The rockefeller university

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. To date, these assemblies have been best created with complex protocols, such as cultured cells that contain a single-haplotype (haploid) genome, single cells where haplotypes are separated, or co-sequencing of parental genomes in a trio-based approach. These approaches are impractical in most situations. To address this issue, we present FALCON-Phase, a phasing tool that uses ultra-long-range Hi-C chromatin interaction data to extend phase blocks of partially-phased diploid assembles to chromosome or scaffold scale. FALCON-Phase uses the inherent phasing information in Hi-C reads, skipping variant calling, and reduces the computational complexity of phasing. Our method is validated on three benchmark datasets generated as part of the Vertebrate Genomes Project (VGP), including human, cow, and zebra finch, for which high-quality, fully haplotype-resolved assemblies are available using the trio-based approach. FALCON-Phase is accurate without having parental data and performance is better in samples with higher heterozygosity. For cow and zebra finch the accuracy is 97% compared to 80-91% for human. FALCON-Phase is applicable to any draft assembly that contains long primary contigs and phased associate contigs. Methods to produce haplotype-resolved genome assemblies often rely on access to family trios. The authors present FALCON-Phase, a tool that combines ultra-long range Hi-C chromatin interaction data with a long read de novo assembly to extend haplotype phasing to the contig or scaffold level.

Background: Psoriasis is associated with increased cardiovascular risk that is not captured by traditional proinflammatory biomarkers. Objective: To investigate the relationship between Psoriasis Area and Severity Index, circulating proinflammatory biomarkers, and vascular health in psoriasis. Methods: In patients with psoriasis and in age and sex-matched controls, 273 proteins were analyzed with the Proseek Multiplex Cardiovascular disease reagents kit and Inflammatory reagents kit (Olink Bioscience), whereas vascular endothelial inflammation and health were measured via direct transcriptomic analysis of brachial vein endothelial cells. Results: In psoriasis, chemokine ligand 20 (CCL20), interleukin (IL) 6, and IL-17A were the top 3 circulating proinflammatory cytokines. Vascular endothelial inflammation correlated with CCL20 (r = 0.55; P < .001) and less so with IL-6 (r = 0.36; P = .04) and IL-17A (r = 0.29; P = .12). After adjustment for potential confounders, the association between CCL20 and vascular endothelial inflammation remained significant (beta = 1.71; P = .02). In nested models, CCL20 added value (chi(2) = 79.22; P < .001) to a model already incorporating the Psoriasis Area and Severity Index, Framingham risk, high-sensitivity C-reactive protein, Il-17A, and IL-6 (chi(2) = 48.18; P < .001) in predicting vascular endothelial inflammation. Limitations: Our study was observational and did not allow for causal inference in the relationship between CCL20 and cardiovascular risk. Conclusion: We demonstrate that CCL20 expression has a strong association with vascular endothelial inflammation, reflects systemic inflammation, and may serve as a potential biomarker of impaired vascular health in psoriasis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models(1,2). Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.

Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary adipocytes and neoplastic cells in the tumor microenvironment (TME), we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes attenuated obesity-driven tumor growth. Similarly, genetic inhibition of creatine import into cancer cells reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals upregulated the fatty acyl-CoA synthetase Acsbg1 to promote creatine-dependent tumor progression. These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the TME necessary for obesity-driven breast cancer progression.

As the world population grows, muscle atrophy leading to muscle wasting could become a bigger risk. Long noncoding RNAs (lncRNAs) are known to play important roles in muscle growth and muscle atrophy. Meanwhile, it has recently come to light that many putative small open reading frames (sORFs) are hidden in lncRNAs; however, their translational capabilities and functions remain unclear. In this study, we uncovered 104 myogenic-associated lncRNAs translated, in at least a small peptide, by integrated transcriptome and proteomic analyses. Furthermore, an upstream ORF (uORF) regulatory network was constructed, and a novel muscle atrophy-associated lncRNA named SMUL (Smad ubiquitin regulatory factor 2 [SMURF2] upstream lncRNA) was identified. SMUL was highly expressed in skeletal muscle, and its expression level was down regulated during myoblast differentiation. SMUL promoted myoblast proliferation and suppressed differentiation in vitro. In vivo, SMUL induced skeletal muscle atrophy and promoted a switch from slow-twitch to fast-twitch fibers. In the meantime, translation of the SMUL sORF disrupted the stability of SMURF2 mRNA. Mechanistically, SMUL restrained SMURF2 production via nonsense-mediated mRNA decay (NMD), participating in the regulation of the transforming growth factor beta (TGF-beta)/SMAD pathway and further regulating myogenesis and muscle atrophy. Taken together, these results suggest that SMUL could be a novel therapeutic target for muscle atrophy.

Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutieres syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.

Purpose: All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the G-protein alpha-q (G alpha(q)) pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of G alpha(q) have shown promising preclinical results, but their therapeutic activity in distinct G alpha(q) mutational contexts and in vivo have remained elusive. Experimental Design: We used an isogenic melanocytic cellular system to systematically examine hotspot mutations in GNAQ (e.g., G48V, R183Q, Q209L) and CYSLTR2 (L129Q) found in human uveal melanoma. This cellular system and human uveal melanoma cell lines were used in vitro and in in vivo xenograft studies to assess the efficacy of G alpha(q) inhibition as a single agent and in combination with MEK inhibition. Results: We demonstrate that the G alpha(q) inhibitor YM-254890 inhibited downstream signaling and in vitro growth in all mutants. In vivo, YM-254890 slowed tumor growth but did not cause regression in human uveal melanoma xenografts. Through comprehensive transcriptome analysis, we observed that YM-254890 caused inhibition of the MAPK signaling with evidence of rebound by 24 hours and combination treatment of YM-254890 and a MEK inhibitor led to sustained MAPK inhibition. We further demonstrated that the combination caused synergistic growth inhibition in vitro and tumor shrinkage in vivo. Conclusions: These data suggest that the combination of G alpha(q) and MEK inhibition provides a promising therapeutic strategy and improved therapeutic window of broadly targeting G alpha(q) in uveal melanoma. See related commentary by Neelature Sriramareddy and Smalley, p. 1217

Objectives: Heat-sensory neurons from the dorsal root ganglia (DRG) play a pivotal role in detecting the cutaneous temperature and transmission of external signals to the brain, ensuring the maintenance of thermoregulation. However, whether these thermoreceptor neurons contribute to adaptive thermogenesis remains elusive. It is also unknown whether these neurons play a role in obesity and energy metabolism. Methods: We used genetic ablation of heat-sensing neurons expressing calcitonin gene-related peptide a (CGRPa) to assess whole-body energy expenditure, weight gain, glucose tolerance, and insulin sensitivity in normal chow and high-fat diet-fed mice. Ex vivo lipolysis and transcriptional characterization were combined with adipose tissue-clearing methods to visualize and probe the role of sensory nerves in adipose tissue. Adaptive thermogenesis was explored using infrared imaging of intrascapular brown adipose tissue (iBAT), tail, and core temperature upon various stimuli including diet, external temperature, and the cooling agent icilin. Results: In this report, we show that genetic ablation of heat-sensing CGRPa neurons promotes resistance to weight gain upon high-fat diet (HFD) feeding and increases energy expenditure in mice. Mechanistically, we found that loss of CGRPa-expressing sensory neurons was associated with reduced lipid deposition in adipose tissue, enhanced expression of fatty acid oxidation genes, higher ex vivo lipolysis in primary white adipocytes, and increased mitochondrial respiration from iBAT. Remarkably, mice lacking CGRPa sensory neurons manifested increased tail cutaneous vasoconstriction at room temperature. This exacerbated cold perception was not associated with reduced core temperature, suggesting that heat production and heat conservation mechanisms were engaged. Specific denervation of CGRPa neurons in intrascapular BAT did not contribute to the increased metabolic rate observed upon global sensory denervation. Conclusions: Taken together, these findings highlight an important role of cutaneous thermoreceptors in regulating energy metabolism by triggering counter-regulatory responses involving energy dissipation processes including lipid fuel utilization and cutaneous vasodilation. (c) 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).