The cis-Regulatory Atlas of the Mouse Immune System.

A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.

The gut microbiota promotes distal tissue regeneration via RORγ+ regulatory T cell emissaries.

Specific microbial signals induce the differentiation of a distinct pool of RORγ+ regulatory T (Treg) cells crucial for intestinal homeostasis. We discovered highly analogous populations of microbiota-dependent Treg cells that promoted tissue regeneration at extra-gut sites, notably acutely injured skeletal muscle and fatty liver. Inflammatory meditators elicited by tissue damage combined with MHC-class-II-dependent T cell activation to drive the accumulation of gut-derived RORγ+ Treg cells in injured muscle, wherein they regulated the dynamics and tenor of early inflammation and helped balance the proliferation vs. differentiation of local stem cells. Reining in IL-17A-producing T cells was a major mechanism underlying the rheostatic functions of RORγ+ Treg cells in compromised tissues. Our findings highlight the importance of gut-trained Treg cell emissaries in controlling the response to sterile injury of non-mucosal tissues.

Maternal gut bacteria drive intestinal inflammation in offspring with neurodevelopmental disorders by altering the chromatin landscape of CD4+ T cells.

Children with autism spectrum disorders often display dysregulated immune responses and related gastrointestinal symptoms. However, the underlying mechanisms leading to the development of both phenotypes have not been elucidated. Here, we show that mouse offspring exhibiting autism-like phenotypes due to prenatal exposure to maternal inflammation were more susceptible to developing intestinal inflammation following challenges later in life. In contrast to its prenatal role in neurodevelopmental phenotypes, interleukin-17A (IL-17A) generated immune-primed phenotypes in offspring through changes in the maternal gut microbiota that led to postnatal alterations in the chromatin landscape of naive CD4+ T cells. The transfer of stool samples from pregnant mice with enhanced IL-17A responses into germ-free dams produced immune-primed phenotypes in offspring. Our study provides mechanistic insights into why children exposed to heightened inflammation in the womb might have an increased risk of developing inflammatory diseases in addition to neurodevelopmental disorders.

The transcription factor FoxP3 can fold into two dimerization states with divergent implications for regulatory T cell function and immune homeostasis.

FoxP3 is an essential transcription factor (TF) for immunologic homeostasis, but how it utilizes the common forkhead DNA-binding domain (DBD) to perform its unique function remains poorly understood. We here demonstrated that unlike other known forkhead TFs, FoxP3 formed a head-to-head dimer using a unique linker (Runx1-binding region [RBR]) preceding the forkhead domain. Head-to-head dimerization conferred distinct DNA-binding specificity and created a docking site for the cofactor Runx1. RBR was also important for proper folding of the forkhead domain, as truncation of RBR induced domain-swap dimerization of forkhead, which was previously considered the physiological form of FoxP3. Rather, swap-dimerization impaired FoxP3 function, as demonstrated with the disease-causing mutation R337Q, whereas a swap-suppressive mutation largely rescued R337Q-mediated functional impairment. Altogether, our findings suggest that FoxP3 can fold into two distinct dimerization states: head-to-head dimerization representing functional specialization of an ancient DBD and swap dimerization associated with impaired functions.

FOXP3 recognizes microsatellites and bridges DNA through multimerization.

FOXP3 is a transcription factor that is essential for the development of regulatory T cells, a branch of T cells that suppress excessive inflammation and autoimmunity1-5. However, the molecular mechanisms of FOXP3 remain unclear. Here we here show that FOXP3 uses the forkhead domain-a DNA-binding domain that is commonly thought to function as a monomer or dimer-to form a higher-order multimer after binding to TnG repeat microsatellites. The cryo-electron microscopy structure of FOXP3 in a complex with T3G repeats reveals a ladder-like architecture, whereby two double-stranded DNA molecules form the two 'side rails' bridged by five pairs of FOXP3 molecules, with each pair forming a 'rung'. Each FOXP3 subunit occupies TGTTTGT within the repeats in a manner that is indistinguishable from that of FOXP3 bound to the forkhead consensus motif (TGTTTAC). Mutations in the intra-rung interface impair TnG repeat recognition, DNA bridging and the cellular functions of FOXP3, all without affecting binding to the forkhead consensus motif. FOXP3 can tolerate variable inter-rung spacings, explaining its broad specificity for TnG-repeat-like sequences in vivo and in vitro. Both FOXP3 orthologues and paralogues show similar TnG repeat recognition and DNA bridging. These findings therefore reveal a mode of DNA recognition that involves transcription factor homomultimerization and DNA bridging, and further implicates microsatellites in transcriptional regulation and diseases.

A naturally occurring variant of SHLP2 is a protective factor in Parkinson's disease.

Mitochondrial DNA single nucleotide polymorphisms (mtSNPs) have been associated with a reduced risk of developing Parkinson's disease (PD), yet the underlying mechanisms remain elusive. In this study, we investigate the functional role of a PD-associated mtSNP that impacts the mitochondrial-derived peptide (MDP) Small Humanin-like Peptide 2 (SHLP2). We identify m.2158 T > C, a mtSNP associated with reduced PD risk, within the small open reading frame encoding SHLP2. This mtSNP results in an alternative form of SHLP2 (lysine 4 replaced with arginine; K4R). Using targeted mass spectrometry, we detect specific tryptic fragments of SHLP2 in neuronal cells and demonstrate its binding to mitochondrial complex 1. Notably, we observe that the K4R variant, associated with reduced PD risk, exhibits increased stability compared to WT SHLP2. Additionally, both WT and K4R SHLP2 show enhanced protection against mitochondrial dysfunction in in vitro experiments and confer protection against a PD-inducing toxin, a mitochondrial complex 1 inhibitor, in a mouse model. This study sheds light on the functional consequences of the m.2158 T > C mtSNP on SHLP2 and provides insights into the potential mechanisms by which this mtSNP may reduce the risk of PD.

Pest population dynamics are related to a continental overwintering gradient.

Overwintering success is an important determinant of arthropod populations that must be considered as climate change continues to influence the spatiotemporal population dynamics of agricultural pests. Using a long-term monitoring database and biologically relevant overwintering zones, we modeled the annual and seasonal population dynamics of a common pest, Helicoverpa zea (Boddie), based on three overwintering suitability zones throughout North America using four decades of soil temperatures: the southern range (able to persist through winter), transitional zone (uncertain overwintering survivorship), and northern limits (unable to survive winter). Our model indicates H. zea population dynamics are hierarchically structured with continental-level effects that are partitioned into three geographic zones. Seasonal populations were initially detected in the southern range, where they experienced multiple large population peaks. All three zones experienced a final peak between late July (southern range) and mid-August to mid-September (transitional zone and northern limits). The southern range expanded by 3% since 1981 and is projected to increase by twofold by 2099 but the areas of other zones are expected to decrease in the future. These changes suggest larger populations may persist at higher latitudes in the future due to reduced low-temperature lethal events during winter. Because H. zea is a highly migratory pest, predicting when populations accumulate in one region can inform synchronous or lagged population development in other regions. We show the value of combining long-term datasets, remotely sensed data, and laboratory findings to inform forecasting of insect pests.

Mitochondrial-derived microprotein MOTS-c attenuates immobilization-induced skeletal muscle atrophy by suppressing lipid infiltration.

Mitochondrial open reading frame of the 12S ribosomal RNA type-c (MOTS-c), a mitochondrial microprotein, has been described as a novel regulator of glucose and lipid metabolism. In addition to its role as a metabolic regulator, MOTS-c prevents skeletal muscle atrophy in high fat-fed mice. Here, we examined the preventive effect of MOTS-c on skeletal muscle mass, using an immobilization-induced muscle atrophy model, and explored its underlying mechanisms. Male C57BL/6J mice (10 wk old) were randomly assigned to one of the three experimental groups: nonimmobilization control group (sterilized water injection), immobilization control group (sterilized water injection), and immobilization and MOTS-c-treated group (15 mg/kg/day MOTS-c injection). We used casting tape for the immobilization experiment. After 8 days of the experimental period, skeletal muscle samples were collected and used for Western blotting, RNA sequencing, and lipid and collagen assays. Immobilization reduced ∼15% of muscle mass, whereas MOTS-c treatment attenuated muscle loss, with only a 5% reduction. MOTS-c treatment also normalized phospho-AKT, phospho-FOXO1, and phospho-FOXO3a expression levels and reduced circulating inflammatory cytokines, such as interleukin-1b (IL-1ß), interleukin-6 (IL-6), chemokine C-X-C motif ligand 1 (CXCL1), and monocyte chemoattractant protein 1 (MCP-1), in immobilized mice. Unbiased RNA sequencing and its downstream analyses demonstrated that MOTS-c modified adipogenesis-modulating gene expression within the peroxisome proliferator-activated receptor (PPAR) pathway. Supporting this observation, muscle fatty acid levels were lower in the MOTS-c-treated group than in the casted control mice. These results suggest that MOTS-c treatment inhibits skeletal muscle lipid infiltration by regulating adipogenesis-related genes and prevents immobilization-induced muscle atrophy.NEW & NOTEWORTHY MOTS-c, a mitochondrial microprotein, attenuates immobilization-induced skeletal muscle atrophy. MOTS-c treatment improves systemic inflammation and skeletal muscle AKT/FOXOs signaling pathways. Furthermore, unbiased RNA sequencing and subsequent assays revealed that MOTS-c prevents lipid infiltration in skeletal muscle. Since lipid accumulation is one of the common pathologies among other skeletal muscle atrophies induced by aging, obesity, cancer cachexia, and denervation, MOTS-c treatment could be effective in other muscle atrophy models as well.

Selective depletion of HBV-infected hepatocytes by class A capsid assembly modulators requires high levels of intrahepatic HBV core protein.

Capsid assembly mediated by hepatitis B virus (HBV) core protein (HBc) is an essential part of the HBV replication cycle, which is the target for different classes of capsid assembly modulators (CAMs). While both CAM-A ("aberrant") and CAM-E ("empty") disrupt nucleocapsid assembly and reduce extracellular HBV DNA, CAM-As can also reduce extracellular HBV surface antigen (HBsAg) by triggering apoptosis of HBV-infected cells in preclinical mouse models. However, there have not been substantial HBsAg declines in chronic hepatitis B (CHB) patients treated with CAM-As to date. To investigate this disconnect, we characterized the antiviral activity of tool CAM compounds in HBV-infected primary human hepatocytes (PHHs), as well as in HBV-infected human liver chimeric mice and mice transduced with adeno-associated virus-HBV. Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced a dose-dependent aggregation of HBc in the nucleus which is negatively regulated by the ubiquitin-binding protein p62. We confirmed that CAM-A, but not CAM-E, induced HBc-positive cell death in both mouse models via induction of apoptotic and inflammatory pathways and demonstrated that the degree of HBV-positive cell loss was positively correlated with intrahepatic HBc levels. Importantly, we determined that there is a significantly lower level of HBc per hepatocyte in CHB patient liver biopsies than in either of the HBV mouse models. Taken together, these data confirm that CAM-As have a unique secondary mechanism with the potential to kill HBc-positive hepatocytes. However, this secondary mechanism appears to require higher intrahepatic HBc levels than is typically observed in CHB patients, thereby limiting the therapeutic potential.

Bumble bee responses to climate and landscapes: Investigating habitat associations and species assemblages across geographic regions in the United States of America.

Bumble bees are integral pollinators of native and cultivated plant communities, but species are undergoing significant changes in range and abundance on a global scale. Climate change and land cover alteration are key drivers in pollinator declines; however, limited research has evaluated the cumulative effects of these factors on bumble bee assemblages. This study tests bumble bee assemblage (calculated as richness and abundance) responses to climate and land use by modeling species-specific habitat requirements, and assemblage-level responses across geographic regions. We integrated species richness, abundance, and distribution data for 18 bumble bee species with site-specific bioclimatic, landscape composition, and landscape configuration data to evaluate the effects of multiple environmental stressors on bumble bee assemblages throughout 433 agricultural fields in Florida, Indiana, Kansas, Kentucky, Maryland, South Carolina, Utah, Virginia, and West Virginia from 2018 to 2020. Distinct east versus west groupings emerged when evaluating species-specific habitat associations, prompting a detailed evaluation of bumble bee assemblages by geographic region. Maximum temperature of warmest month and precipitation of driest month had a positive impact on bumble bee assemblages in the Corn Belt/Appalachian/northeast, southeast, and northern plains regions, but a negative impact on the mountain region. Further, forest land cover surrounding agricultural fields was highlighted as supporting more rich and abundant bumble bee assemblages. Overall, climate and land use combine to drive bumble bee assemblages, but how those processes operate is idiosyncratic and spatially contingent across regions. From these findings, we suggested regionally specific management practices to best support rich and abundant bumble bee assemblages in agroecosystems. Results from this study contribute to a better understanding of climate and landscape factors affecting bumble bees and their habitats throughout the United States.

Aire regulates chromatin looping by evicting CTCF from domain boundaries and favoring accumulation of cohesin on superenhancers.

Aire controls immunological tolerance by driving promiscuous expression of a large swath of the genome in medullary thymic epithelial cells (mTECs). Its molecular mechanism remains enigmatic. High-resolution chromosome-conformation capture (Hi-C) experiments on ex vivo mTECs revealed Aire to have a widespread impact on higher-order chromatin structure, disfavoring architectural loops while favoring transcriptional loops. In the presence of Aire, cohesin complexes concentrated on superenhancers together with mediator complexes, while the CCCTC-binding factor (CTCF) was relatively depleted from structural domain boundaries. In particular, Aire associated with the cohesin loader, NIPBL, strengthening this factor's affiliation with cohesin's enzymatic subunits. mTEC transcripts up-regulated in the presence of Aire corresponded closely to those down-regulated in the absence of one of the cohesin subunits, SA-2. A mechanistic model incorporating these findings explains many of the unusual features of Aire's impact on mTEC transcription, providing molecular insight into tolerance induction.

Metastatic merkel cell carcinoma to the thyroid gland: Case report and review of the literature.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive and rare neuroendocrine tumor, accounting for less than 1% of skin cancers. Metastasis primarily manifests in the cervical lymph nodes but rarely affect the thyroid. METHODS: We report a case of primary head and neck cutaneous MCC with metastasis to the thyroid gland. A review of the literature of MCC with thyroid metastasis was conducted. RESULTS: We identified five cases of MCC with thyroid metastasis. Primary sites included the distal upper and lower extremities, axilla, buttock, and groin. Treatment courses varied including thyroidectomy, immunotherapy, and expectant palliative measures. Time from initial diagnosis to thyroid metastasis ranged from four months to four years. Tissue diagnosis was achieved in 5 of 6 cases. CONCLUSIONS: MCC with thyroid metastasis is rare and likely represents aggressive disease. Despite advances in treatment and surveillance, outcomes for MCC remain poor. Ongoing research may establish predictors for treatment response.

The Use of Aspirin for Venous Thromboembolism Prophylaxis in Patients Who Have Morbid Obesity Undergoing Primary and Revision Total Joint Arthroplasty.

BACKGROUND: Venous thromboembolism (VTE) poses a major clinical concern due to its life-threatening nature, and obese and morbidly obese patients are thought to be at an increased risk for VTE. The aims of this study were twofold; first, to explore VTE rates in patients who have a body mass index (BMI) > 40 undergoing primary and revision total joint arthroplasty, and second, to investigate aspirin (ASA) efficacy and safety. METHODS: We identified all patients (n = 4,672) who had a BMI > 40 who underwent primary and revision total joint arthroplasty from 2016 to 2022 at a single academic tertiary care center. Patients were stratified by BMI groups: 40 to 44.9 (n = 3,462), 45 to 49.9 (n = 935), and 50+ (n = 275). The primary outcome was any VTE event within 90 days postoperatively. The secondary outcome consisted of wound complications within 90 days postoperatively. RESULTS: The total VTE rate was 0.4% (n = 21) and did not differ statistically between the BMI groups (0.4 versus 0.4 versus 0.7%, P = .669). The VTEs consisted of 6 deep venous thromboses (DVTs), 14 pulmonary embolisms, and one concomitant deep venous thrombosis and pulmonary embolism. The VTE rates were not statistically different between patients who received aspirin 325 mg 0.5% (n = 9), aspirin 81 mg 0.2% (n = 1), aspirin + anticoagulant (AC) 0.5% (n = 6), and AC alone 0.4% (n = 5) (P = .954). In addition, wound complications did not differ significantly between patients who received ASA 325 mg, ASA 81mg, ASA + AC, or AC alone (1.6 versus 1.0 versus 1.8 versus 1.1%, P = .351). CONCLUSIONS: The use of aspirin 325 and 81 mg was found to have similar VTE rates as aspirin + ACs and ACs alone, with no significant increase in wound complications. In patients who have a BMI > 40, the use of aspirin is a safe option for VTE prophylaxis and should be prescribed in the context of the patient who has other risk factors for VTE.

Perioperative Cefazolin for Total Joint Arthroplasty Patients Who Have a Penicillin Allergy: Is It Safe?

BACKGROUND: Cefazolin is the standard of care for perioperative antibiotic prophylaxis in total joint arthroplasty (TJA) in the United States. The potential allergic cross-reactivity between cefazolin and penicillin causes uncertainty regarding optimal antibiotic choice in patients who have a reported penicillin allergy (rPCNA). The purpose of this study was to determine the safety of perioperative cefazolin in PCNA patients undergoing primary TJA. METHODS: We identified all patients (n = 49,842) undergoing primary total hip arthroplasty (n = 25,659) or total knee arthroplasty (n = 24,183) from 2016 to 2022 who received perioperative intravenous antibiotic prophylaxis. Patients who had an rPCNA (n = 5,508) who received cefazolin (n = 4,938, 89.7%) were compared to rPCNA patients who did not (n = 570, 10.3%), and to patients who did not have an rPCNA (n = 43,359). The primary outcome was the rate of allergic reactions within 72 hours postoperatively. Secondary outcomes included the rates of superficial infections, deep infections, and Clostridioides difficile infections within 90 days. RESULTS: The rate of allergic reactions was 0.1% (n = 5) in rPCNA patients who received cefazolin, compared to 0.2% (n = 1) in rPCNA patients who did not (P = .48) and 0.02% (n = 11) in patients who have no rPCNA (P = .02). Allergic reactions were mild in all 5 rPCNA patients who received cefazolin and were characterized by cutaneous symptoms (n = 4) or dyspnea in the absence of respiratory distress (n = 1) that resolved promptly with antibiotic discontinuation and administration of antihistamines and/or corticosteroids. We observed no differences in the rates of superficial infections (0.1 versus 0.2%, P = .58), deep infections (0.3 versus 0.4%, P = .68), or C difficile infections (0.04% versus 0%, P = .99) within 90 days in rPCNA patients who received cefazolin versus alternative perioperative antibiotics. CONCLUSIONS: In this series of more than 5,500 patients who had an rPCNA undergoing primary TJA, perioperative prophylaxis with cefazolin resulted in a 0.1% incidence of allergic reactions that were clinically indolent. Cefazolin can be safely administered to most patients, independent of rPCNA severity. LEVEL OF EVIDENCE: III.

Concerted cis and trans effects underpin heightened defense gene expression in multi-herbivore-resistant maize lines.

In maize (Zea mays ssp. mays), agriculturally damaging herbivores include lepidopteran insects, such as the European corn borer (Ostrinia nubilalis), and distantly related arthropods, like the two-spotted spider mite (Tetranychus urticae). A small number of maize lines, including B96 and B75, are highly resistant to both herbivores, and B96 is also resistant to thrips. Using T. urticae as a representative pest that causes significant leaf tissue damage, we examined the gene expression responses of these lines to herbivory in comparison with each other and with the susceptible line B73. Upon herbivory, the most resistant line, B96, showed the strongest gene expression response, with a dramatic upregulation of genes associated with jasmonic acid biosynthesis and signaling, as well as the biosynthesis of specialized herbivore deterrent compounds, such as death acids and benzoxazinoids. Extending this work with allele-specific expression analyses in F1 hybrids, we inferred that the concerted upregulation of many defense genes, including the majority of benzoxazinoid biosynthetic genes in B96, as compared with B73, for the herbivore treatment, resulted from an assemblage of trans control and multiple cis effects acting with similar directionality on gene expression. Further, at the level of individual and potentially rate limiting genes in several major defense pathways, cis and trans effects acted in a reinforcing manner to result in exceptionally high expression in B96. Our study provides a comprehensive resource of cis elements for maize lines important in breeding efforts for herbivore resistance, and reveals potential genetic underpinnings of the origins of multi-herbivore resistance in plant populations.

Identification of Dp140 and α1-syntrophin as novel molecular interactors of the neuronal CaV2.1 channel.

The primary function of dystrophin is to form a link between the cytoskeleton and the extracellular matrix. In addition to this crucial structural function, dystrophin also plays an essential role in clustering and organizing several signaling proteins, including ion channels. Proteomic analysis of the whole rodent brain has stressed the role of some components of the dystrophin-associated glycoprotein complex (DGC) as potential interacting proteins of the voltage-gated Ca2+ channels of the CaV2 subfamily. The interaction of CaV2 with signaling and scaffolding proteins, such as the DGC components, may influence their function, stability, and location in neurons. This work aims to study the interaction between dystrophin and CaV2.1. Our immunoprecipitation data showed the presence of a complex formed by CaV2.1, CaVα2δ-1, CaVß4e, Dp140, and α1-syntrophin in the brain. Furthermore, proximity ligation assays (PLA) showed that CaV2.1 and CaVα2δ-1 interact with dystrophin in the hippocampus and cerebellum. Notably, Dp140 and α1-syntrophin increase CaV2.1 protein stability, half-life, permanence in the plasma membrane, and current density through recombinant CaV2.1 channels. Therefore, we have identified the Dp140 and α1-syntrophin as novel interaction partners of CaV2.1 channels in the mammalian brain. Consistent with previous findings, our work provides evidence of the role of DGC in anchoring and clustering CaV channels in a macromolecular complex.

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.

LD block disorder-specific pleiotropic roles of novel CRHR1 in type 2 diabetes and depression disorder comorbidity.

Major depressive disorder (MDD) and type 2 diabetes (T2D) are complex disorders whose comorbidity can be due to hypercortisolism and may be explained by dysfunction of the corticotropin-releasing hormone receptor 1 (CRHR1) and cortisol feedback within the hypothalamic-pituitary-adrenal axis (HPA axis). To investigate the role of the CRHR1 gene in familial T2D, MDD, and MDD-T2D comorbidity, we tested 152 CRHR1 single-nucleotide-polymorphisms (SNPs), via 2-point parametric linkage and linkage disequilibrium (LD; i.e., association) analyses using 4 models, in 212 peninsular families with T2D and MDD. We detected linkage/LD/association to/with MDD and T2D with 122 (116 novel) SNPs. MDD and T2D had 4 and 3 disorder-specific novel risk LD blocks, respectively, whose risk variants reciprocally confirm one another. Comorbidity was conferred by 3 novel independent SNPs. In silico analyses reported novel functional changes, including the binding site of glucocorticoid receptor-alpha [GR-α] on CRHR1 for transcription regulation. This is the first report of CRHR1 pleiotropic linkage/LD/association with peninsular familial MDD and T2D. CRHR1 contribution to MDD is stronger than to T2D and may antecede T2D onset. Our findings suggest a new molecular-based clinical entity of MDD-T2D and should be replicated in other ethnic groups.

Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo.

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.

Deep learning of immune cell differentiation.

Although we know many sequence-specific transcription factors (TFs), how the DNA sequence of cis-regulatory elements is decoded and orchestrated on the genome scale to determine immune cell differentiation is beyond our grasp. Leveraging a granular atlas of chromatin accessibility across 81 immune cell types, we asked if a convolutional neural network (CNN) could learn to infer cell type-specific chromatin accessibility solely from regulatory DNA sequences. With a tailored architecture and an ensemble approach to CNN parameter interpretation, we show that our trained network ("AI-TAC") does so by rediscovering ab initio the binding motifs for known regulators and some unknown ones. Motifs whose importance is learned virtually as functionally important overlap strikingly well with positions determined by chromatin immunoprecipitation for several TFs. AI-TAC establishes a hierarchy of TFs and their interactions that drives lineage specification and also identifies stage-specific interactions, like Pax5/Ebf1 vs. Pax5/Prdm1, or the role of different NF-κB dimers in different cell types. AI-TAC assigns Spi1/Cebp and Pax5/Ebf1 as the drivers necessary for myeloid and B lineage fates, respectively, but no factors seemed as dominantly required for T cell differentiation, which may represent a fall-back pathway. Mouse-trained AI-TAC can parse human DNA, revealing a strikingly similar ranking of influential TFs and providing additional support that AI-TAC is a generalizable regulatory sequence decoder. Thus, deep learning can reveal the regulatory syntax predictive of the full differentiative complexity of the immune system.