Scalable Hypothalamic Arcuate Neuron Differentiation from Human Pluripotent Stem Cells Suitable for Modeling Metabolic and Reproductive Disorders.

The hypothalamus, composed of several nuclei, is essential for maintaining our body's homeostasis. The arcuate nucleus (ARC), located in the mediobasal hypothalamus, contains neuronal populations with eminent roles in energy and glucose homeostasis as well as reproduction. These neuronal populations are of great interest for translational research. To fulfill this promise, we used a robotic cell culture platform to provide a scalable and chemically defined approach for differentiating human pluripotent stem cells (hPSCs) into pro-opiomelanocortin (POMC), somatostatin (SST), tyrosine hydroxylase (TH) and gonadotropin-releasing hormone (GnRH) neuronal subpopulations with an ARC-like signature. This robust approach is reproducible across several distinct hPSC lines and exhibits a stepwise induction of key ventral diencephalon and ARC markers in transcriptomic profiling experiments. This is further corroborated by direct comparison to human fetal hypothalamus, and the enriched expression of genes implicated in obesity and type 2 diabetes (T2D). Genome-wide chromatin accessibility profiling by ATAC-seq identified accessible regulatory regions that can be utilized to predict candidate enhancers related to metabolic disorders and hypothalamic development. In depth molecular, cellular, and functional experiments unveiled the responsiveness of the hPSC-derived hypothalamic neurons to hormonal stimuli, such as insulin, neuropeptides including kisspeptin, and incretin mimetic drugs such as Exendin-4, highlighting their potential utility as physiologically relevant cellular models for disease studies. In addition, differential glucose and insulin treatments uncovered adaptability within the generated ARC neurons in the dynamic regulation of POMC and insulin receptors. In summary, the establishment of this model represents a novel, chemically defined, and scalable platform for manufacturing large numbers of hypothalamic arcuate neurons and serves as a valuable resource for modeling metabolic and reproductive disorders.

Single-cell transcriptomic profiling of human pancreatic islets reveals genes responsive to glucose exposure over 24 h.

AIMS/HYPOTHESIS: Disruption of pancreatic islet function and glucose homeostasis can lead to the development of sustained hyperglycaemia, beta cell glucotoxicity and subsequently type 2 diabetes. In this study, we explored the effects of in vitro hyperglycaemic conditions on human pancreatic islet gene expression across 24 h in six pancreatic cell types: alpha; beta; gamma; delta; ductal; and acinar. We hypothesised that genes associated with hyperglycaemic conditions may be relevant to the onset and progression of diabetes. METHODS: We exposed human pancreatic islets from two donors to low (2.8 mmol/l) and high (15.0 mmol/l) glucose concentrations over 24 h in vitro. To assess the transcriptome, we performed single-cell RNA-seq (scRNA-seq) at seven time points. We modelled time as both a discrete and continuous variable to determine momentary and longitudinal changes in transcription associated with islet time in culture or glucose exposure. Additionally, we integrated genomic features and genetic summary statistics to nominate candidate effector genes. For three of these genes, we functionally characterised the effect on insulin production and secretion using CRISPR interference to knock down gene expression in EndoC-ßH1 cells, followed by a glucose-stimulated insulin secretion assay. RESULTS: In the discrete time models, we identified 1344 genes associated with time and 668 genes associated with glucose exposure across all cell types and time points. In the continuous time models, we identified 1311 genes associated with time, 345 genes associated with glucose exposure and 418 genes associated with interaction effects between time and glucose across all cell types. By integrating these expression profiles with summary statistics from genetic association studies, we identified 2449 candidate effector genes for type 2 diabetes, HbA1c, random blood glucose and fasting blood glucose. Of these candidate effector genes, we showed that three (ERO1B, HNRNPA2B1 and RHOBTB3) exhibited an effect on glucose-stimulated insulin production and secretion in EndoC-ßH1 cells. CONCLUSIONS/INTERPRETATION: The findings of our study provide an in-depth characterisation of the 24 h transcriptomic response of human pancreatic islets to glucose exposure at a single-cell resolution. By integrating differentially expressed genes with genetic signals for type 2 diabetes and glucose-related traits, we provide insights into the molecular mechanisms underlying glucose homeostasis. Finally, we provide functional evidence to support the role of three candidate effector genes in insulin secretion and production. DATA AVAILABILITY: The scRNA-seq data from the 24 h glucose exposure experiment performed in this study are available in the database of Genotypes and Phenotypes (dbGap; https://www.ncbi.nlm.nih.gov/gap/ ) with accession no. phs001188.v3.p1. Study metadata and summary statistics for the differential expression, gene set enrichment and candidate effector gene prediction analyses are available in the Zenodo data repository ( https://zenodo.org/ ) under accession number 11123248. The code used in this study is publicly available at https://github.com/CollinsLabBioComp/publication-islet_glucose_timecourse .

The methods and baseline characteristics of a multi-site randomized controlled trial evaluating mindfulness-based relapse prevention in conjunction with peer support to improve adherence to medications for opioid use disorders.

Introduction: Medications for opioid use disorders (MOUD) remain the gold standard for treating OUD, but treatment initiation and adherence remain challenging. Exclusive utilization of pharmacotherapy as a treatment modality for OUD is sub-optimal, and a combination of psychotherapies and pharmacotherapies is recommended. General trends indicate the benefits of peer mentoring and MBRP separately. Therefore, we hypothesize that the combined effect of MBRP and Peer mentoring will produce synergistic improvements in MOUD adherence compared to an enhanced twelve-step facilitation (TSF). Methods: This paper describes the methods and baseline characteristics of a multi-site randomized controlled trial evaluating the effectiveness of a combination of MBRP and peer support (MiMP) compared to an enhanced TSF in improving adherence to MOUD. Both MiMP and TSF are 12-week manualized protocols that utilize licensed therapists. The interventions are delivered in weekly group sessions that last about 75-90 minutes per session. The primary outcome is MOUD adherence. Secondary and exploratory outcomes include relapse, cravings, depression, anxiety, stress, quality of life, and pain catastrophizing. Results: The participants' ages ranged from 21 years to 77 years, with a mean age of 44.5 (SD ± 11.5 years). There was an almost equal distribution of gender and place of residence. Overall, 51.9% (n=54) of participants identified as female and 48.1% (n=50) were male. Similarly, 51.9% (n=54) of participants resided in urban areas, while 48.1% (n=50) resided in rural areas. Participants identified as either black or white, with over three-quarters identifying as white (77.9%, n= 81) and 22.1% (n= 23) as black. Most participants randomized to the 12-step facilitation group were white (93.1%). Relationships and employment status were well distributed between categories. Over half of the participants reported some college or higher education. Over 90% of the participants made less than $75,000 per year. Some participants indicated that they had both public and private health insurance. Discussion and conclusion: This study is innovative in several ways including combining MBRP and peer support, addressing comorbid mental health issues among individuals with OUD, utilizing manualized protocols, and evaluating of both physiological and self-reported measures in assessing cortisol reactivity as a predictor of relapse and treatment outcomes.

Forskolin reverses the O-GlcNAcylation dependent decrease in GABAAR current amplitude at hippocampal synapses possibly at a neurosteroid site on GABAARs.

GABAergic transmission is influenced by post-translational modifications, like phosphorylation, impacting channel conductance, allosteric modulator sensitivity, and membrane trafficking. O-GlcNAcylation is a post-translational modification involving the O-linked attachment of ß-N-acetylglucosamine on serine/threonine residues. Previously we reported an acute increase in O-GlcNAcylation elicits a long-term depression of evoked GABAAR inhibitory postsynaptic currents (eIPSCs) onto hippocampal principal cells. Importantly, O-GlcNAcylation and phosphorylation can co-occur or compete for the same residue; whether they interact in modulating GABAergic IPSCs is unknown. We tested this by recording IPSCs from hippocampal principal cells and pharmacologically increased O-GlcNAcylation, before or after increasing serine phosphorylation using the adenylate cyclase activator, forskolin. Although forskolin had no significant effect on baseline eIPSC amplitude, we found that a prior increase in O-GlcNAcylation unmasks a forskolin-dependent increase in eIPSC amplitude, reversing the O-GlcNAc-induced eIPSC depression. Inhibition of adenylate cyclase or protein kinase A did not prevent the potentiating effect of forskolin, indicating serine phosphorylation is not the mechanism. Surprisingly, increasing O-GlcNAcylation also unmasked a potentiating effect of the neurosteroids 5α-pregnane-3α,21-diol-20-one (THDOC) and progesterone on eIPSC amplitude in about half of the recorded cells, mimicking forskolin. Our findings show that under conditions of heightened O-GlcNAcylation, the neurosteroid site on synaptic GABAARs is possibly accessible to agonists, permitting strengthening of synaptic inhibition.

In a circuit necessary for cognition and emotional affect, Alzheimer's-like pathology associates with neuroinflammation, cognitive and motivational deficits in the young adult TgF344-AD rat.

In addition to extracellular amyloid plaques, intracellular neurofibrillary tau tangles, and inflammation, cognitive and emotional affect perturbations are characteristic of Alzheimer's disease (AD). The cognitive and emotional domains impaired by AD include several forms of decision making (such as intertemporal choice), blunted motivation (increased apathy), and impaired executive function (such as working memory and cognitive flexibility). However, the interaction between these domains of the mind and their supporting neurobiological substrates at prodromal stages of AD, or whether these interactions can be predictive of AD severity (individual variability), remain unclear. In this study, we employed a battery of cognitive and emotional tests in the young adult (5-7 mo) transgenic Fisher-344 AD (TgF344-AD; TgAD) rat model of AD. We also assessed whether markers of inflammation or AD-like pathology in the prelimbic cortex (PrL) of the medial prefrontal cortex (mPFC), basolateral amygdala (BLA), or nucleus accumbens (NAc), all structures that directly support the aforementioned behaviors, were predictive of behavioral deficits. We found TgAD rats displayed maladaptive decision making, greater apathy, and impaired working memory that was indeed predicted by AD-like pathology in the relevant brain structures, even at an early age. Moreover, we report that the BLA is an early epicenter of inflammation, and notably, AD-like pathology in the PrL, BLA, and NAc was predictive of BLA inflammation. These results suggest that operant-based battery testing may be sensitive enough to determine pathology trajectories, including neuroinflammation, from early stages of AD.

A hierarchy of processing complexity and timescales for natural sounds in human auditory cortex.

Efficient behavior is supported by humans' ability to rapidly recognize acoustically distinct sounds as members of a common category. Within auditory cortex, there are critical unanswered questions regarding the organization and dynamics of sound categorization. Here, we performed intracerebral recordings in the context of epilepsy surgery as 20 patient-participants listened to natural sounds. We built encoding models to predict neural responses using features of these sounds extracted from different layers within a sound-categorization deep neural network (DNN). This approach yielded highly accurate models of neural responses throughout auditory cortex. The complexity of a cortical site's representation (measured by the depth of the DNN layer that produced the best model) was closely related to its anatomical location, with shallow, middle, and deep layers of the DNN associated with core (primary auditory cortex), lateral belt, and parabelt regions, respectively. Smoothly varying gradients of representational complexity also existed within these regions, with complexity increasing along a posteromedial-to-anterolateral direction in core and lateral belt, and along posterior-to-anterior and dorsal-to-ventral dimensions in parabelt. When we estimated the time window over which each recording site integrates information, we found shorter integration windows in core relative to lateral belt and parabelt. Lastly, we found a relationship between the length of the integration window and the complexity of information processing within core (but not lateral belt or parabelt). These findings suggest hierarchies of timescales and processing complexity, and their interrelationship, represent a functional organizational principle of the auditory stream that underlies our perception of complex, abstract auditory information.

Post-traumatic stress disorder: the role of the amygdala and potential therapeutic interventions - a review.

Introduction: Post-traumatic stress disorder (PTSD) is a psychiatric disorder triggered by exposure to a life-threatening or sexually violent traumatic event, and is characterized by symptoms involving intrusive re-experiencing, persistent avoidance of associated stimuli, emotional and cognitive disturbances, and hyperarousal for long periods after the trauma has occurred. These debilitating symptoms induce occupational and social impairments that contribute to a significant clinical burden for PTSD patients, and substantial socioeconomic costs, reaching approximately $20,000 dollars per individual with PTSD each year in the US. Despite increased translational research focus in the field of PTSD, the development of novel, effective pharmacotherapies for its treatment remains an important unmet clinical need. Observations: In this review, we summarize the evidence implicating dysfunctional activity of the amygdala in the pathophysiology of PTSD. We identify the transient receptor potential canonical (TRPC) ion channels as promising drug targets given their distribution in the amygdala, and evidence from animal studies demonstrating their role in fear response modulation. We discuss the evidence-based pharmacotherapy and psychotherapy treatment approaches for PTSD. Discussion: In view of the prevalence and economic burden associated with PTSD, further investigation is warranted into novel treatment approaches based on our knowledge of the involvement of brain circuitry and the role of the amygdala in PTSD, as well as the potential added value of combined pharmacotherapy and psychotherapy to better manage PTSD symptoms.

A one-man bilingual cocktail party: linguistic and non-linguistic effects on bilinguals' speech recognition in Mandarin and English.

Multilingual speakers can find speech recognition in everyday environments like restaurants and open-plan offices particularly challenging. In a world where speaking multiple languages is increasingly common, effective clinical and educational interventions will require a better understanding of how factors like multilingual contexts and listeners' language proficiency interact with adverse listening environments. For example, word and phrase recognition is facilitated when competing voices speak different languages. Is this due to a "release from masking" from lower-level acoustic differences between languages and talkers, or higher-level cognitive and linguistic factors? To address this question, we created a "one-man bilingual cocktail party" selective attention task using English and Mandarin speech from one bilingual talker to reduce low-level acoustic cues. In Experiment 1, 58 listeners more accurately recognized English targets when distracting speech was Mandarin compared to English. Bilingual Mandarin-English listeners experienced significantly more interference and intrusions from the Mandarin distractor than did English listeners, exacerbated by challenging target-to-masker ratios. In Experiment 2, 29 Mandarin-English bilingual listeners exhibited linguistic release from masking in both languages. Bilinguals experienced greater release from masking when attending to English, confirming an influence of linguistic knowledge on the "cocktail party" paradigm that is separate from primarily energetic masking effects. Effects of higher-order language processing and expertise emerge only in the most demanding target-to-masker contexts. The "one-man bilingual cocktail party" establishes a useful tool for future investigations and characterization of communication challenges in the large and growing worldwide community of Mandarin-English bilinguals.

Sex, racial, and APOE-ε4 allele differences in longitudinal white matter microstructure in multiple cohorts of aging and Alzheimer's disease.

INTRODUCTION: The effects of sex, race, and Apolipoprotein E (APOE) - Alzheimer's disease (AD) risk factors - on white matter integrity are not well characterized. METHODS: Diffusion MRI data from nine well-established longitudinal cohorts of aging were free-water (FW)-corrected and harmonized. This dataset included 4,702 participants (age=73.06 ± 9.75) with 9,671 imaging sessions over time. FW and FW-corrected fractional anisotropy (FAFWcorr) were used to assess differences in white matter microstructure by sex, race, and APOE-ε4 carrier status. RESULTS: Sex differences in FAFWcorr in association and projection tracts, racial differences in FAFWcorr in projection tracts, and APOE-ε4 differences in FW limbic and occipital transcallosal tracts were most pronounced. DISCUSSION: There are prominent differences in white matter microstructure by sex, race, and APOE-ε4 carrier status. This work adds to our understanding of disparities in AD. Additional work to understand the etiology of these differences is warranted.

Proteomic analysis of Alzheimer's disease cerebrospinal fluid reveals alterations associated with APOE ε4 and atomoxetine treatment.

Alzheimer's disease (AD) is currently defined by the aggregation of amyloid-ß (Aß) and tau proteins in the brain. Although biofluid biomarkers are available to measure Aß and tau pathology, few markers are available to measure the complex pathophysiology that is associated with these two cardinal neuropathologies. Here, we characterized the proteomic landscape of cerebrospinal fluid (CSF) changes associated with Aß and tau pathology in 300 individuals using two different proteomic technologies-tandem mass tag mass spectrometry and SomaScan. Integration of both data types allowed for generation of a robust protein coexpression network consisting of 34 modules derived from 5242 protein measurements, including disease-relevant modules associated with autophagy, ubiquitination, endocytosis, and glycolysis. Three modules strongly associated with the apolipoprotein E ε4 (APOE ε4) AD risk genotype mapped to oxidant detoxification, mitogen-associated protein kinase signaling, neddylation, and mitochondrial biology and overlapped with a previously described lipoprotein module in serum. Alterations of all three modules in blood were associated with dementia more than 20 years before diagnosis. Analysis of CSF samples from an AD phase 2 clinical trial of atomoxetine (ATX) demonstrated that abnormal elevations in the glycolysis CSF module-the network module most strongly correlated to cognitive function-were reduced by ATX treatment. Clustering of individuals based on their CSF proteomic profiles revealed heterogeneity of pathological changes not fully reflected by Aß and tau.

The Influence of a Genetic Variant in CCDC78 on LMNA-Associated Skeletal Muscle Disease.

Mutations in the LMNA gene-encoding A-type lamins can cause Limb-Girdle muscular dystrophy Type 1B (LGMD1B). This disease presents with weakness and wasting of the proximal skeletal muscles and has a variable age of onset and disease severity. This variability has been attributed to genetic background differences among individuals; however, such variants have not been well characterized. To identify such variants, we investigated a multigeneration family in which affected individuals are diagnosed with LGMD1B. The primary genetic cause of LGMD1B in this family is a dominant mutation that activates a cryptic splice site, leading to a five-nucleotide deletion in the mature mRNA. This results in a frame shift and a premature stop in translation. Skeletal muscle biopsies from the family members showed dystrophic features of variable severity, with the muscle fibers of some family members possessing cores, regions of sarcomeric disruption, and a paucity of mitochondria, not commonly associated with LGMD1B. Using whole genome sequencing (WGS), we identified 21 DNA sequence variants that segregate with the family members possessing more profound dystrophic features and muscle cores. These include a relatively common variant in coiled-coil domain containing protein 78 (CCDC78). This variant was given priority because another mutation in CCDC78 causes autosomal dominant centronuclear myopathy-4, which causes cores in addition to centrally positioned nuclei. Therefore, we analyzed muscle biopsies from family members and discovered that those with both the LMNA mutation and the CCDC78 variant contain muscle cores that accumulated both CCDC78 and RyR1. Muscle cores containing mislocalized CCDC78 and RyR1 were absent in the less profoundly affected family members possessing only the LMNA mutation. Taken together, our findings suggest that a relatively common variant in CCDC78 can impart profound muscle pathology in combination with a LMNA mutation and accounts for variability in skeletal muscle disease phenotypes.

N-(4-Bromo-2,5-Dimethoxyphenethyl)-6-(4-Phenylbutoxy)Hexan-1-Amine (XOB): A Novel Phenylalkylamine Antagonist of Serotonin 2A Receptors and Voltage-Gated Sodium Channels.

Bipolar disorder impacts millions of patients in the United States but the mechanistic understanding of its pathophysiology and therapeutics is incomplete. Atypical antipsychotic serotonin2A (5-HT2A) receptor antagonists, such as quetiapine and olanzapine, and mood-stabilizing voltage-gated sodium channel (VGSC) blockers, such as lamotrigine, carbamazepine, and valproate, show therapeutic synergy and are often prescribed in combination for the treatment of bipolar disorder. Combination therapy is a complex task for clinicians and patients, often resulting in unexpected difficulties with dosing, drug tolerances, and decreased patient compliance. Thus, an unmet need for bipolar disorder treatment is to develop a therapeutic agent that targets both 5-HT2A receptors and VGSCs. Toward this goal, we developed a novel small molecule that simultaneously antagonizes 5-HT2A receptors and blocks sodium current. The new compound, N-(4-bromo-2,5-dimethoxyphenethyl)-6-(4-phenylbutoxy)hexan-1-amine (XOB) antagonizes 5-HT-stimulated, Gq-mediated, calcium flux at 5-HT2A receptors at low micromolar concentrations while displaying negligible affinity and activity at 5-HT1A, 5-HT2B, and 5-HT2C receptors. At similar concentrations, XOB administration inhibits sodium current in heterologous cells and results in reduced action potential (AP) firing and VGSC-related AP properties in mouse prefrontal cortex layer V pyramidal neurons. Thus, XOB represents a new, proof-of-principle tool that can be used for future preclinical investigations and therapeutic development. This polypharmacology approach of developing a single molecule to act upon two targets, which are currently independently targeted by combination therapies, may lead to safer alternatives for the treatment of psychiatric disorders that are increasingly being found to benefit from the simultaneous targeting of multiple receptors. SIGNIFICANCE STATEMENT: The authors synthesized a novel small molecule (XOB) that simultaneously antagonizes two key therapeutic targets of bipolar disorder, 5-HT2A receptors and voltage-gated sodium channels, in heterologous cells, and inhibits the intrinsic excitability of mouse prefrontal cortex layer V pyramidal neurons in brain slices. XOB represents a valuable new proof-of-principle tool for future preclinical investigations and provides a novel molecular approach to the pharmacological treatment of complex neuropsychiatric disease, which often requires a combination of therapeutics for sufficient patient benefit.

Forskolin reverses the O-GlcNAcylation dependent decrease in GABAAR current amplitude at hippocampal synapses possibly at a neurosteroid site on GABAARs.

GABAergic transmission is influenced by post-translational modifications, like phosphorylation, impacting channel conductance, allosteric modulator sensitivity, and membrane trafficking. O-GlcNAcylation is a post-translational modification involving the O-linked attachment of ß-N-acetylglucosamine on serine/threonine residues. Previously we reported an acute increase in O-GlcNAcylation elicits a long-term depression of evoked GABAAR inhibitory post synaptic currents (eIPSCs) onto hippocampal principal cells. Importantly, O-GlcNAcylation and phosphorylation can co-occur or compete for the same residue; whether they interact in modulating GABAergic IPSCs is unknown. We tested this by recording IPSCs from hippocampal principal cells and pharmacologically increased O-GlcNAcylation, before or after increasing serine phosphorylation using the adenylate cyclase activator, forskolin. Although forskolin had no significant effect on baseline eIPSC amplitude, we found that a prior increase in O-GlcNAcylation unmasks a forskolin-dependent increase in eIPSC amplitude, reversing the O-GlcNAc-induced eIPSC depression. Inhibition of adenylate cyclase or protein kinase A did not prevent the potentiating effect of forskolin, indicating serine phosphorylation is not the mechanism. Surprisingly, increasing O-GlcNAcylation also unmasked a potentiating effect of the neurosteroids 5α-pregnane-3α,21-diol-20-one (THDOC) and progesterone on eIPSC amplitude, mimicking forskolin. Our findings show under conditions of heightened O-GlcNAcylation, the neurosteroid site on synaptic GABAARs is accessible to agonists, permitting strengthening of synaptic inhibition.

Intracranial Mapping of Response Latencies and Task Effects for Spoken Syllable Processing in the Human Brain.

Prior lesion, noninvasive-imaging, and intracranial-electroencephalography (iEEG) studies have documented hierarchical, parallel, and distributed characteristics of human speech processing. Yet, there have not been direct, intracranial observations of the latency with which regions outside the temporal lobe respond to speech, or how these responses are impacted by task demands. We leveraged human intracranial recordings via stereo-EEG to measure responses from diverse forebrain sites during (i) passive listening to /bi/ and /pi/ syllables, and (ii) active listening requiring /bi/-versus-/pi/ categorization. We find that neural response latency increases from a few tens of ms in Heschl's gyrus (HG) to several tens of ms in superior temporal gyrus (STG), superior temporal sulcus (STS), and early parietal areas, and hundreds of ms in later parietal areas, insula, frontal cortex, hippocampus, and amygdala. These data also suggest parallel flow of speech information dorsally and ventrally, from HG to parietal areas and from HG to STG and STS, respectively. Latency data also reveal areas in parietal cortex, frontal cortex, hippocampus, and amygdala that are not responsive to the stimuli during passive listening but are responsive during categorization. Furthermore, multiple regions-spanning auditory, parietal, frontal, and insular cortices, and hippocampus and amygdala-show greater neural response amplitudes during active versus passive listening (a task-related effect). Overall, these results are consistent with hierarchical processing of speech at a macro level and parallel streams of information flow in temporal and parietal regions. These data also reveal regions where the speech code is stimulus-faithful and those that encode task-relevant representations.

The Challenges of Advance Care Planning for Acute Care Registered Nurses.

STUDY BACKGROUND: The practice of acute care nurses is shaped by organizational factors such as lack of privacy, heavy workloads, unclear roles, lack of time, and lack of specific policies and procedures. We know little about the social and organizational structures and processes that influence nurses' uptake of valuable patient-centered discussions like advance care planning (ACP). ACP is beneficial for patients, their substitute decision makers, and healthcare providers. PURPOSE: To describe the operational, organizational, and societal influences shaping nurses' ACP work in acute care settings. METHODS: This ethnographic study purposively sampled 14 registered nurses and 9 administrators who worked in two acute care hospitals in Northeastern Ontario. Methods consisted of 23 open-ended, semi-structured interviews, 20 hours of observational fieldwork, and a collection of publicly available organizational documents. Data were inductively analyzed using an iterative constant comparative approach. RESULTS: Nurses were challenged to meet multiple competing demands, leaving them to scramble to manage complex and critically ill acute care patients while also fulfilling organizational tasks aligned with funding metrics, accreditation, and strategic planning priorities. Such factors limited nurses' capacity to engage their patients in ACP. CONCLUSIONS: Acute care settings that align patient values and medical treatment need to foster ACP practices by revising organizational policies and processes to support this outcome, analyzing the tasks of healthcare providers to determine who might best address it, and budgeting how to support it with additional resources.

Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform.

Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.

Empirical assessment of the assumptions of ComBat with diffusion tensor imaging.

Purpose: Diffusion tensor imaging (DTI) is a magnetic resonance imaging technique that provides unique information about white matter microstructure in the brain but is susceptible to confounding effects introduced by scanner or acquisition differences. ComBat is a leading approach for addressing these site biases. However, despite its frequent use for harmonization, ComBat's robustness toward site dissimilarities and overall cohort size have not yet been evaluated in terms of DTI. Approach: As a baseline, we match N=358 participants from two sites to create a "silver standard" that simulates a cohort for multi-site harmonization. Across sites, we harmonize mean fractional anisotropy and mean diffusivity, calculated using participant DTI data, for the regions of interest defined by the JHU EVE-Type III atlas. We bootstrap 10 iterations at 19 levels of total sample size, 10 levels of sample size imbalance between sites, and 6 levels of mean age difference between sites to quantify (i) ßAGE, the linear regression coefficient of the relationship between FA and age; (ii) Î³/f*, the ComBat-estimated site-shift; and (iii) Î´/f*, the ComBat-estimated site-scaling. We characterize the reliability of ComBat by evaluating the root mean squared error in these three metrics and examine if there is a correlation between the reliability of ComBat and a violation of assumptions. Results: ComBat remains well behaved for ßAGE when N>162 and when the mean age difference is less than 4 years. The assumptions of the ComBat model regarding the normality of residual distributions are not violated as the model becomes unstable. Conclusion: Prior to harmonization of DTI data with ComBat, the input cohort should be examined for size and covariate distributions of each site. Direct assessment of residual distributions is less informative on stability than bootstrap analysis. We caution use ComBat of in situations that do not conform to the above thresholds.

DeepN4: Learning N4ITK Bias Field Correction for T1-weighted Images.

T1-weighted (T1w) MRI has low frequency intensity artifacts due to magnetic field inhomogeneities. Removal of these biases in T1w MRI images is a critical preprocessing step to ensure spatially consistent image interpretation. N4ITK bias field correction, the current state-of-the-art, is implemented in such a way that makes it difficult to port between different pipelines and workflows, thus making it hard to reimplement and reproduce results across local, cloud, and edge platforms. Moreover, N4ITK is opaque to optimization before and after its application, meaning that methodological development must work around the inhomogeneity correction step. Given the importance of bias fields correction in structural preprocessing and flexible implementation, we pursue a deep learning approximation / reinterpretation of the N4ITK bias fields correction to create a method which is portable, flexible, and fully differentiable. In this paper, we trained a deep learning network "DeepN4" on eight independent cohorts from 72 different scanners and age ranges with N4ITK-corrected T1w MRI and bias field for supervision in log space. We found that we can closely approximate N4ITK bias fields correction with naïve networks. We evaluate the peak signal to noise ratio (PSNR) in test dataset against the N4ITK corrected images. The median PSNR of corrected images between N4ITK and DeepN4 was 47.96 dB. In addition, we assess the DeepN4 model on eight additional external datasets and show the generalizability of the approach. This study establishes that incompatible N4ITK preprocessing steps can be closely approximated by naïve deep neural networks, facilitating more flexibility. All code and models are released at https://github.com/MASILab/DeepN4 .

Establishing an infrastructure to optimize the integration of genomics into research: Results from a precision health needs assessment.

Researchers across the translational research continuum have emphasized the importance of integrating genomics into their research program. To date capacity and resources for genomics research have been limited; however, a recent population-wide genomic screening initiative launched at the Medical University of South Carolina in partnership with Helix has rapidly advanced the need to develop appropriate infrastructure for genomics research at our institution. We conducted a survey with researchers from across our institution (n = 36) to assess current knowledge about genomics health, barriers, and facilitators to uptake, and next steps to support translational research using genomics. We also completed 30-minute qualitative interviews with providers and researchers from diverse specialties (n = 8). Quantitative data were analyzed using descriptive analyses. A rapid assessment process was used to develop a preliminary understanding of each interviewee's perspective. These interviews were transcribed and coded to extract themes. The codes included types of research, alignment with precision health, opportunities to incorporate precision health, examples of researchers in the field, barriers, and facilitators to uptake, educational activity suggestions, questions to be answered, and other observations. Themes from the surveys and interviews inform implementation strategies that are applicable not only to our institution, but also to other organizations interested in making genomic data available to researchers to support genomics-informed translational research.

Researchers have recognized the significance of integrating genomics into their studies across the translational research continuum. However, limited capacity and resources have hindered progress in genomics research. We conducted a survey and qualitative interviews with researchers and healthcare providers from our institution to assess their understanding of genomics in health, identify barriers, and facilitators to its adoption, and determine next steps for supporting translational research using genomics. Themes identified included different types of research, alignment with precision health, opportunities to incorporate precision health, examples of researchers in the field, barriers, and facilitators to adoption, educational recommendations, unanswered questions, and other valuable observations. The insights gathered from the surveys and interviews informed the development of implementation strategies. These strategies can benefit not only our institution but also other researchers who are interested in providing access to genomic data to support genomics-informed translational research.

Plasma proteomic signature of human longevity.

The identification of protein targets that exhibit anti-aging clinical potential could inform interventions to lengthen the human health span. Most previous proteomics research has been focused on chronological age instead of longevity. We leveraged two large population-based prospective cohorts with long follow-ups to evaluate the proteomic signature of longevity defined by survival to 90 years of age. Plasma proteomics was measured using a SOMAscan assay in 3067 participants from the Cardiovascular Health Study (discovery cohort) and 4690 participants from the Age Gene/Environment Susceptibility-Reykjavik Study (replication cohort). Logistic regression identified 211 significant proteins in the CHS cohort using a Bonferroni-adjusted threshold, of which 168 were available in the replication cohort and 105 were replicated (corrected p value <0.05). The most significant proteins were GDF-15 and N-terminal pro-BNP in both cohorts. A parsimonious protein-based prediction model was built using 33 proteins selected by LASSO with 10-fold cross-validation and validated using 27 available proteins in the validation cohort. This protein model outperformed a basic model using traditional factors (demographics, height, weight, and smoking) by improving the AUC from 0.658 to 0.748 in the discovery cohort and from 0.755 to 0.802 in the validation cohort. We also found that the associations of 169 out of 211 proteins were partially mediated by physical and/or cognitive function. These findings could contribute to the identification of biomarkers and pathways of aging and potential therapeutic targets to delay aging and age-related diseases.