Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics.

Ebola virus (EBOV) causes epidemics with high mortality yet remains understudied due to the challenge of experimentation in high-containment and outbreak settings. Here, we used single-cell transcriptomics and CyTOF-based single-cell protein quantification to characterize peripheral immune cells during EBOV infection in rhesus monkeys. We obtained 100,000 transcriptomes and 15,000,000 protein profiles, finding that immature, proliferative monocyte-lineage cells with reduced antigen-presentation capacity replace conventional monocyte subsets, while lymphocytes upregulate apoptosis genes and decline in abundance. By quantifying intracellular viral RNA, we identify molecular determinants of tropism among circulating immune cells and examine temporal dynamics in viral and host gene expression. Within infected cells, EBOV downregulates STAT1 mRNA and interferon signaling, and it upregulates putative pro-viral genes (e.g., DYNLL1 and HSPA5), nominating pathways the virus manipulates for its replication. This study sheds light on EBOV tropism, replication dynamics, and elicited immune response and provides a framework for characterizing host-virus interactions under maximum containment.

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues.

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.

Single-Cell RNA-Seq Reveals AML Hierarchies Relevant to Disease Progression and Immunity.

Acute myeloid leukemia (AML) is a heterogeneous disease that resides within a complex microenvironment, complicating efforts to understand how different cell types contribute to disease progression. We combined single-cell RNA sequencing and genotyping to profile 38,410 cells from 40 bone marrow aspirates, including 16 AML patients and five healthy donors. We then applied a machine learning classifier to distinguish a spectrum of malignant cell types whose abundances varied between patients and between subclones in the same tumor. Cell type compositions correlated with prototypic genetic lesions, including an association of FLT3-ITD with abundant progenitor-like cells. Primitive AML cells exhibited dysregulated transcriptional programs with co-expression of stemness and myeloid priming genes and had prognostic significance. Differentiated monocyte-like AML cells expressed diverse immunomodulatory genes and suppressed T cell activity in vitro. In conclusion, we provide single-cell technologies and an atlas of AML cell states, regulators, and markers with implications for precision medicine and immune therapies. VIDEO ABSTRACT.

The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control.

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies.

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

Prevention of tuberculosis in macaques after intravenous BCG immunization.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide1. The only available vaccine, BCG (Bacillus Calmette-Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission1,2. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography-computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.

International Union of Basic and Clinical Pharmacology CXIII: Nuclear Receptor Superfamily-Update 2023.

The NR superfamily comprises 48 transcription factors in humans that control a plethora of gene network programs involved in a wide range of physiologic processes. This review will summarize and discuss recent progress in NR biology and drug development derived from integrating various approaches, including biophysical techniques, structural studies, and translational investigation. We also highlight how defective NR signaling results in various diseases and disorders and how NRs can be targeted for therapeutic intervention via modulation via binding to synthetic lipophilic ligands. Furthermore, we also review recent studies that improved our understanding of NR structure and signaling. SIGNIFICANCE STATEMENT: Nuclear receptors (NRs) are ligand-regulated transcription factors that are critical regulators of myriad physiological processes. NRs serve as receptors for an array of drugs, and in this review, we provide an update on recent research into the roles of these drug targets.

A structural mechanism of nuclear receptor biased agonism.

Efforts to decrease the adverse effects of nuclear receptor (NR) drugs have yielded experimental agonists that produce better outcomes in mice. Some of these agonists have been shown to cause different, not just less intense, on-target transcriptomic effects; however, a structural explanation for such agonist-specific effects remains unknown. Here, we show that partial agonists of the NR peroxisome proliferator-associated receptor γ (PPARγ), which induce better outcomes in mice compared to clinically utilized type II diabetes PPARγ-binding drugs thiazolidinediones (TZDs), also favor a different group of coactivator peptides than the TZDs. We find that PPARγ full agonists can also be biased relative to each other in terms of coactivator peptide binding. We find differences in coactivator-PPARγ bonding between the coactivator subgroups which allow agonists to favor one group of coactivator peptides over another, including differential bonding to a C-terminal residue of helix 4. Analysis of all available NR-coactivator structures indicates that such differential helix 4 bonding persists across other NR-coactivator complexes, providing a general structural mechanism of biased agonism for many NRs. Further work will be necessary to determine if such bias translates into altered coactivator occupancy and physiology in cells.

Single-Cell RNA Sequencing Reveals That Adaptation of Human Aortic Endothelial Cells to Antiproliferative Therapies Is Modulated by Flow-Induced Shear Stress.

BACKGROUND: Endothelial cells (ECs) are capable of quickly responding in a coordinated manner to a wide array of stresses to maintain vascular homeostasis. Loss of EC cellular adaptation may be a potential marker for cardiovascular disease and a predictor of poor response to endovascular pharmacological interventions such as drug-eluting stents. Here, we report single-cell transcriptional profiling of ECs exposed to multiple stimulus classes to evaluate EC adaptation. METHODS: Human aortic ECs were costimulated with both pathophysiological flows mimicking shear stress levels found in the human aorta (laminar and turbulent, ranging from 2.5 to 30 dynes/cm2) and clinically relevant antiproliferative drugs, namely paclitaxel and rapamycin. EC state in response to these stimuli was defined using single-cell RNA sequencing. RESULTS: We identified differentially expressed genes and inferred the TF (transcription factor) landscape modulated by flow shear stress using single-cell RNA sequencing. These flow-sensitive markers differentiated previously identified spatially distinct subpopulations of ECs in the murine aorta. Moreover, distinct transcriptional modules defined flow- and drug-responsive EC adaptation singly and in combination. Flow shear stress was the dominant driver of EC state, altering their response to pharmacological therapies. CONCLUSIONS: We showed that flow shear stress modulates the cellular capacity of ECs to respond to paclitaxel and rapamycin administration, suggesting that while responding to different flow patterns, ECs experience an impairment in their transcriptional adaptation to other stimuli.

Allergic inflammatory memory in human respiratory epithelial progenitor cells.

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases1. Specialized subsets of epithelial cells-including secretory and ciliated cells-differentiate from basal stem cells to collectively protect the upper airway2-4. Allergic inflammation can develop from persistent activation5 of type 2 immunity6 in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps7. Basal cell hyperplasia is a hallmark of severe disease7-9, but it is not known how these progenitor cells2,10,11 contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing12, report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic13, epigenetic14,15 and extrinsic factors11,16,17 that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories.

Lateral Axillary Exposure for Antegrade Access during Endovascular Repair of Complex Abdominal Aortic and Thoracoabdominal Aneurysms.

BACKGROUND: During endovascular treatment of pararenal aortic aneurysms (PAA) and thoracoabdominal aortic aneurysms (TAAA), our antegrade vascular access of choice is a lateral axillary exposure (LAE). We directly access the axillary artery with multiple sheaths followed by primary closure of the axillary artery at case completion. The aim of this study is to describe our technique and to report our results with this approach. METHODS: This study is a single-institution, retrospective review of 53 patients who were treated with parallel grafts for endovascular repair of PAA and TAAA from 2006 to 2018. The aortic repairs requiring LAE included: 9 cases of endo-leaks from prior endovascular repair, 20 TAAAs, and 24 PAAs. The axillary artery was exposed with a vertical axillary skin incision followed by retraction of the lateral border of the pectoralis major to expose the axillary artery distal to the pectoralis minor. A 5-French (F) through 12F sheaths were used to directly access the axillary artery for delivery of endovascular devices. RESULTS: Two hundred and sixty reno-visceral stents were delivered through 125 axillary sheaths in an antegrade fashion to 114 arteries without intraoperative complications or technical failures. Two postoperative complications included an access-site hematoma managed conservatively (1.9%) and a left brachial vein thrombosis treated with anticoagulation (1.9%). There were no cases of cerebrovascular or peripheral neurologic events, upper extremity ischemia, or reoperation related to LAE. CONCLUSIONS: LAE is a valid approach for upper extremity access during the endovascular repair of complex aortic aneurysms requiring simultaneous delivery of multiple reno-visceral devices. It does not require the use of a prosthetic conduit. There were no neurologic events or upper extremity ischemia in our series.

Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput.

Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.

Professional fee payments by specialty for inpatient open ventral hernia repair: who gets paid for treating comorbidities and complications?

BACKGROUND: The purpose of this study was to determine perioperative professional fee payments to providers from different specialties for the care of patients undergoing inpatient open ventral hernia repair (VHR). METHODS: Perioperative data of patients undergoing VHR at a single center over 3 years were selected from our NSQIP database. 180-day follow-up data were obtained via retrospective review of records and phone calls to patients. Professional fee payments (PFPs) to all providers were obtained from our physician billing system for the VHR hospitalization, the 180 days prior to operation (180Prior) and the 180 days post-discharge (180Post). RESULTS: PFPs for 283 cases were analyzed. Average total 360-day PFPs per patient were $3409 ± SD 3294, with 14.5% ($493 ± 1546) for services in the 180Preop period, 72.5% ($2473 ± 1881) for the VHR hospitalization, and 13.0% ($443 ± 1097) in the 180Postop period. The surgical service received 62% of PFPs followed by anesthesia (18%), medical specialties (9%), radiology (6%), and all other provider services (5%). Medical specialties received increased PFPs for care of patients with COPD and HCT < 38% ($90 and $521, respectively) and for the pulmonary complications ($2471) and sepsis ($2714) that correlated with those patient comorbidities; surgeons did not. Operative duration, mesh size, and separation of components were associated with increased surgeon PFPs (p < .05). At 6 months, wound complications were associated with increased surgeon and radiology payments (p < .01). CONCLUSIONS: Management of acute comorbid conditions and the associated higher postoperative morbidity is not reimbursed to the surgeon under the 90-day global fee. These represent opportunity costs of care that pressure busy surgeons to select against these patients or to delegate more management to their medical specialty colleagues, thereby increasing total system costs. A comorbid risk adjustment of procedural reimbursement is warranted. In negotiating bundled payments, surgeon groups should keep in mind that surgeon reimbursement, unlike medical specialty and hospital reimbursement, have been bundled since the 1990s.

Harnessing single-cell genomics to improve the physiological fidelity of organoid-derived cell types.

BACKGROUND: Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity. RESULTS: Here, we present a generally applicable framework that utilizes massively parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in PC physiology. CONCLUSIONS: Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation, relative to that in vivo, of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types.

Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists.

Synchronizing rhythms of behaviour and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERB-α and REV-ERB-ß have an integral role in regulating the expression of core clock proteins driving rhythms in activity and metabolism. Here we describe the identification of potent synthetic REV-ERB agonists with in vivo activity. Administration of synthetic REV-ERB ligands alters circadian behaviour and the circadian pattern of core clock gene expression in the hypothalami of mice. The circadian pattern of expression of an array of metabolic genes in the liver, skeletal muscle and adipose tissue was also altered, resulting in increased energy expenditure. Treatment of diet-induced obese mice with a REV-ERB agonist decreased obesity by reducing fat mass and markedly improving dyslipidaemia and hyperglycaemia. These results indicate that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may be beneficial in the treatment of sleep disorders as well as metabolic diseases.

Genetic heterogeneity within the HLA region in three distinct clinical subgroups of myasthenia gravis.

This study aims to investigate genetic susceptibility to early-onset and late-onset anti-acetylcholine receptor antibody positive myasthenia gravis (EOMG and LOMG) and anti-muscle specific kinase antibody positive MG (MuSK-MG) at genome-wide level in a single population. Using a custom-designed array and imputing additional variants and the classical HLA alleles in 398 patients, we detected distinct associations. In EOMG, rs113519545 in the HLA class I region (OR=5.71 [3.77-8.66], P=2.24×10(-16)), HLA-B*08:01 (OR=7.04 [3.95-12.52], P=3.34×10(-11)) and HLA-C*07:01 (OR=2.74 [1.97-3.81], P=2.07(-9)), in LOMG, rs111256513 in the HLA class II region (OR=2.22 [1.59-3.09], P=2.48×10(-6)) and in MuSK-MG, an intronic variant within HLA-DQB1 (rs68081734, OR=5.86, P=2.25×10(-14)) and HLA-DQB1*05:02 (OR=8.56, P=6.88×10(-13)) revealed the most significant associations for genome-wide significance. Differential genetic susceptibility within the HLA to EOMG, LOMG and MuSK-MG has been established in a population from Turkey.

Identification of multiple genetic susceptibility loci in Takayasu arteritis.

Takayasu arteritis is a rare inflammatory disease of large arteries. The etiology of Takayasu arteritis remains poorly understood, but genetic contribution to the disease pathogenesis is supported by the genetic association with HLA-B*52. We genotyped ~200,000 genetic variants in two ethnically divergent Takayasu arteritis cohorts from Turkey and North America by using a custom-designed genotyping platform (Immunochip). Additional genetic variants and the classical HLA alleles were imputed and analyzed. We identified and confirmed two independent susceptibility loci within the HLA region (r(2) < 0.2): HLA-B/MICA (rs12524487, OR = 3.29, p = 5.57 × 10(-16)) and HLA-DQB1/HLA-DRB1 (rs113452171, OR = 2.34, p = 3.74 × 10(-9); and rs189754752, OR = 2.47, p = 4.22 × 10(-9)). In addition, we identified and confirmed a genetic association between Takayasu arteritis and the FCGR2A/FCGR3A locus on chromosome 1 (rs10919543, OR = 1.81, p = 5.89 × 10(-12)). The risk allele in this locus results in increased mRNA expression of FCGR2A. We also established the genetic association between IL12B and Takayasu arteritis (rs56167332, OR = 1.54, p = 2.18 × 10(-8)).

Nuclear receptors and their selective pharmacologic modulators.

Nuclear receptors are ligand-activated transcription factors and include the receptors for steroid hormones, lipophilic vitamins, sterols, and bile acids. These receptors serve as targets for development of myriad drugs that target a range of disorders. Classically defined ligands that bind to the ligand-binding domain of nuclear receptors, whether they are endogenous or synthetic, either activate receptor activity (agonists) or block activation (antagonists) and due to the ability to alter activity of the receptors are often termed receptor "modulators." The complex pharmacology of nuclear receptors has provided a class of ligands distinct from these simple modulators where ligands display agonist/partial agonist/antagonist function in a tissue or gene selective manner. This class of ligands is defined as selective modulators. Here, we review the development and pharmacology of a range of selective nuclear receptor modulators.

Structure of REV-ERBß ligand-binding domain bound to a porphyrin antagonist.

REV-ERBα and REV-ERBß are members of the nuclear receptor (NR) superfamily of ligand-regulated transcription factors that play important roles in the regulation of circadian physiology, metabolism, and immune function. Although the REV-ERBs were originally characterized as orphan receptors, recent studies have demonstrated that they function as receptors for heme. Here, we demonstrate that cobalt protoporphyrin IX (CoPP) and zinc protoporphyrin IX (ZnPP) are ligands that bind directly to the REV-ERBs. However, instead of mimicking the agonist action of heme, CoPP and ZnPP function as antagonists of REV-ERB function. This was unexpected because the only distinction between these ligands is the metal ion that is coordinated. To understand the structural basis by which REV-ERBß can differentiate between a porphyrin agonist and antagonist, we characterized the interaction between REV-ERBß with heme, CoPP, and ZnPP using biochemical and structural approaches, including x-ray crystallography and NMR. The crystal structure of CoPP-bound REV-ERBß indicates only minor conformational changes induced by CoPP compared with heme, including the porphyrin ring of CoPP, which adopts a planar conformation as opposed to the puckered conformation observed in the heme-bound REV-ERBß crystal structure. Thus, subtle changes in the porphyrin metal center and ring conformation may influence the agonist versus antagonist action of porphyrins and when considered with other studies suggest that gas binding to the iron metal center heme may drive alterations in REV-ERB activity.