The recent decade has seen an increasing recognition of the importance of sleep in substance use disorders (SUD) in both research and clinical settings. Not only is sleep disturbance a common comorbidity in SUD, but it may provide a causal link and druggable targets for complementary treatment. The interpersonal variation in sleep also provides opportunities for developing biomarkers and individualized medicine. This review is focused on the key neurotransmitter and neuromodulator systems through which sleep loss may bias reward seeking, increasing the risks for initial drug exposure as well as relapse after drug withdrawal. The review summarizes sleep changes following acute or long-term drug exposure and withdrawal, and current understanding of sleep-mediated regulation of glutamatergic, dopaminergic, and peptidergic transmissions importantly indicated in SUD research.

Background:Acute sleep loss has been linked to increased reward-seeking behaviors in both humans and animals, including increased food consumption, greater risk taking for reward, and increased substance use in previous substance users. During adolescence, developing reward systems are already particularly vulnerable to substance use. Therefore, any acute sleep loss that occurs during adolescence may further heighten reward-seeking behaviors, increasing susceptibility to substance misuse and substance use disorder. Methods:Male and female heterogeneous stock rats underwent a 6-hour acute sleep deprivation session prior to testing motivation for a sucrose pellet reward under a progressive ratio schedule or transcriptomic analysis of nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) gene expression relative to control sleep. Results:Rats of both sexes exhibited increased motivation for sucrose reward for at least 3 days following sleep deprivation and substantial changes in gene transcription that were largely conserved across brain regions, although the mPFC showed more transcriptional changes than the NAc. Pathway analyses indicated major changes related to cellular development, immune signaling, and rhythmic processes. The orexin system, particularly the orexin 2 receptor (OX2R), was strongly upregulated in the mPFC and NAc, so we determined the effect of the OX2R antagonist seltorexant (10 mg/kg) on sleep deprivation-induced motivation for reward and found that seltorexant acutely reduced responding for sucrose under both sleep deprivation and sleep recovery conditions. Conclusions:These results suggest that acute sleep deprivation induces profound changes in the reward system and cellular function in adolescents, including the orexin system, that may modulate motivation for reward.

Correcting for library size is an essential step in bulk RNA-seq analyses, as differences in sequencing depth across samples can obscure biological signal with technical noise. While numerous normalization methods and model-based strategies have been proposed, we demonstrate here that library size-normalized counts and differential expression results obtained from such widely adopted approaches often remain strongly correlated with library size in large-scale RNA-seq experiments. Through a systematic analysis of over 100 publicly available GEO and TCGA RNA-seq datasets with raw count data, we show that library size association is observed for a substantial proportion of genes even after state-of-the-art library size correction approaches recommended by leading normalization tools. To address this issue, we propose gecco , a gene-specific exponent-corrected normalization method for RNA-seq counts that incorporates library size directly into the statistical framework via a gene-specific correction term, rather than applying a uniform adjustment factor across all genes. This formulation generalizes existing normalization approaches and yields normalized counts that are free of residual library size effects. Using both simulation studies and real large-scale RNA-seq datasets, we show that our method mitigates library size bias while preserving biological signal across a range of parameter settings. We further demonstrate that our approach leads to higher detection accuracy and more biologically meaningful pathway enrichment results in downstream differential expression and rhythmicity analyses without compromising false discovery rate control. Our method is implemented in R and is fully compatible with the widely used differential expression analysis methods DESeq2 and edgeR .

Chronotype describes an individual's day or night preference and is thought to exert health effects through circadian timing, yet continuous circadian measures related to chronotype have never been examined in large human cohorts. We sought to quantify wearable-derived heart rate phase (HRP) as an indicator of chronotype-relevant circadian shifts and determine its associations with human disease. Heart rate was aggregated over 5 min intervals in All of Us participants with wearable data and fit to a sine curve with 24 h period to determine HRP. Associations between HRP and circadian genomic variants were determined by linear regression. A phenome-wide association study (PheWAS) was performed by multiple logistic regression. One-sample Mendelian randomization (MR) was performed by two-stage residual inclusion. Average HRP was 9.48 ± 1.57 h (n = 15,960). PheWAS identified phenome-wide associations of later HRP with addiction, mood, sleep and metabolic disorders, while certain conditions of pregnancy were associated with earlier HRP. In focused analyses of type 2 diabetes mellitus (T2DM), later HRP was associated with increased T2DM risk [odds ratio (OR) 1.09 [1.06,1.13], P = 1.58 × 10-7). The morningness genomic variant rs1144566(T) was associated with earlier HRP (P = 1.38 × 10-7) and decreased risk of T2DM (OR 0.69 [0.55,0.88], P = 2.67 × 10-3). MR analysis suggested a causal effect of rs1144566 on T2DM risk through HRP (P = 2.70 × 10-3). We introduce the use of a quantitative longitudinal circadian metric, HRP. We combine HRP with phenomics, genomics and electronic health records data to provide evidence for a relationship between circadian shifts and disease. HRP may hold value in the management and surveillance of human health. KEY POINTS: Chronotype is a genetically influenced innate preference for the timing of daily activity and is believed to affect health outcomes through shifts in circadian rhythms. Measurement of chronotype-relevant circadian changes may be possible through wearable technology. We show that the timing of daily heart rate rhythms, which we call heart rate phase (HRP), is associated with chronotype-relevant genetic and demographic factors. We report associations of HRP with addiction, mood, sleep and metabolism disorders as well as certain pregnancy conditions. We show that HRP is associated with type 2 diabetes mellitus (T2DM) risk and that a morning genetic chronotype variant may causally influence T2DM risk through HRP.

The brain’s complex network relies on both electrical and chemical signaling to support its physiological and cognitive functions. To fully understand neural circuit dynamics and their dysfunctions, it is crucial to simultaneously detect neurotransmitters and modulators alongside electrophysiological signals. The striatal dopamine circuits are integral to neurological processes such as movement, reward, learning, and circadian rhythm regulation, making it highly desirable to monitor both neural activity and dopamine (DA) levels in freely behaving animals. One promising approach involves the implantation of multimodal microelectrode arrays (MEAs). However, chronic electrochemical sensing of DA in freely moving animals faces significant challenges, including biofouling of sensing electrodes and the instability of Ag/AgCl reference electrodes. In this study, we developed two complementary strategies—surface grafting and photo crosslinking—to coat the MEA and implanted Ag/AgCl reference electrodes, respectively, with zwitterionic poly(sulfobetaine methacrylate) (PSB). The surface-grafted thin PSB coating effectively inhibits protein fouling and inflammatory responses to the MEA, while the PSB hydrogel protects the Ag/AgCl electrodes from delamination in vivo, ensuring a stable reference potential. By coating both the Ag/AgCl reference electrodes and flexible polyimide MEAs with PSB and PEDOT/CNT, we achieved stable DA detection and electrophysiological recordings in freely moving mice over a four-week period. Weekly electrochemical impedance spectroscopy confirmed the long-term stability of the implanted electrodes. Our method enables multidimensional analysis of behavioral patterns, electrophysiological activity, and DA dynamics, providing a comprehensive approach for neuroscience research. This work advances neurochemical and electrophysiological methodologies by offering reliable tools for longitudinal investigations of brain function in freely behaving animals.

20% of Americans are at risk for environmental circadian rhythm disruptions (CRD) due to shift work, leading to substantial negative health outcomes. However, females are especially affected with greater vulnerability for substance use (SU) and adverse outcomes associated with pregnancy, including for offspring at birth and later in life. In mice, prenatal CRD (pCRD) recapitulates these risks, but it is unknown whether pCRD affects SU in mature offspring. To investigate this, C57BL/6J dams were disrupted by reversing the light/dark cycle during gestation. Following pCRD, reward- (cocaine conditioned place preference, intravenous self-administration) and mood-related behaviors (open field, elevated plus maze, light/dark box, forced swim) were measured in adult offspring. Adult female offspring of dams exposed to CRD developed an anhedonic-like phenotype with decreased food self-administration, cocaine intake and reinforcing properties of cocaine. Opposingly, pCRD male offspring showed a SU-like phenotype with increased cocaine preference, higher order food self-administration and cocaine reinforcement. Interestingly, these divergent behavioral outcomes were not specific to reward. While female pCRD mice showed increased anxiety-like behavior, pCRD males showed decreased anxiety/increased risk-taking behavior, as well as decreased immobility in the forced swim test. Rhythms in corticosterone were also sex-specifically affected by pCRD. These results suggest that pCRD may predispose individuals to distinct psychiatric disorders based on sex with mood disorders developing in females and SU disorders developing in males. By better understanding how disrupted rhythms during pregnancy affect behavior in adulthood, we can develop novel therapeutic approaches for SU and mood disorders in adults.

INTRODUCTION:Circadian dysfunction is involved in the pathophysiology of bipolar disorders (BD), and circadian-based interventions are gaining recognition in their management. Moreover, basic and epidemiologic research has generated findings inspiring circadian-informed self- and clinician-management strategies. Despite these gains, many Clinical Practice Guidelines and clinical training programs have not incorporated this evidence in their recommendations and curricula. This International Society for Bipolar Disorders (ISBD) Chronobiology and Chronotherapy Task Force position paper reports a Delphi-based expert consensus on what is essential for mental health clinicians to know about the chronobiology and chronotherapy of BD. METHODS:An initial pool of statements was extracted from academic and grey literature, and experts could suggest additional statements. Statements were rated on a 5-point scale ('essential'; 'important'; 'don't know/depends'; 'unimportant'; 'should not be included'). Consensus was reached when statements were rated as essential or important by ≥ 80% of experts. RESULTS:Thirty experts from 15 countries in Europe, North and South America, and the Asia Pacific participated (mean age of 55.3 years [SD = 11.8]; 40% female; 83% psychiatrists; mean clinical experience of 26 years [SD = 10.8]). Eight-hundred-and-thirty-seven statements were rated across three rounds. Consensus was reached on 342 statements spanning four major themes: basic circadian science; circadian health and disruption; chronobiology of BD; and six chronotherapies (e.g., protocols, outcomes, risks/contraindications). CONCLUSIONS:An expert consensus was obtained on the essential information about the chronobiology and chronotherapy of BD, intended to help clinicians optimise their management of BD. Dissemination of this knowledge is expected to enhance the training and efficacy of clinicians.

Chronopsychiatry refers to a research and clinical approach which studies psychiatric illness through the lens of circadian rhythms and aims to reset the body’s biological clock to counteract circadian rhythm abnormalities in psychiatric disorders. This review highlights the use of circadian biomarkers collected from wearable devices and other methods to help the development of personalized treatment plans based on patients’ individual circadian functioning. Circadian biomarkers, such as activity, heart rate, skin temperature, and light exposure recorded over 24 h, has shown promise in diagnosing and assessing the severity of various neuropsychiatric disorders. Wearable devices capable of recording multiple circadian parameters have greatly aided data collection, and when integrated into treatment, have shown promise for revolutionizing circadian medicine. Therapies that entrain or shift the circadian system can help stabilize sleep-wake rhythms or better align rhythms with the environment. These approaches support the emerging field of precision psychiatry, moving beyond a one-size-fits-all approach and addressing the heterogeneity and complexity of psychiatric disorders.

Suicide is a leading cause of death worldwide, yet the biological mechanisms underlying suicide remain poorly understood. A clearer understanding at the molecular level is essential for developing objective biomarkers and targeted interventions. In this study, we used transcriptomic profiling to investigate gene expression patterns associated with suicidal thoughts and behaviors across peripheral blood (n=264) and postmortem brain tissue from two prefrontal regions (dorsolateral prefrontal cortex, DLPFC; subgenual anterior cingulate cortex, sgACC) of individuals with and without psychiatric illness (n=249). Peripheral analyses revealed broad transcriptional changes associated with suicidal thoughts and behaviors, marked by dysregulated immune-related and inflammatory processes. Longitudinal modeling further revealed gene co-expression modules that predicted future suicide attempts over a 12-month follow-up, highlighting processes related to apoptosis, mitochondrial function, and immune regulation. By contrast, transcriptomic analyses of postmortem tissue derived from the DLPFC and sgACC revealed largely suppressed neuroimmune activity. Gene co-expression analyses in the brain identified suicide-associated modules enriched for synaptic plasticity, oxidative stress, and neuroimmune function, some of which displayed regional specificity. Cross-tissue comparison showed minimal gene-level overlap between brain and blood, although shared pathway-level themes emerged in immune, sensory, and cellular stress processes. Taken together, these findings suggest that suicide is associated with distinct but functionally convergent transcriptional alterations across brain and blood. By integrating tissue-specific and systems-level molecular signatures, this work provides insight into the biological architecture of suicide and lays the groundwork for developing novel biomarkers and therapeutic targets to improve prevention and treatment outcomes.

Chronically implanted neural electrodes for neural recording, stimulation, and chemical sensing are essential tools in studying and treating neurological disorders. However, their performances are limited by implant-induced inflammation, causing a loss in signal quality over time. Because disease states can alter inflammatory processes, it is critical to characterize the tissue response to neural implants within relevant disease models. Circadian rhythm disruption is a hallmark of many psychiatric and neurodegenerative disorders, yet its influence on neural implant performance remains poorly understood. The Circadian Locomotor Output Cycles Kaput (Clock) gene is a core regulator of circadian rhythms and a key modulator of inflammatory pathways, including NF-κB signaling. The ClockΔ19 mutant mouse is a well-established model of circadian disruption with documented immunological abnormalities. Here, we evaluated striatal electrophysiological recording performance and quantified the host tissue response to microelectrode implants in ClockΔ19 (MU) and wild-type (WT) mice over four weeks. Silicon microelectrode arrays (MEAs) were implanted into the striatum, followed by weekly electrochemical impedance spectroscopy and neuronal recording measurements. Electrodes implanted in MU mice exhibited significantly lower impedance, noise, and peak-to-peak amplitude compared to WT mice, while signal-to-noise ratio and channel yield were comparable between groups. Endpoint immunohistological analyses revealed significantly reduced microglia and astrocyte activity, as indicated by lower Iba-1 and GFAP intensities around the implant site in MU mice. Additionally, ClockΔ19 tissue showed elevated 4- hydroxynonenal (HNE) levels and reduced nuclear NF-κB expression following implantation. Morphological analysis further identified baseline and injury-induced differences in microglia phenotypes between MU and WT animals. Notably, decreased neurofilament expression, together with a non-significant trend toward reduced neuronal density, suggests compromised neuronal health surrounding the implant in MU animals. Together, these findings demonstrate that circadian disruption alters both neural and immune responses to chronic neural implants, resulting in heightened oxidative stress and an impaired reparative inflammatory response. This work highlights the importance of circadian regulation in neuroimmune responses, providing insights into how circadian dysfunction may impact long-term neural interface performance with implications for both basic research and clinical neurotechnology development.

Abstract Inter-individual variation in human molecular and behavioral circadian rhythms motivates genetic dissection in model systems with human-like diversity. We quantified cellular clock phenotypes from primary skin fibroblasts of several hundred Diversity Outbred (DO) mice-each carrying a unique mosaic of eight founder genomes-by longitudinal bioluminescence recordings of a Bmal1-luciferase reporter (LumiCycle). Canonical rhythm parameters (period, phase, amplitude, damping) were extracted and exhibited broad variability (heritability ≈13–35%), exceeding the ranges of founder strains. We performed genome-wide QTL mapping with R/qtl2 (linear mixed models with sex and experimental group covariates, kinship control, permutation-based significance, 1.5-LOD support intervals). A suggestive QTL for amplitude localized to chromosome 12 (LOD 6.9; ∼7.5–12.0 Mb), with founder effects indicating higher amplitude for C57BL/6J and lower for PWK/PhJ. Among 21 protein-coding genes in this interval, Apob (apolipoprotein B), a clock-regulated determinant of lipoprotein assembly, emerged as a strong candidate for amplitude control. A phase QTL mapped to chromosome 1 (support interval ∼1.36 Mb) with divergent founder effects (C57BL/6J, NOD/ShiLtJ, WSB/EiJ: delayed; PWK/PhJ, CAST/EiJ: advanced) and prioritized candidates including Epha4 (an Eph receptor tyrosine kinase implicated in photic entrainment) and Acsl3 . Integrative analysis in GeneWeaver connected QTL gene sets to prior loci for voluntary alcohol consumption and circadian period on proximal chromosome 12, and highlighted overlaps with GWAS signals for adolescent idiopathic scoliosis and schizophrenia, suggesting shared pathways between circadian regulation, metabolism, and neurobehavioral traits. Together, these findings define reproducible genomic loci for cellular clock phenotypes in a highly recombinant population, nominate tractable candidate genes ( Apob, Epha4/Acsl3 ) for mechanistic follow-up, and illustrate how high-diversity mouse genetics bridges cellular circadian variation with complex disease biology.

The circadian rhythm regulates physiological and behavioral processes, with disruptions linked to metabolic and neuropsychiatric disorders. Circadian genes play a crucial role in the regulation of dopaminergic signaling, yet the underlying molecular mechanisms remain unclear. This study investigates how the Clock gene modulates dopamine (DA) dynamics using in vivo electrochemical DA sensing and molecular profiling. Utilizing carbon fiber electrodes (CFEs) with poly(3,4-ethylenedioxythiophene)/carbon nanotube (PEDOT/CNT) coatings, we measured extracellular DA levels in the striatum of wild-type (WT) and ClockΔ19 mutant mice via square wave voltammetry (SWV). Pharmacological perturbation with raclopride (D2/D3 receptor antagonist) and nomifensine (DA reuptake inhibitor) revealed an increased DA receptor sensitivity in ClockΔ19 mice, with a significantly faster DA response to raclopride. Molecular profiling via qRT-PCR showed elevated tyrosine hydroxylase (TH) expression in the ventral tegmental area (VTA) of ClockΔ19 mice, suggesting increased DA synthesis. Additionally, ClockΔ19 mice exhibited higher expression of D2 DA receptors and glutamate decarboxylase 67 (Gad67) in the VTA and of D3 DA receptors in the nucleus accumbens (NAc), implicating altered dopaminergic and γ-aminobutyric acid (GABA)ergic regulation. These findings highlight the Clock gene's role in DA homeostasis, revealing its impact on neurotransmission.

While previous studies have found rhythms in gene expression in the prefrontal cortex (PFC), the contribution of different cell types and potential variation by sex has not been determined. Of interest are excitatory pyramidal cells and inhibitory parvalbumin (PV) interneurons, as the interaction between these cells is thought to underlie gamma oscillations and play a role in schizophrenia. We identify cell-type-specific rhythms in ribosome-associated transcripts from PV and pyramidal cells in the mouse PFC and assess rhythms in PV cell electrophysiology. We find that while core molecular clock genes are synchronized between cell types, pyramidal cells have nearly twice as many rhythmic transcripts as PV cells (35% vs. 18%). Moreover, in contrast to PV cells, rhythmic transcripts in pyramidal cells show substantial overlap between sexes. Additionally, there is a sex-specific reduction in action potential amplitude and spike frequency adaptation during the dark phase in PV cells from females. This study demonstrates that rhythms in gene expression and electrophysiological properties in the PFC vary by cell type and by sex. Moreover, the biological processes associated with rhythmic transcripts may provide insight into the unique functions of rhythms in these cells, as well as their selective vulnerabilities to circadian disruption.

Psychosis is a hallmark symptom of schizophrenia and highly prevalent in bipolar disorder. Previous work has shown altered gene expression within subregions of the striatum in subjects with psychosis, but it is unclear if these alterations differ across subregions. Moreover, despite known sex differences in the presentation of psychosis (such as age of onset and disease course), it is unclear if there are sex differences in gene expression across subregions of the human striatum in the context of psychosis. Using RNA-sequencing data from human postmortem nucleus accumbens (NAc), caudate, and putamen, we first performed differential expression analyses across these striatal subregions in unaffected (n = 60) and psychosis (n = 36) subjects. For analysis of sex differences, we used equal numbers of males and females in each subject group and evaluated sex and psychosis effects within each brain region. We found that the NAc is the most transcriptionally unique region compared to the caudate and the putamen in both psychosis and unaffected subjects. We also found distinct patterns in gene expression across the three striatal subregions, with an altered pattern of cilia-related genes in subjects with psychosis. Our sex-based analyses showed a striking discordant expression pattern, with opposite effect directions between male and female subjects with psychosis in all three subregions, including a reversal of sex differences in immune- and angiogenesis-related pathways. Overall, we identified regional and sex differences in gene expression across the human striatum that may underlie sex-specific striatal dysfunction and symptomatology in psychosis.

Abstract This chapter describes findings from cell culture, animal, and human studies that have shed light on the molecular and cellular basis of bipolar disorder. It summarizes the most replicated genetic findings and the contribution of environmental influences. It also details the various cellular and brain circuit abnormalities that are thought to play a role in the development of this disorder. Sleep–wake and other circadian rhythm disruptions are central to the disorder; thus, a good portion of the chapter details how disruptions to normal 24-hr rhythms in a variety of cellular processes could contribute to bipolar disorder pathophysiology and treatment. In addition, the chapter discusses ways in which mood-stabilizing medications are thought to work therapeutically and how this has enhanced knowledge of the causes of this disease and how it may vary from individual to individual.

IntroductionAlterations in multiple subregions of the human prefrontal cortex (PFC) have been heavily implicated in psychiatric diseases. Moreover, emerging evidence suggests that circadian rhythms in gene expression are present across the brain, including in the PFC, and that these rhythms are altered in disease. However, investigation into the potential circadian mechanisms underlying these diseases in animal models must contend with the fact that the human PFC is highly evolved and specialized relative to that of rodents.MethodsHere, we use RNA sequencing to lay the groundwork for translational studies of molecular rhythms through a sex-specific, cross species comparison of transcriptomic rhythms between the mouse medial PFC (mPFC) and two subregions of the human PFC, the anterior cingulate cortex (ACC) and the dorsolateral PFC (DLPFC).ResultsWe find that while circadian rhythm signaling is conserved across species and subregions, there is a phase shift in the expression of core clock genes between the mouse mPFC and human PFC subregions that differs by sex. Furthermore, we find that the identity of rhythmic transcripts is largely unique between the mouse mPFC and human PFC subregions, with the most overlap (20%, 236 transcripts) between the mouse mPFC and the human ACC in females. Nevertheless, we find that basic biological processes are enriched for rhythmic transcripts across species, with key differences between regions and sexes.DiscussionTogether, this work highlights both the evolutionary conservation of transcriptomic rhythms and the advancement of the human PFC, underscoring the importance of considering cross-species differences when using animal models.

High-dimensional omics data often contain intricate and multifaceted information, resulting in the coexistence of multiple plausible sample partitions based on different subsets of selected features. Conventional clustering methods typically yield only one clustering solution, limiting their capacity to fully capture all facets of cluster structures in high-dimensional data. To address this challenge, we propose a model-based multifacet clustering (MFClust) method based on a mixture of Gaussian mixture models, where the former mixture achieves facet assignment for gene features and the latter mixture determines cluster assignment of samples. We demonstrate superior facet and cluster assignment accuracy of MFClust through simulation studies. The proposed method is applied to three transcriptomic applications from postmortem brain and lung disease studies. The result captures multifacet clustering structures associated with critical clinical variables and provides intriguing biological insights for further hypothesis generation and discovery.

BACKGROUNDSleep is increasingly recognized as essential to human health, yet the adverse health consequences of acute sleep deprivation are unknown. We hypothesized that acute sleep deprivation is associated with health outcomes and modulated by sleep-associated genotypes.METHODSLocally estimated scatterplot smoothing (LOESS) was performed on sleep estimates from Fitbit users (n = 14,681) between June 1, 2016, and July 1, 2022. Dates when population minutes slept were less than the 90% confidence interval of the LOESS regression were named acute sleep deprivation events (ASDEs). Phenome-wide disease incidence among the All of Us Research Program population (n = 287,012) in the 10 days after ASDE was compared with a preceding reference period by McNemar's test. Circadian rhythm-associated and sleep duration-associated SNPs were screened to identify genotypes associated with shorter ASDE sleep duration. Influences of sleep and circadian genotype on post-ASDE influenza risk were modeled using binomial family generalized estimating equations.RESULTSWe identified 32 ASDEs spanning major national events. A phenome-wide screen found increased risk of influenza (odds ratio = 1.54 [1.40, 1.70], P = 1.00 × 10-18) following ASDEs. Fifty-six SNPs were associated with decreased sleep duration on ASDEs. Higher quantiles of ASDE-related SNP genotype burden were associated with less ASDE sleep duration and a greater risk of influenza-associated health care visits.CONCLUSIONMajor national events are associated with acute sleep deprivation and greater influenza risk, which is amplified by sleep genotypes. These findings should inform public health vigilance surrounding major national events.FUNDINGWoodNext Foundation; NIH grants T32HL129964, K08ES037420, R01HL124021, R01HL122596, and R01HL151228; American Heart Association grants 24SFRNCCN1276089 and 24SFRNPCN1280228; and the United Therapeutics Jenesis Innovative Research Awards, the Pulmonary Hypertension Association, the McKamish Family Foundation, the Hemophilia Center of Western Pennsylvania, and the Institute for Transfusion Medicine.