answer question from science paper

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Hello,

In the attachment below there is an article and 5 questions. I want to answer those questions depending in the article. It must be your words (no quotation) and the answer must be clear. It should not be long answer for example Q1 just I want to mention the purpose of writing this article as the author's idea and the important of it. However, Q2 needs to explain the all process in figure 3(a,b,c,d,f,g) then the result(overview). And question 3 needs to summaries the benefit of Figure 3 in the big project(How do the experiments fit into their project).Q4 it is like what do you think the authors will focus on after this article?(basic but clear) the Grammar/Spelling Count!

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Corrected 13 March 2017. See full text. R ES E A RC H REPORT ◥ VASCULAR DISEASE Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice José J. Fuster,1* Susan MacLauchlan,1 María A. Zuriaga,1 Maya N. Polackal,1 Allison C. Ostriker,2 Raja Chakraborty,2 Chia-Ling Wu,1 Soichi Sano,1 Sujatha Muralidharan,1 Cristina Rius,3 Jacqueline Vuong,1 Sophia Jacob,1 Varsha Muralidhar,1 Avril A. B. Robertson,4 Matthew A. Cooper,4 Vicente Andrés,3 Karen K. Hirschi,5 Kathleen A. Martin,2 Kenneth Walsh1* C ardiovascular disease (CVD) is the leading cause of death in the elderly, but almost 60% of elderly patients with atherosclerotic CVD have either no conventional risk factors (e.g., hypertension, hypercholesterolemia, etc.) or just one risk factor (1). Furthermore, increasing evidence suggests that most middle-aged individuals at low risk of CVD, based on conventional risk factors, exhibit subclinical atherosclerosis (2, 3). These clinical data suggest that unidentified age-dependent risk factors contribute to the development of CVD. The accumulation of somatic DNA mutations is a hallmark of aging, particularly in proliferating tissues, which over time may become a mosaic of cells with different genotypes due to the clonal expansion of single de novo mutations (4). However, though human studies suggest that somatic mutations may be associated 1 Molecular Cardiology, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118, USA. 2Yale Cardiovascular Research Center, Vascular Biology and Therapeutics Program, and Departments of Medicine and Pharmacology, Yale University School of Medicine, New Haven, CT 06511, USA. 3Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) and CIBER de Enfermedades Cardiovasculares, Madrid, Spain. 4Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. 5Yale Cardiovascular Research Center and Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT 06511, USA. *Corresponding author. Email: jjfuster@bu.edu (J.J.F.); kxwalsh@bu.edu (K.W.) Fuster et al., Science 355, 842–847 (2017) with a broad spectrum of human disease (5–7), there is little information on the potential causal role of somatic mutations in age-associated disorders other than cancer. Recent human studies have shown that normal aging is associated with an increased frequency of somatic mutations in the hematopoietic system, which provide a competitive growth advantage to the mutant cell and allow its progressive clonal expansion (clonal hematopoiesis) (7–11). This acquired clonal mosaicism in the hematopoietic system of healthy individuals correlates with an increased risk of subsequent hematologic cancer (7–9), but it has also been associated with higher prevalence of vascular complications of diabetes, greater incidence of atherosclerotic conditions (i.e., coronary heart disease, stroke), and increased frequency of CVD-related deaths (6, 7). Although these human studies suggest an unexpected connection between somatic mutations in hematopoietic cells, clonal hematopoiesis, and atherosclerosis, their descriptive nature does not allow cause-effect relationships, or even directionality, to be established. Most of the reported somatic mutations associated with age-related clonal hematopoiesis occur in a small number of genes encoding epigenetic regulators (7–10). The present study focuses on one of these genes, TET2 (ten-eleven translocation 2), the first gene reported to exhibit somatic mutations in blood cells in individuals with clonal hematopoiesis without hematological malignancies 24 February 2017 1 of 6 Downloaded from http://science.sciencemag.org/ on October 16, 2018 Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2-mutant cells in atherosclerosis-prone, low-density lipoprotein receptor–deficient (Ldlr–/–) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome–mediated interleukin-1b secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis. (10). More than 70 different mutations have been reported in this gene (7–10, 12). The protein encoded by TET2 is an epigenetic regulatory enzyme that catalyzes the oxidation of 5-methylcytosine (5mc) in DNA to 5-hydroxymethylcytosine (5hmc) and also exerts noncatalytic actions. TET2 modulates hematopoietic stem and progenitor cell (HSPC) self-renewal (13–16), but its role in CVD remains largely unexplored. To mimic the human scenario of clonal hematopoiesis and test whether clonal expansion of TET2-deficient hematopoietic cells contributes to atherosclerosis, we used a competitive bone marrow transplantation (BMT) strategy to generate atherosclerosis-prone, low-density lipoprotein receptor–deficient (Ldlr−/−) chimeric mice with a small proportion of TET2-deficient HSPCs. Lethally irradiated Ldlr−/− recipients were transplanted with suspensions of bone marrow (BM) cells containing 10% Tet2−/− cells and 90% Tet2+/+ cells [10% knockout (KO)– BMT mice] and then fed a normal diet (ND) or a high-fat/high-cholesterol (HFHC) diet for 9 weeks to induce atherosclerosis development (fig. S1). To distinguish donor Tet2−/− and Tet2+/+ cells in this experimental setting, Tet2+/+ cells were obtained from mice carrying the CD45.1 variant of the CD45 hematopoietic antigen, whereas Tet2−/− cells were obtained from mice carrying the CD45.2 variant of this protein. Control mice [10% wild-type (WT)–BMT] were transplanted with 10% CD45.2+ Tet2+/+ cells and 90% CD45.1+ Tet2+/+ cells. Flow cytometry analysis of CD45.2+ blood cells established that this BMT strategy led to the clonal expansion of Tet2−/− hematopoietic cells to an extent consistent with variant allelic fractions for somatic TET2 mutations observed in human studies linking clonal hematopoiesis to accelerated CVD (7). At the start of ND or HFHC diet feeding (4 weeks after BMT), CD45.2+ cells represented ~28% of blood cells in 10% KO-BMT mice, and they expanded further over time, reaching 42% of blood cells 6 weeks after BMT and 56% 12 weeks after BMT (Fig. 1, A and B). This clonal expansion of TET2-deficient hematopoietic cells is similar to that observed when human cells carrying somatic TET2 mutations are transplanted into immune-deficient mice (17). TET2 ablation in CD45.2+ cells of HFHC-fed 10% KO-BMT mice was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis of CD45.2+ white blood cell (WBC) fractions (Fig. 1C and fig. S2A). No changes were observed in the expression of TET1 or TET3, two related epigenetic modulators. Consistent with the enzymatic activity of TET2, ablation of the gene was paralleled by a decrease in 5hmC levels in WBCs and macrophages (fig. S2, B and C). Although the absolute number of HSPCs [defined as lineage–, Sca1+, c-Kit+ (LSK) cells] was comparable between genotypes (fig. S3A), CD45.2+ cells represented 69% of LSK cells in the BM (fig. S3B) and 61% in the spleen (fig. S3C) of 10% KO-BMT mice at 13 weeks post-BMT, consistent with previous studies reporting that TET2 inactivation enhances HSPC self-renewal (13–16). R ES E A RC H | R E PO R T 60 10% WT BMT 48% CD45.1+ 88% CD45.1+ *** *** 40 *** 52% CD45.2+ 12% CD45.2+ 4 weeks 6 weeks 12 weeks BLOOD *** *** 10% WT BMT Plaque size (x10³ µm²) 80 *** 60 40 *** 20 0 B T 0.6 0.4 0.2 0.0 Tet2 Tet3 10% WT BMT 10% KO BMT 10% KO BMT 600 400 200 0 Neu Mon AORTA *** n.d. Tet1 p=0.0002 10% WT BMT 10% KO BMT AORTIC MACROPHAGES AORTIC T CELLS 10% KO BMT 10% KO BMT 35% CD45.1+ 63% CD45.1+ 60 20 0 CD45+ Macs T cells 64% CD45.2+ CD45.2 CD45.1 * 40 10% WT BMT 10% KO BMT CD45.1 % CD45.2 cells *** 0.8 800 10% WT BMT 10% KO BMT 80 *** 1.0 CD45.2 CD45.2 Time after BMT 100 CD45.1 20 0 % CD45.2+ cells 10% KO BMT CD45.1 % CD45.2+ cells 10% WT BMT 10% KO BMT Relative mRNA levels BLOOD 80 34% CD45.2+ CD45.2 Fig. 1. Clonal expansion of TET2-deficient cells accelerates atherosclerosis in Ldlr−/− mice. 10% KO-BMT mice and 10% WT-BMT controls were fed a high-fat/high-cholesterol (HFHC) diet for 9 weeks, starting 4 weeks after BMT. (A) Percentage of CD45.2+ WBCs in blood, evaluated by flow cytometry (n = 9 mice per genotype). (B) Representative images of CD45.1/CD45.2 flow cytometry analysis of WBC populations. (C) qRT-PCR analysis of TET2 transcript levels in CD45.2+ WBCs from 10% WT-BMT (n = 14 mice) and 10% KO-BMTmice (n = 15 mice). (D) Percentage of CD45.2+ cells within main blood cell lineages 13 weeks after BMT, measured by flow cytometry (n = 11 10% WT-BMTmice per genotype; n = 14 10% KOBMT mice per genotype). (E) Aortic root plaque size. Representative images of hematoxylin and eosin (H&E)–stained sections are shown; atherosclerotic plaques are delineated by dashed lines. Scale bars, 100 mm. (F) Percentage of CD45.2+ cells within the CD45+ immune cell population, F4/80+ macrophages (Macs), and CD3+ Tcells in the aortic arch (n = 4 pools of two aortic arches per genotype). (G) Representative images of CD45.1/CD45.2 flow cytometry analysis of aortic macrophages and Tcells. Statistical significance was evaluated by two-way analysis of variance (ANOVA) with Sidak multiple comparison tests (*P < 0.05, ***P < 0.001) [(A), (C), (D), and (F)] and by two-tailed unpaired Student’s t test (E). n.d., not detected. Error bars indicate SEM. Transplanted Tet2−/− BM cells expanded into all blood cell lineages, regardless of type of diet, although with a slight myeloid bias and a reduced expansion into the T-lymphoid lineage in the BM, spleen, and blood (Fig. 1D and fig. S3, D to F), in agreement with previous studies with TET2-deficient mice (13–16). The expansion of Tet2−/− HSPCs did not affect blood cell counts (fig. S3G), consistent with findings in cancer-free individuals carrying TET2 mutations in blood cells (7, 10). Having demonstrated that the competitive BMT strategy leads to the clonal expansion of TET2-deficient HSPCs and mimics the human scenario of clonal hematopoiesis associated with TET2 mutations, we next evaluated whether the clonal expansion of TET2-deficient HSPCs Fuster et al., Science 355, 842–847 (2017) affects atherogenesis and related metabolic abnormalities. We observed no effects on body weight (fig. S4A), spleen weight (fig. S4B), blood glucose levels (fig. S4C), systemic insulin sensitivity (fig. S4D), or plasma cholesterol levels (fig. S4E). ND-fed mice developed no aortic atherosclerosis, regardless of BM genotype (fig. S4F). In contrast, clonal expansion of TET2-deficient BM cells had a profound effect on HFHC-induced atherosclerosis, as 10% KO-BMT mice exhibited 60% larger plaques in the aortic root than did WT controls (Fig. 1E). Competitive BMT experiments with Tet2+/− cells revealed that TET2 heterozygosity is sufficient to accelerate atherosclerosis, despite the slower kinetics of TET2heterozygous cell expansion (fig. S5). Increased atherogenesis in 10% KO-BMT mice was paral- 24 February 2017 leled by an increase in total macrophage content in the intima, although this parameter was not statistically significant when normalized to plaque size (fig. S6). BM genotype did not affect lesional content of collagen or vascular smooth muscle cells (fig. S6), apoptosis (fig. S7A), necrotic core extension (fig. S7B), or proliferation rates of total plaque cells or lesional macrophages (fig. S7C). Overall, these data demonstrate that clonal expansion of TET2-deficient hematopoietic cells accelerates atherogenesis in a manner independent of alterations in systemic metabolism, changes in blood cell counts, or macrophage proliferation or apoptosis in the plaque. Consistent with their above-mentioned preferential differentiation into myeloid cells, TET2deficient HSPCs expanded preferentially into the macrophage population in the atherosclerotic vascular wall. CD45.2+ cells represented 58% of total immune cells, 62% of macrophages, and 35% of T cells present in the aortic wall of 10% KO-BMT mice (Fig. 1, F and G, and fig. S8). On the basis of these findings, we hypothesized that TET2-deficient hematopoietic cells accelerate atherosclerosis mainly by generating a pool of macrophages with enhanced proatherogenic activities. To test this possibility, we used BMT and LysM-Cre/LoxP strategies to generate atherosclerosis-prone mice exhibiting TET2 deficiency restricted to myeloid cells (Mye-Tet2KO mice). Although this strategy led to a partial (∼80%) inactivation of Tet2 in BM-derived macrophages (Fig. 2A), it was sufficient to increase plaque size in the aortic root of HFHC-fed mice (Fig. 2B), with no differences in body or spleen weight, blood monocyte counts, or glucose and cholesterol levels (fig. S9). These results demonstrate that TET2 deficiency in myeloid cells is sufficient to promote atherogenesis and suggest that macrophages play a major role in the accelerated atherosclerosis associated with expansion of TET2-deficient HSPCs. However, they do not rule out a potential contribution from other BM-derived cells. Analysis of aorta and aorta-draining mediastinal lymph nodes showed that the expansion of TET2deficient HSPCs does not affect T cell numbers or aortic expression of T cell activation markers (fig. S10, A to C), although it leads to modest changes in the frequency of various T cell subsets (fig. S10, D to G), consistent with recent studies (18, 19). Such changes were not observed in Mye-Tet2-KO mice (fig. S10, H to J). Therefore, although a contribution of TET2deficient T cells to the atherogenic effects of the expansion of TET2-deficient HSPCs cannot be excluded, these data demonstrate that changes in T cells are not essential for the accelerated atherosclerosis associated with TET2 loss of function and suggest instead a predominant role of macrophages in this context. We next evaluated the effects of TET2 deficiency on the function of macrophages in culture. Consistent with the in vivo observations, TET2 deficiency did not affect macrophage proliferation (fig. S11A), apoptosis (fig. S11B), 2 of 6 R ES E A RC H | R E PO R T 1.5 WT p<0.05 1.0 0.5 0.0 ing conditions and after treatment with a combination of lipopolysaccharide (LPS) and interferon-g (IFN-g). Whereas no genes were differentially expressed in unstimulated macrophages (q value < 0.05; fig. S12), a widespread alteration in gene expression was found in Tet2−/− macrophages after a 10-hour treatment with Plaque size (x10³ µm²) Relative Tet2 mRNA levels oxidized low-density lipoprotein (oxLDL) uptake (fig. S11C), or the expression of cholesterol trafficking regulators (fig. S11, D and E). To evaluate whether TET2 deficiency affects proinflammatory macrophage activation, we performed Affymetrix microarray analysis on Tet2−/− macrophages and WT controls in rest- 800 Mye-Tet2-KO p<0.05 600 400 200 0 WT Mye WT Mye Tet2-KO Tet2-KO relative relative 0 Enrichment score 4 6 8 10 2 12 Cytokine (PC00083) Signaling molecule (PC00207) 2 3 4 5 6 7 -Log (P value) IL-6 WT KO *** p<0.0001 5000 4000 20000 ** *** 8 IL-1β WT KO * *** p<0.0001 *** 3000 * 2000 10000 9 1000 0 4 8 12 16 hours LPS/IFNγ 0 4 8 12 16 hours Receptor 30000 1 Signaling molecule/ others Relative mRNA expression 0 Cytokine/ chemokine Chemokine (PC00074) LPS/IFNγ Fig. 2. TET2 deficiency in macrophages promotes inflammation and aggravates atherosclerosis. (A and B) Ldlr−/− Mye-Tet2-KO mice (LysM-Cre+ Tet2flox/flox BMT) and WT controls (LysM-Cre– Tet2flox/flox BMT) were fed a HFHC diet for 10 weeks. (A) qRT-PCR analysis of TET2 transcript levels in BMderived macrophages isolated from Mye-Tet2-KO mice and WT controls (n = 6 mice per genotype). (B) Aortic root plaque size. Representative images of H&E-stained sections are shown; atherosclerotic plaques are delineated by dashed lines. Scale bars, 100 mm. (C to F) Peritoneal macrophages were isolated from Tet2−/− mice or WT controls [n = 3 mice per genotype in (C) to (E); n = 4 mice per genotype in (F)] and treated with 10 ng/ml LPS and 2 ng/ml IFN-g to induce proinflammatory activation. (C) Heat map of genes with expression change exceeding a factor of 1.5 (q < 0.05) after 10 hours of LPS/IFN-g stimulation, from a genome-wide expression profiling by microarray. (D) PANTHER analysis of genome-wide expression profiling by microarray. Three overrepresented classes were identified in Tet2−/− macrophages compared with all genes in Mus musculus (Bonferroni correction P < 0.05). (E) Heat map of selected genes up-regulated in Tet2−/− macrophages with expression change exceeding a factor of 1.5 (q < 0.05) from the genome-wide expression profiling by microarray. (F) qRT-PCR analysis of transcript levels of proinflammatory cytokines (IL-6 and IL-1b). Unt, untreated. Statistical significance was evaluated by two-tailed unpaired Student’s t test with Welsh’s correction (A), by two-tailed unpaired Student’s t test (B), and by two-way ANOVA (P value for effect of genotype shown in graph) with Sidak multiple comparison tests (*P < 0.05, **P < 0.01, ***P < 0.001) (F). Error bars indicate SEM. Fuster et al., Science 355, 842–847 (2017) 24 February 2017 LPS/IFN-g. Expression of 475 genes was altered by more than a factor of 1.5 when compared with WT macrophages (q < 0.05; Fig. 2C). PANTHER functional annotation software revealed that transcripts encoding cytokine, chemokine, and signaling molecules were the top overrepresented classes altered in the transcriptome of LPS/IFN-g–treated TET2-deficient macrophages (Fig. 2D). Genes in these classes with known proinflammatory actions were mostly up-regulated in TET2-deficient macrophages (Fig. 2E). Consistent with this observation, qRT-PCR analysis revealed that TET2-deficient macrophages exhibit markedly increased expression of proinflammatory cytokines (Fig. 2F and fig. S13, A and B), chemokines (fig. S13C), and enzymes (fig. S13D). This pattern of gene expression was also evident in Tet2+/− macrophages and macrophages isolated from Mye-Tet2-KO mice (fig. S13, E and F). TET2 deficiency also resulted in increased interleukin-6 (IL-6) protein levels in macrophage culture supernatants (fig. S13G). These data suggest that TET2 acts as a negative transcriptional regulator of proinflammatory responses and are consistent with a previous study reporting that TET2 represses LPS-induced IL-6 expression (20). However, the situation in vivo in the atherosclerotic plaque is particularly complex, as lesional macrophages are exposed to multiple signals simultaneously. Therefore, the anti-inflammatory actions of TET2 in cultured macrophages were further evaluated by testing their effect on macrophage response to a cocktail of low doses of oxLDL, tumor necrosis factor (TNF), and IFN-g, three stimuli present in atherosclerotic plaques. These conditions minimized the impact of TET2 deficiency on cytokine and chemokine expression (fig. S14), with the exception of IL-1b, which was markedly up-regulated in TET2-deficient macrophages at all time points of oxLDL/TNF/IFN-g stimulation (Fig. 3A). Supporting a p ...
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ACADEMICARSENAL
School: Cornell University

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21st October, 2018

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Biol 547 Fall 18
Scientific Paper Summary #5 (Fuster et al.)

Q1/
Identify the major goal of the authors in this paper and explain why this project is
important.

The major goal of the project is to find out if the clonal expansion of the hematopoietic
cells that do not have the TET2 gene are prone to atherosclerosis. This research finding
could inform research that will lead to develop therapies ad preventive measures for
atherosclerosis.
Q2/
Explain the figure 3 assigned to you both in terms of the experimental design and
the results

The experimental design involved analyzing th...

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