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The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action., This study set out to identify novel gene variants that may contribute to human obesity, by combining human exosome sequencing analyses with systematic functional screening in Drosophila. This identifies a number of novel obesity-associated genes which control adiposity in flies, and uncovers a potential role for the Hippo signaling pathway in obesity.

There is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution., In this study, Qin et al. present a murine-adapted SARS-CoV-2 strain, MASCp36, as a model for studying the pathogenicity, evolution and adaptation of the virus to human and animal hosts.

Significance To date, due to the insufficient expression level of reporter genes from previous recombinant (r)SARS-CoV-2, in which the viral open reading frame (ORF) 7a gene was replaced by reporter genes, tracking the SARS-CoV-2 infection dynamic has been challenging. Here, we engineered rSARS-CoV-2 expressing fluorescent (Venus) or luciferase (Nano luciferase) reporter genes from the viral nucleocapsid (N) locus, without deleting any viral gene. These novel reporter-expressing rSARS-CoV-2, which give a higher expression level of reporter genes, allow us to monitor SARS-CoV-2 replication dynamic both in vitro and in vivo. These new reporter-expressing rSARS-CoVs-2 represent an excellent option to assess viral replication, tropisms, and pathogenicity as well as the rapid in vivo evaluation of effective countermeasures for the treatment of SARS-CoV-2 infection., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

Objective— Leptin is an adipokine initially thought to be a metabolic factor. Recent publications have shown its roles in inflammation and vascular disease, to which Sca-1 + vascular progenitor cells within the vessel wall may contribute. We sought to elucidate the effects of leptin on Sca-1 + progenitor cells migration and neointimal formation and to understand the underlying mechanisms. Approach and Results— Sca-1 + progenitor cells from the vessel wall of Lepr +/+ and Lepr −/− mice were cultured and purified. The migration of Lepr +/+ Sca-1 + progenitor cells in vitro was markedly induced by leptin. Western blotting and kinase assays revealed that leptin induced the activation of phosphorylated signal transducer and activator of transcription 3, phosphorylated extracellular signal–regulated kinases 1/2, pFAK (phosphorylated focal adhesion kinase), and Rac1 (ras-related C3 botulinum toxin substrate 1)/Cdc42 (cell division control protein 42 homolog). In a mouse femoral artery guidewire injury model, an increased expression of leptin in both injured vessels and serum was observed 24 hours post-surgery. RFP (red fluorescent protein)-Sca-1 + progenitor cells in Matrigel were applied to the adventitia of the injured femoral artery. RFP + cells were observed in the intima 24 hours post-surgery, subsequently increasing neointimal lesions at 2 weeks when compared with the arteries without seeded cells. This increase was reduced by pre-treatment of Sca-1 + cells with a leptin antagonist. Guidewire injury could only induce minor neointima in Lepr −/− mice 2 weeks post-surgery. However, transplantation of Lepr +/+ Sca-1 + progenitor cells into the adventitial side of injured artery in Lepr −/− mice significantly enhanced neointimal formation. Conclusions— Upregulation of leptin levels in both the vessel wall and the circulation after vessel injury promoted the migration of Sca-1 + progenitor cells via leptin receptor–dependent signal transducer and activator of transcription 3- Rac1/Cdc42-ERK (extracellular signal–regulated kinase)-FAK pathways, which enhanced neointimal formation.

Supplemental Digital Content is available in the text., Background: Dickkopf-related protein 3 (DKK3) is a secreted protein that is involved in the regulation of cardiac remodeling and vascular smooth muscle cell differentiation, but little is known about its role in atherosclerosis. Methods: We tested the hypothesis that DKK3 is atheroprotective using both epidemiological and experimental approaches. Blood DKK3 levels were measured in the Bruneck Study in 2000 (n=684) and then in 2005 (n=574). DKK3-deficient mice were crossed with apolipoprotein E-/- mice to evaluate atherosclerosis development and vessel injury-induced neointimal formation. Endothelial cell migration and the underlying mechanisms were studied using in vitro cell culture models. Results: In the prospective population-based Bruneck Study, the level of plasma DKK3 was inversely related to carotid artery intima-media thickness and 5-year progression of carotid atherosclerosis independently from standard risk factors for atherosclerosis. Experimentally, we analyzed the area of atherosclerotic lesions, femoral artery injury-induced reendothelialization, and neointima formation in both DKK3-/-/apolipoprotein E-/- and DKK3+/+/apolipoprotein E-/- mice. It was demonstrated that DKK3 deficiency accelerated atherosclerosis and delayed reendothelialization with consequently exacerbated neointima formation. To explore the underlying mechanisms, we performed transwell and scratch migration assays using cultured human endothelial cells, which exhibited a significant induction in cell migration in response to DKK3 stimulation. This DKK3-induced migration activated ROR2 and DVL1, activated Rac1 GTPases, and upregulated JNK and c-jun phosphorylation in endothelial cells. Knockdown of the ROR2 receptor using specific siRNA or transfection of a dominant-negative form of Rac1 in endothelial cells markedly inhibited cell migration and downstream JNK and c-jun phosphorylation. Conclusions: This study provides the evidence for a role of DKK3 in the protection against atherosclerosis involving endothelial migration and repair, with great therapeutic potential implications against atherosclerosis.

Gut microbe–derived metabolites influence human physiology and disease. However, establishing mechanistic links between gut microbial metabolites and disease pathogenesis in animal models remains challenging. The major route of absorption for microbe-derived small molecules is venous drainage via the portal vein to the liver. In the event of presystemic hepatic metabolism, the route of metabolite administration becomes critical. To our knowledge, we describe here a novel portal vein cannulation technique using a s.c. implanted osmotic pump to achieve continuous portal vein infusion in mice. We first administered the microbial metabolite trimethylamine (TMA) over 4 weeks, during which increased peripheral plasma levels of TMA and its host liver-derived cometabolite, trimethylamine-N-oxide, were observed when compared with a vehicle control. Next, 4-hydroxyphenylacetic acid (4-HPAA), a microbial metabolite that undergoes extensive presystemic hepatic metabolism, was administered intraportally to examine effects on hepatic gene expression. As expected, hepatic levels of 4-HPAA were elevated when compared with the control group while peripheral plasma 4-HPAA levels remained the same. Moreover, significant changes in the hepatic transcriptome were revealed by an unbiased RNA-Seq approach. Collectively, to our knowledge this work describes a novel method for administering gut microbe–derived metabolites via the portal vein, mimicking their physiologic delivery in vivo.

Endothelial damage and dysfunction are implicated in cardiovascular pathological changes and the development of vascular diseases. In view of the fact that the spontaneous endothelial cell (EC) regeneration is a slow and insufficient process, it would be of great significance to explore alternative cell sources capable of generating functional ECs to repair damaged endothelium. Indeed, recent achievements of cell reprogramming to convert somatic cells to other cell types provide new powerful approaches to study endothelial regeneration. Based on progress in the research field, the present review aims to explore the strategies and mechanisms of generating endothelial cells through reprogramming from somatic cells, and to examine what this means for the potential application of cell therapy in the clinic.