral pallidum, and prefrontal cortex. They also include brain regions previously unknown to shape bonding, such as ventromedial hypothalamus, medial preoptic area, and the medial amygdala, but that play essential roles in bonding-relevant processes, such as sexual behavior, social reward, and territorial aggression. Contrary to some hypotheses, we found that circuits active during mating and bonding were largely sexually monomorphic. Moreover, c-Fos induction across regions was strikingly consistent between members of a pair, with activity best predicted by rates of ejaculation. A novel cluster of regions centered in the amygdala remained coordinated after bonds had formed, suggesting novel substrates for bond maintenance. Our tools and results provide an unprecedented resource for elucidating the networks that translate sexual experience into an enduring bond.

ABSTRACT Influenza A virus infection during pregnancy can cause adverse maternal and fetal outcomes but the mechanism responsible remains elusive. Infection of outbred mice with 2009 H1N1 at embryonic day (E) 10 resulted in significant maternal morbidity, placental tissue damage and inflammation, fetal growth restriction, and developmental delays that lasted through weaning. Restriction of pulmonary virus replication was not inhibited during pregnancy, but infected dams had suppressed circulating and placental progesterone (P4) concentrations that were caused by H1N1-induced upregulation of pulmonary cyclooxygenase (COX)-1-, but not COX-2-, dependent synthesis and secretion of prostaglandin (PG) F2α. Treatment with 17-α-hydroxyprogesterone caproate (17-OHPC), a synthetic progestin that is safe to use in pregnancy, ameliorated the adverse maternal and fetal outcomes from H1N1 infection and prevented placental cell death and inflammation. These findings highlight the therapeutic potential of progestin treatments for influenza during pregnancy.IMPORTANCEPregnant individuals are at risk of severe outcomes from both seasonal and pandemic influenza A viruses. Influenza infection during pregnancy is associated with adverse fetal outcomes at birth and adverse consequences for offspring into adulthood. When outbred dams, with semi-allogenic fetuses, were infected with 2009 H1N1, in addition to pulmonary virus replication, lung damage, and inflammation, the placenta showed evidence of transient cell death and inflammation that was mediated by increased activity along the arachidonic acid pathway leading to suppression of circulating progesterone. Placental damage and suppressed progesterone were associated with detrimental effects on perinatal growth and developmental delays in offspring. Treatment of H1N1-infected pregnant mice with 17-OHPC, a synthetic progestin treatment that is safe to use in pregnancy, prevented placental damage and inflammation and adverse fetal outcomes. This novel therapeutic option for the treatment of influenza during pregnancy should be explored clinically.

Introduction: Individuals living with sickle cell disease experience high levels of morbidity that result in frequent utilization of the emergency department. The objective of this study was to provide updated national estimates of emergency department utilization associated with sickle cell disease in the U.S. Methods: Data from the National Hospital Ambulatory Medical Care Survey for the years 1999–2020 were analyzed. Complex survey analysis was utilized to produce national estimates overall and by patient age groups. Results: On average, approximately 222,612 emergency department visits occurred annually among individuals with sickle cell disease, a nearly 13% increase from prior estimates. The annual volume of emergency department visits steadily increased over time, and pain remains the most common patient-cited reason for visiting the emergency department. Patient-reported pain levels for individuals with sickle cell disease were high, with 64% of visits associated with severe pain and 21% associated with moderate pain. Public insurance sources continue to cover most visits, with Medicaid paying for 60% of visits and Medicare paying for 12% of visits. The average time spent in the emergency department increased from previous estimates by about an hour, rising to approximately 6 hours. The average wait time to see a provider was 53 minutes. Conclusions: Utilization of the emergency department by individuals living with sickle cell disease remains high, especially for pain. With more than half of patients with sickle cell disease reporting severe pain levels, emergency department staff should be prepared to assess and treat sickle cell disease–related pain following evidence-based guidelines and recommendations. The findings of this study can help improve care in this population.

Currently available pain assessment scales focus on pain-related symptoms and limitations imposed by pain. Validated assessment tools that measure how pain is regulated by those who live well with pain are missing. This study seeks to fill this gap by describing the development and preliminary validation of the Biobehavior Life Regulation (BLR) scale. The BLR scale assesses engagement, social relatedness, and self-growth in the presence of chronic pain and the unpredictability of chronic pain. Sources for items included survivor strategies, patient experiences, existing scales, and unpredictable pain research. Review for suitability yielded 52 items. Validation measures were identified for engagement, social relatedness, self-growth, and unpredictability of pain. The study sample (n = 202) represented patients treated in the Phoenix VA Health Care System (n = 112) and two community clinics (n = 90). Demographic characteristics included average age of 52.5, heterogeneous in ethnicity and race at the VA, mainly Non-Hispanic White at the community clinics, 14 years of education, and pain duration of 18 years for the VA and 15.4 years for community clinics. Exploratory factor analysis using Oblimin rotation in the VA sample (n = 112) yielded a two-factor solution that accounted for 48.23% of the total variance. Confirmatory factor analysis (CFA) in the same sample showed high correlations among items in Factor 1, indicating redundancy and the need to further reduce items. The final CFA indicated a 2-factor solution with adequate fit to the data. The 2-factor CFA was replicated in Sample 2 from the community clinics (n = 90) with similarly adequate fit to the data. Factor 1, Pain Regulation, covered 8 items of engagement, social relatedness, and self-growth while Factor 2, Pain Unpredictability, covered 6 items related to the experience of unpredictable pain. Construct validity showed moderate to higher Pearson correlations between BLR subscales and relevant well-established constructs that were consistent across VA and community samples. The BLR scale assesses adaptive regulation strategies in unpredictable pain as a potential tool for evaluating regulation resources and pain unpredictability.

Episodic increases in cerebral blood flow (CBF) are thought to contribute to improved cerebrovascular function and health. Head-out water immersion (HOWI) may be a useful modality to increase CBF secondary to the hydrostatic pressure placed on the body. However, it is unclear whether water temperatures common to the general public elicit similar cerebrovascular responses. We tested the hypothesis that mean middle cerebral artery blood velocity (MCAvmean) and cerebrovascular reactivity to CO2 (CVRCO2) would be higher during an acute bout of thermoneutral (TN; 35°C) vs. cool (COOL; 25°C) HOWI. Ten healthy participants (age: 23±3 y; 4 women) completed two randomized HOWI visits. Right MCAvmean, end-tidal CO2 (PETCO2) mean arterial pressure (MAP), and MCA conductance (MCAvmean/MAP) were continuously recorded. CVRCO2 was assessed using a stepped hypercapnia protocol before (PRE), at 30 minutes of HOWI (HOWI), immediately after HOWI (POST-1), and 45 minutes after HOWI (POST-2). Absolute values are reported as mean ± SD. MCAvmean, PETCO2, MAP, and CVRCO2 were not different between conditions at any timepoint (all P≥0.17). In COOL, MCAvmean increased from PRE (61±9 cm/s) during HOWI (68±11 cm/s), at POST-1 (69±11 cm/s), and POST-2 (72±8 cm/s) (all P

In urban and human-modified landscapes, animals face novel selection pressures resulting from differences in habitat structure and increased anthropogenic noise. Urban noise pollution can negatively impact songbirds because low-frequency noise often masks portions of birds’ mating signals and reduces signal transmission. Previous research has demonstrated that the songs of birds in more urban habitats have structural differences that can enhance signal transmission when noise is present. The majority of these studies have focused on species that deliver short, stereotyped songs and have limited repertoires. Gray catbirds (Dumetella carolinensis, family: Mimidae) sing long bouts containing imitated, improvised, and invented song elements, and therefore may have an increased ability to vary songs in response to noise. We hypothesized that aspects of developed habitats including loud anthropogenic noise and changes to land cover would impact catbirds’ song structural parameters, including song minimum, peak, and maximum frequency, frequency bandwidth, and entropy. We recorded and processed songs from 42 male catbirds and analyzed over 18,000 song elements from sites along an urban gradient from western Virginia to the Washington, DC metropolitan region. We quantified the urban intensity at each site-centroid based on percent canopy cover, percent impervious surface, and noise level. Song features such as minimum, maximum, and peak frequency increased significantly as noise levels increased, demonstrating that catbirds in noisier areas sing higher frequency songs compared to individuals in quieter habitats. Land cover variables also significantly predicted certain song features such as maximum frequency (impervious surface) or entropy (canopy cover). These structural differences in catbird song can limit the negative effects of environmental noise-masking, even for their long song bouts, and suggest that vocal mimics respond to anthropogenic noise. Future studies could investigate repertoire size and composition along an urban gradient and if these structural differences lead to functional consequences for the songs of vocal mimics.

Background: Perioperative cardiac arrest continues to occur. This study aims to identify risk factors for perioperative cardiac arrest in children presenting for noncardiac surgery and characterise its outcomes. Methods: Using the National Surgical Quality Improvement Program (NSQIP) Pediatric Database 2019 and 2020, 261 276 patients were included. Patients ≥18 yr and cardiac surgical procedures were excluded. Exploratory multivariable analysis was performed to identify independent predictors of perioperative cardiac arrest and associated outcomes. Results: The overall rate of cardiac arrest was 0.1%, with an intraoperative rate of 0.05% and 48-h postoperative rate of 0.06%. Significant risk factors for perioperative cardiac arrest included age

Abstract Background Many snakes are low-energy predators that use crypsis to ambush their prey. Most of these species feed very infrequently, are sensitive to the presence of larger vertebrates, such as humans, and spend large portions of their lifetime hidden. This makes direct observation of feeding behaviour challenging, and previous methodologies developed for documenting predation behaviours of free-ranging snakes have critical limitations. Animal-borne accelerometers have been increasingly used by ecologists to quantify activity and moment-to-moment behaviour of free ranging animals, but their application in snakes has been limited to documenting basic behavioural states (e.g., active vs. non-active). High-frequency accelerometry can provide new insight into the behaviour of this important group of predators, and here we propose a new method to quantify key aspects of the feeding behaviour of three species of viperid snakes (Crotalus spp.) and assess the transferability of classification models across those species. Results We used open-source software to create species-specific models that classified locomotion, stillness, predatory striking, and prey swallowing with high precision, accuracy, and recall. In addition, we identified a low cost, reliable, non-invasive attachment method for accelerometry devices to be placed anteriorly on snakes, as is likely necessary for accurately classifying distinct behaviours in these species. However, species-specific models had low transferability in our cross-species comparison. Conclusions Overall, our study demonstrates the strong potential for using accelerometry to document critical feeding behaviours in snakes that are difficult to observe directly. Furthermore, we provide an ‘end-to-end’ template for identifying important behaviours involved in the foraging ecology of viperids using high-frequency accelerometry. We highlight a method of attachment of accelerometers, a technique to simulate feeding events in captivity, and a model selection procedure using biologically relevant window sizes in an open-access software for analyzing acceleration data (AcceleRater). Although we were unable to obtain a generalized model across species, if more data are incorporated from snakes across different body sizes and different contexts (i.e., moving through natural habitat), general models could potentially be developed that have higher transferability.

Even slight structural differences between phytocannabinoid isomers are usually enough to cause a change in their biological properties. In this study, we used in vitro CB1 agonism/antagonism assays to compare the receptor binding functionality of THCV (tetrahydrocannabivarin) and HHC (hexahydrocannabinol) isomers and applied molecular docking to provide an explanation for the difference in the activities. No CB1 agonism was observed for ∆9- and ∆8-THCV. Instead, both isomers antagonized CP 55940, with ∆9-THCV being approximately two times more potent than the ∆8 counterpart (IC50 = 52.4 nM and 119.6 nM for ∆9- and ∆8-THCV, respectively). Docking simulations found two binding poses for THCV isomers, one very similar to ∆9-THC and one newly discovered pose involving the occupation of side pocket 1 of the CB1 receptor by the alkyl chain of the ligand. We suggested the latter as a potential antagonist pose. In addition, our results established 9R-HHC and 9S-HHC among partial agonists of the CB1 receptor. The 9R-HHC (EC50 = 53.4 nM) isomer was a significantly more potent agonist than 9S (EC50 = 624.3 nM). ∆9-THC and 9R-HHC showed comparable binding poses inside the receptor pocket, whereas 9S-HHC adopted a new and different binding posture that can explain its weak agonist activity.

Abstract Prostate cancer is the second leading cause of malignancy‐related deaths among American men. Active surveillance is a safe option for many men with less aggressive disease, yet definitively determining low‐risk cancer is challenging with biopsy alone. Herein, we sought to identify prostate‐derived microRNAs in patient sera and serum extracellular vesicles, and determine if those microRNAs improve upon the current clinical risk calculators for prostate cancer prognosis before and after biopsy. Prostate‐derived intracellular and extracellular vesicle‐contained microRNAs were identified by small RNA sequencing of prostate cancer patient explants and primary cells. Abundant microRNAs were included in a custom microRNA PCR panel that was queried in whole serum and serum extracellular vesicles from a diverse cohort of men diagnosed with prostate cancer. The levels of these circulating microRNAs significantly differed between indolent and aggressive disease and improved the area under the curve for pretreatment nomograms of prostate cancer disease risk. The microRNAs within the extracellular vesicles were the most informative and improved the AUC to 0.739 compared to the existing nomogram alone, which has an AUC of 0.561. The microRNAs in the whole serum improved it to AUC 0.675. In summary, quantifying microRNAs circulating in extracellular vesicles is a clinically feasible assay that may provide additional information for assessing prostate cancer risk stratification.

Previous research indicated that inherently present electron donors and carriers contribute to nonenzymatic metmyoglobin reduction. However, limited knowledge is currently available on how light and oxygen influence nonenzymatic metmyoglobin reduction. The objective of this study was to evaluate the effect of light and oxygen levels on nonenzymatic metmyoglobin reduction in vitro. Two experiments were conducted using equine metmyoglobin solution at pH 5.6 (represents typical postmortem muscle pH) with different electron donors and electron carriers in a 96-well plate. A gas blend of 80% oxygen and 20% carbon dioxide or a pure 99.9% nitrogen gas was bubbled in phosphate buffers and myoglobin solutions to create 2 oxygen levels (low oxygen [4%] and high oxygen [160%]). The desired dissolved oxygen content was measured using a NeoFox oxygen probe (atmospheric oxygen level was 20% oxygen). There were significant effects of electron donors, carriers, and light on nonenzymatic metmyoglobin reduction. Ascorbate and nicotinamide adenine dinucleotide reduced form (NADH) in the presence of methylene blue and ethylenediaminetetraacetic acid (EDTA) exhibited a greater nonenzymatic metmyoglobin reduction in light than dark. An increase in nonenzymatic metmyoglobin-reducing activity (P

Newly collected material from northern Namibia’s Otjihipa Mountains and west-central Angola allowed us to revisit the Afroedura bogerti Loveridge, 1944 group. The employment of additional gene markers, including nuclear markers, allowed us to identify two new species in the group and infer species boundaries and potential speciation events in Afroedura from southwestern Africa. The new Namibian material is recovered as a sister species to A. donveae, from which it differs mostly by the colour of the iris (copper versus black) and dorsal colouration. Material from the first elevational gradient of the escarpment in Benguela Province, Angola was found to be more closely related to A. bogerti than A. wulfhaackei. The differences between these two species are more subtle, although the new species exhibits higher mid-body scale rows (79.5 versus 74.8), different dorsal colouration and supranasal scales always in contact (versus 57% in contact).

Sarah Bentley,1 Lucy Morgan,1 Elizabeth Exall,1 Rob Arbuckle,1 Rebecca C Rossom,2 Nicholas Roche,3 Kamlesh Khunti,4 Victoria Higgins,5 James Piercy5 1Patient-Centered Outcomes, Adelphi Values, Bollington, Cheshire, UK; 2HealthPartners Institute, University of Minnesota Medical School, Minneapolis, MN, USA; 3Respiratory Medicine, Cochin Hospital, APHP Centre University Paris Cité, Institut Cochin (UMR1016), Paris, France; 4Diabetes Research Centre, Leicester University, Leicester, UK; 5Adelphi Real World, Bollington, Cheshire, UKCorrespondence: Rob Arbuckle, Patient-Centered Outcomes, Adelphi Values, Bollington, Cheshire, UK, Tel +44 7720 880884, Email rob.arbuckle@Adelphivalues.comPurpose: The Adelphi Adherence Questionnaire (ADAQ©) is a newly developed generic patient-reported outcome (PRO) assessment of medication adherence. The aim was to assess its content validity by conducting cognitive debriefing (CD) interviews with patients prescribed medication(s) of various treatment modalities in a range of therapy areas.Materials and Methods: Targeted literature/instrument review and concept elicitation interviews informed development of the ADAQ©. CD interviews were conducted with 57 adults from the United States of America (USA; n = 21), Spain (n = 18), and Germany (n = 18) who prescribed medication for hypertension, diabetes, depression, schizophrenia, asthma, multiple myeloma, psoriasis, and/or multiple sclerosis. Interviews were conducted in two rounds to explore the relevance and understanding of the item wording, instructions, recall period and response options. Verbatim transcripts were analysed in ATLAS.Ti using thematic analysis. Three expert clinicians provided guidance throughout the study.Results: ADAQ© items/instructions were well understood and relevant to participants. Key modifications following round 1 included revising instructions to refer to current medication(s) for one condition to reduce cognitive burden, removing two items with lower relevance (specifically those assessing running out of medication and social discouragement), and adding a response option for participants to indicate if they had stopped taking a medication. Minor wording modifications were made following round 2. Subgroup differences in item relevance were explored based on clinical characteristics. Cost of medication was more relevant amongst US participants.Conclusion: Content validity of the ADAQ© was confirmed in demographically and clinically diverse participants. Psychometric properties of the ADAQ© will be explored in future studies.Keywords: patient-reported outcome development, qualitative research, adherence, cognitive debriefing

The Tonian to Cryogenian (ca. 1000–635 Ma) marks a crucial turning point in Earth’s history, where tectonic reorganisation and fluctuating oceanic and atmospheric geochemistry plunged the globe into icehouse conditions. This was followed by a postglacial warming period that delivered large volumes of nutrients to the oceans and stimulated eukaryotic evolution. The Adelaide Superbasin in South Australia hosts a thick repository of Neoproterozoic and lower Cambrian sedimentary successions that preserve the depositional conditions during this unique time. In this study, detailed sedimentological data were collected from over 8,350 m of measured section at seven locations across the northern Flinders Ranges. Tonian deposits reveal a carbonate platform setting, where deposition was controlled by basin geometry and proximity to uplifted source areas. In the early Cryogenian, sedimentary successions were affected by the Sturtian glaciation, characterised by two glacial advance-retreat phases that coincide with climatically driven regression. The end of the Sturtian glaciation was marked by basin subsidence and widespread transgression into a more distal subaqueous environment. Despite the lithostratigraphic and sequence stratigraphic similarity between Tonian–Cryogenian successions globally, their correlation remains contentious. The influence of local tectonic regimes during the Tonian created a potential oceanic restriction between developing basins, which challenges the chemostratigraphic correlation between these deposits. Further, limited geochronological ages and opposing interpretations of glacial cyclicity puts into question the timing and extent of the Sturtian glaciation. Conversely, the post glacial transgression appears to be the most globally consistent as it results from climatically controlled sea level rise that was driven by melting ice sheets.

Background: The purpose of the present study was to investigate the relationship between socioeconomic status and Patient-Reported Outcomes Measurement Information System Global Health (PROMIS-GH) scores before and after primary total knee arthroplasty (TKA). We hypothesized that patients with greater social deprivation would have lower PROMIS-GH scores at 3 months and 1 year following primary TKA. Methods: We retrospectively reviewed data from patients who underwent unilateral primary TKA and completed PROMIS-GH preoperatively and at 3 months (n = 257) or 1 year (n = 154) postoperatively. Area Deprivation Index (ADI), calculated from 9-digit zip codes, was used to measure social deprivation. Participants were grouped into quartiles by ADI score. Minimal clinically important difference in PROMIS-GH mental (PROMIS-MH) and physical health (PROMIS-PH) component scores were compared between ADI groups. Results: Participants in the highest ADI quartile (most disadvantaged) had significantly lower PROMIS-MH and PROMIS-PH scores at every time point relative to the lowest ADI quartile (least disadvantaged) (P < .05 for all). Both ADI groups experienced significant improvements in PROMIS-PH following TKA (P < .001 for all), but not in PROMIS-MH (P > .05 for all) at 3-months and 1-year postoperatively. Magnitude of improvement in PROMIS-PH and rates of achievement of minimal clinically important difference did not significantly differ between ADI groups (P > .05 for all). Conclusions: Socially disadvantaged patients benefit equally from primary TKA but are more likely to have persistently lower 1-year postoperative PROMIS-GH scores relative to less disadvantaged patients. Social deprivation should be accounted for when using PROMIS-GH to assess clinical outcomes for research and quality measures. Level of Evidence: IV, retrospective cohort study.

A key objective for the future exploration of the icy moon Enceladus is the characterization of the habitable conditions in its internal ocean. Radio science instrumentation on board a spacecraft in a low-altitude orbit about Enceladus would enable gravity measurements that are fundamental to providing constraints on its internal structure. We present here the concept of operations and expected results of the gravity investigation for a New Frontiers–class mission. Numerical simulations are carried out to determine the gravity field in spherical harmonics to degree and order 30 and the Love number k _2 and its phase. By combining Enceladus’s shape measured by Cassini and the geophysical constraints obtained through the processing of the simulated radio science data, a Bayesian inference network is used for the interior model inversion. Our results indicate that the gravity investigation would enable tight constraints on core radius and density, ocean depth and density, and ice shell rigidity. By assuming a high core rigidity and a preliminary modeling of dissipation in the ice shell, our interior model inversion also yields information on the ice shell viscosity. Further data on the hydrosphere properties might be gathered through optical navigation data by accurately measuring Enceladus’s orientation model.

To detect new and changing SARS-CoV-2 variants, we investigated candidate Delta–Omicron recombinant genomes from Centers for Disease Control and Prevention national genomic surveillance. Laboratory and bioinformatic investigations identified and validated 9 genetically related SARS-CoV-2 viruses with a hybrid Delta–Omicron spike protein.

Background: We designed a prospective feasibility study to assess the 5x-multiplier (5x) calculation (eg, 3 pills in last 24 hours × 5 = 15) to standardize discharge opioid prescriptions compared to usual care. Methods: Faculty-based surgical teams volunteered for either 5x or usual care arms. Patients undergoing inpatient (≥48 hours) surgery and discharged by surgical teams were included. The primary end point was discharge oral morphine equivalents. Secondary end points were opioid-free discharges and 30-day refill rates. Results: Median last 24-hour oral morphine equivalents was similar between arms (7.5 mg 5x vs 10 mg usual care, P = .830). Median discharge oral morphine equivalents were less in the 5x arm (50 mg 5x vs 75 mg usual care, P

Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC-replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC-integrated CAR+ T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells.

Introduction and aimsDietary Rational Gene Targeting (DRGT) is a therapeutic dietary strategy that uses healthy dietary agents to modulate the expression of disease-causing genes back toward the normal. Here we use the DRGT approach to (1) identify human studies assessing gene expression after ingestion of healthy dietary agents with an emphasis on whole foods, and (2) use this data to construct an online dietary guide app prototype toward eventually aiding patients, healthcare providers, community and researchers in treating and preventing numerous health conditions.MethodsWe used the keywords “human”, “gene expression” and separately, 51 different dietary agents with reported health benefits to search GEO, PubMed, Google Scholar, Clinical trials, Cochrane library, and EMBL-EBI databases for related studies. Studies meeting qualifying criteria were assessed for gene modulations. The R-Shiny platform was utilized to construct an interactive app called “Eat4Genes”.ResultsFifty-one human ingestion studies (37 whole food related) and 96 key risk genes were identified. Human gene expression studies were found for 18 of 41 searched whole foods or extracts. App construction included the option to select either specific conditions/diseases or genes followed by food guide suggestions, key target genes, data sources and links, dietary suggestion rankings, bar chart or bubble chart visualization, optional full report, and nutrient categories. We also present user scenarios from physician and researcher perspectives.ConclusionIn conclusion, an interactive dietary guide app prototype has been constructed as a first step towards eventually translating our DRGT strategy into an innovative, low-cost, healthy, and readily translatable public resource to improve health.

BackgroundAnxiety and depression are common in the perinatal period and negatively affect the health of the mother and baby. Our group has developed “Happy Mother-Healthy Baby” (HMHB), a cognitive behavioral therapy–based psychosocial intervention to address risk factors specific to anxiety during pregnancy in low- and middle-income countries (LMICs). ObjectiveThe purpose of this study is to examine biological mechanisms that may be linked to perinatal anxiety in conjunction with a randomized controlled trial of HMHB in Pakistan. MethodsWe are recruiting 120 pregnant women from the Holy Family Hospital, a public facility in Rawalpindi, Pakistan. Participants are assessed for at least mild anxiety symptoms using the Hospital Anxiety and Depression Scale (ie, a score ≥8 on the anxiety scale is necessary for inclusion in the anxiety groups and

Summary: Biology is tuned to the Earth’s diurnal cycle by the circadian clock, a transcriptional/translational negative feedback loop that regulates physiology via transcriptional activation and other post-transcriptional mechanisms. We hypothesize that circadian post-transcriptional regulation might stem from conformational shifts in the intrinsically disordered proteins that comprise the negative arm of the feedback loop to coordinate variation in negative-arm-centered macromolecular complexes. This work demonstrates temporal conformational fluidity in the negative arm that correlates with 24-h variation in physiologically diverse macromolecular complex components in eukaryotic clock proteins. Short linear motifs on the negative-arm proteins that correspond with the interactors localized to disordered regions and known temporal phosphorylation sites suggesting changes in these macromolecular complexes could be due to conformational changes imparted by the temporal phospho-state. Interactors that oscillate in the macromolecular complexes over circadian time correlate with post-transcriptionally regulated proteins, highlighting how time-of-day variation in the negative-arm protein complexes may tune cellular physiology.

Abstract Neflamapimod, a selective inhibitor of p38 mitogen activated protein kinase alpha (MAPKα), is under clinical investigation for its efficacy in Alzheimer’s disease (AD) and dementia with Lewy Bodies (DLB). Here, we investigated if neflamapimod-mediated acute inhibition of p38 MAPKα could induce vasodilation in resistance-size rat mesenteric arteries. Our pressure myography data demonstrated that neflamapimod produced a dose-dependent vasodilation in mesenteric arteries. Our Western blotting data revealed that acute neflamapimod treatment significantly reduced the phosphorylation of p38 MAPKα and its downstream target heat-shock protein 27 (Hsp27) involved in cytoskeletal reorganization and smooth muscle contraction. Likewise, non-selective inhibition of p38 MAPK by SB203580 attenuated p38 MAPKα and Hsp27 phosphorylation, and induced vasodilation. Endothelium denudation or pharmacological inhibition of endothelium-derived vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) had no effect on such vasodilation. Neflamapimod-evoked vasorelaxation remained unaltered by the inhibition of smooth muscle cell K+ channels. Altogether, our data for the first time demonstrates that in resistance mesenteric arteries, neflamapimod inhibits p38 MAPKα and phosphorylation of its downstream actin-associated protein Hsp27, leading to vasodilation. This novel finding may be clinically significant and is likely to improve systemic blood pressure and cognitive deficits in AD and DLB patients for which neflamapimod is being investigated.

A cure for HIV-1 (HIV) remains unrealized due to a reservoir of latently infected cells that persist during antiretroviral therapy (ART), with reservoir size associated with adverse health outcomes and inversely with time to viral rebound upon ART cessation. Once established during ART, the HIV reservoir decays minimally over time; thus, understanding factors that impact the size of the HIV reservoir near its establishment is key to improving the health of people living with HIV and for the development of novel cure strategies. Yet, to date, few correlates of HIV reservoir size have been identified, particularly in pediatric populations. Here, we employed a cross-subtype intact proviral DNA assay (CS-IPDA) to quantify HIV provirus between one- and two-years post-ART initiation in a cohort of Kenyan children (n = 72), which had a median of 99 intact (range: 0–2469), 1340 defective (range: 172–3.84 × 104), and 1729 total (range: 178–5.11 × 104) HIV proviral copies per one million T cells. Additionally, pre-ART plasma was tested for HIV Env-specific antibody-dependent cellular cytotoxicity (ADCC) activity. We found that pre-ART gp120-specific ADCC activity inversely correlated with defective provirus levels (n = 68, r = −0.285, p = 0.0214) but not the intact reservoir (n = 68, r = −0.0321, p-value = 0.800). Pre-ART gp41-specific ADCC did not significantly correlate with either proviral population (n = 68; intact: r = −0.0512, p-value = 0.686; defective: r = −0.109, p-value = 0.389). This suggests specific host immune factors prior to ART initiation can impact proviruses that persist during ART.