B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response – Science Advances

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Abstract

Activation-induced cytidine deaminase (AID) mediates immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM), critical processes for maturation of the antibody response. Epigenetic factors, such as histone deacetylases (HDACs), would underpin B cell differentiation stagespecific AID expression. Here, we showed that NAD+-dependent class III HDAC sirtuin 1 (Sirt1) is highly expressed in resting B cells and down-regulated by stimuli inducing AID. B cell Sirt1 down-regulation, deprivation of NAD+ cofactor, or genetic Sirt1 deletion reduced deacetylation of Aicda promoter histones, Dnmt1, and nuclear factorB (NF-B) p65 and increased AID expression. This promoted class-switched and hypermutated T-dependent and T-independent antibody responses or led to generation of autoantibodies. Genetic Sirt1 overexpression, Sirt1 boost by NAD+, or allosteric Sirt1 enhancement by SRT1720 repressed AID expression and CSR/SHM. By deacetylating histone and nonhistone proteins (Dnmt1 and NF-B p65), Sirt1 transduces metabolic cues into epigenetic changes to play an important B cellintrinsic role in modulating antibody and autoantibody responses.

As the master molecule for immunoglobulin (Ig) class switch DNA recombination (CSR) and somatic hypermutation (SHM), activation-induced cytidine deaminase (AID) is central to the maturation of the antibody response (1). Physiologically, AID expression is restricted to B cells only and at a specific B cell differentiation stage only. AID is undetectable in resting B cells, and it is induced at high levels in B cells activated by T-dependent and T-independent stimuli, including CD154:CD40, Toll-like receptor (TLR) ligand:TLR, and select cytokine:cytokine receptors engagement, and then subsides to undetectable levels in resting memory B cells and plasma cells (24). AID is also greatly up-regulated in activated B cells that make class-switched and somatically hypermutated autoantibodies in autoimmune diseases, such as systemic lupus erythematosus (SLE) (3, 4). AID induction is mediated through activation of the Aicda (AID gene) promoter and regulatory regions by transcription factor nuclear factorB (NF-B) as complemented by HoxC4, as well as by Aicda histones acetylation and DNA demethylation (58). Select microRNAs and Aicda cis-elements have been shown to prevent AID expression in nonactivated B cells (7, 9). However, the mechanisms underpinning the suppression of Aicda transcription, which is required to avoid AID expression in B cells either resting or in response to subliminal and/or nonspecific stimuli and to control prolonged AID activation, have remained virtually unexplored.

We contend here that B cellintrinsic regulation of AID expression is mediated by epigenetic mechanisms (3, 4, 10). Epigenetic modifications and factors comprise epigenetic marks, such as DNA methylation/demethylation and histone posttranslational modifications, and epigenetic mediators, including histone methyltransferases and demethylases, histone acyltransferases, and histone deacetylases (HDACs comprise classical class I, II, and IV HDACs and nonclassical class III HDACs or Sirtuins) as well as noncoding RNAs (e.g., microRNAs and long noncoding RNAs) (4, 10). Epigenetic modifications and factors are known to influence gene expression and modulate critical B cell processes, including CSR/SHM and plasma cell differentiation, thereby informing the antibody response (4, 10). By using well-characterized short-chain fatty acid HDAC inhibitors valproic acid and butyrate, which inhibit the classical class I, II, and IV HDACs, we have shown that these HDACs regulate intrinsic B cell functions that are critical in shaping effective antibody and autoantibody responses (11). Sirt1, a nonclassical class III HDAC, has been implicated in innate and adaptive immune responses (12). Sirt1 has been suggested to function as an immune regulator, suppressing the antinuclear autoantibody response in murine lupus and type 1 diabetes through modulation of T cell functions (1214). Also, Sirt1-null mice develop an autoimmune condition with characteristic antinuclear autoantibodies (15).

Sirt1 actively deacetylates acetyl-lysine in multiple histones, including H3K9Ac and H3K14Ac, to facilitate chromatin compaction and silence gene transcription (16). In addition to deacetylating histones, Sirt1 deacetylates select nonhistone proteins. These are generally molecules involved in signal transduction, metabolism, or gene transcription. By deacetylating the p65 subunit at lysine 310, Sirt1 inactivates NF-B (17); by deacetylating the catalytic domain of DNA methyltransferase 1 (Dnmt1), Sirt1 boosts this enzymes activity (18). Unlike class I, II, and IV HDACs that are Zn2+ dependent for their function, Sirt1, like the other class III deacetylases, depends on nicotinamide adenosine dinucleotide (NAD+) as cofactor (19). Increased cellular NAD+ boosts Sirt1 activity. Resveratrol, a natural polyphenolic compound found mainly in grape skin and red wine, which increases Sirt1 activity by facilitating NADH oxidation to NAD+ and, likely, by increasing Sirt1 affinity for both NAD+ and acetylated substrate, has been shown to affect protection in lupus mice (20, 21). By contrast, reduced cellular NAD+ concentration, resulting from conversion of NAD+ to NADH by the glucose metabolic pathway (glycolysis), leads to decreased Sirt1 activity (22). As a result of its dependence on NAD+ and, therefore, the cellular NAD+/NADH ratio, Sirt1 has emerged as a key metabolic sensor in various tissues (23). By deacetylating histone and nonhistone proteins, including transcription factors, Sirt1 couples the cell metabolic status (through NAD+) to the modulation of multiple biological processes, such as signal transduction, gene transcription, and DNA repair (24, 25). Although a role for Sirt1 in B cells has remained mostly unexplored, our preliminary findings have suggested that Sirt1 suppresses AID expression in a B cellintrinsic fashion, thereby impacting CSR/SHM in antibody and autoantibody responses.

We hypothesized here that Sirt1 regulates AID expression in a B cellintrinsic fashion to modulate CSR/SHM and the maturation of the antibody response. To test our hypothesis, we generated conditional knockout AicdacreSirt1fl/fl mice and used them together with transgenic mice expressing multiple copies of Sirt1 (Sirt1super mice) to address the B cellintrinsic role of Sirt1 in T-dependent and T-independent antibody responses, namely, the role of Sirt1 in modulating histone acetylation of the Aicda and, for comparison, the Prdm1 (Blimp1 gene) and Xbp1 promoters. In addition, we addressed the potential role of Sirt1 in modulating NF-B acetylation and, therefore, NF-B recruitment to the Aicda promoter for induction of Aicda expression. We also addressed the role of Sirt1 in modulating the methylation status of the Aicda promoter through deacetylation and activation of the DNA methyltransferase Dnmt1. Further, we analyzed the impact of elevated glucose on the cellular NAD+/NADH ratio and Sirt1 activity on Aicda and, for comparison, Prdm1 expression in B cells. Last, we used the small-molecule Sirt1 activator SRT1720, which is 1000-fold more potent than resveratrol, to boost Sirt1 activity in B cells in vitro and in vivo and measured SRT1720 impact on AID levels and CSR/SHM. Overall, our findings outline an important B cellintrinsic role for Sirt1 as an epigenetic modulator of AID and as a regulator of class-switched and hypermutated antibody and autoantibody responses. Sirt1 affects these functions by acetylating histone and nonhistone proteins in response to B cell activation stimuli, the metabolic milieu, or small-molecule activator(s).

Sirt1 has been shown to be expressed in lymphoid tissues (13). We found Sirt1 to be expressed at the highest level among the seven Sirt genes in mouse nave B cells (Fig. 1A). Stimulation of these B cells to induce CSR greatly down-regulated Sirt1 [by 86.5% after stimulation with lipopolysaccharide (LPS) plus interleukin-4 (IL-4) and 87.5% after stimulation with CD154 plus IL-4] while up-regulating Aicda transcripts (Fig. 1B). Like mouse B cells, purified human nave B cells expressed SIRT1 at a high level and down-regulated it by 90.8% after a 72-hour stimulation by CD154 plus IL-4 and IL-21, which up-regulated AICDA, resulting in CSR to IgG (Fig. 1C and fig. S1). By contrast, in B cells activated by cross-linking the B cell antigen receptor (BCR) with anti-Ig monoclonal antibody (mAb), expression of Sirt1 and Aicda was unchanged (Fig. 1D). A reciprocal Sirt1/Aicda expression also occurred in vivo. In B cells isolated from NP-conjugated chicken gamma globulin (NP16-CGG)immunized C57BL/6 mice, in which Aicda expression was greatly increased, Sirt1 expression was significantly reduced, as compared to nonimmunized mice (Fig. 1E). In those B cells, reduced Sirt1 expression was reflected in reduced levels of Sirt1 protein and was concomitant with increased AID protein (Fig. 1F). Sirt1 level in germinal center B cells, which expressed AID, was significantly lower than that in nave B cells or plasma cells, which did not express AID, as shown by intracellular immunofluorescence with anti-Sirt1 and anti-AID Abs. Similarly, in B cells stimulated by LPS plus IL-4 in vitro, Sirt1 protein expression was down-regulated while AID protein was up-regulated, as shown by intracellular immunofluorescence and immunoblotting (Fig. 1, G to I). Thus, Sirt1 is expressed at a high level in resting nave B cells, in which AID expression is virtually nil. Activation of B cells by stimuli that induce CSR down-regulates Sirt1 while reciprocally up-regulating Aicda expression, indicating a role for Sirt1 in modulation of Aicda expression.

(A) Sirt1-7 and Aicda expression in mouse nave B cells before and after stimulation with LPS plus IL-4 for 72 hours, as measured by mRNA-Seq and depicted as RPKM (reads per kilobase of transcripts per million mapped reads; one of two independent experiments yielding comparable results). (B) Sirt1 and Aicda transcript levels [quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis] in mouse B cells stimulated with LPS or CD154 plus IL-4 for 0, 6, 12, 24, 48, or 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates, left panel). Also shown are the inverse correlation scatter plots of Sirt1 and Aicda expression levels (right). (C) SIRT1 and AICDA expression (qRT-PCR analysis) in human nave B cells stimulated with CD154 plus IL-4 and IL-21 for 0, 6, 12, 24, 48, 72, or 96 hours. Data are ratio to the expression in unstimulated B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates, left). Also shown are the inverse correlation scatter plots of SIRT1 and AICDA expression levels (right). (D) Sirt1 and Aicda transcript levels (qRT-PCR analysis) in mouse B cells stimulated with anti-Ig mAb or LPS plus IL-4 for 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (E) Sirt1 and Aicda expression (qRT-PCR analysis) in spleen B cells from C57BL/6 mice immunized with nil or NP16-CGG and analyzed 10 days after immunization. Data are ratios to the expression in nonimmunized mice (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (F) Sirt1 and AID protein levels in CD19+IgDhiGL7CD138 nave B cells, CD19+IgDGL7hiCD138 germinal center (GC) B cells, and CD19loCD138hi plasma cells/plasmablasts in C57BL/6 mice immunized with NP16-CGG, as analyzed by flow cytometry 10 days after immunization. (G to I) Sirt1 and AID protein levels in mouse B cells before (nil) and after stimulation with LPS plus IL-4 for 72 hours analyzed by flow cytometry (G), intracellular immunofluorescence (H), and immunoblotting (I). Densitometry quantification of immunoblotting signals normalized to -actin levels and depicted as ratios of readings in LPS plus IL-4 stimulated B cells to those in unstimulated (0 hours) B cells (I, right). Data in (F) to (I) are one of two independent experiments yielding similar results. ***P < 0.001; ns, not significant, unpaired two-tailed Students t test. Scale bar, 5 m. DAPI, 4,6-diamidino-2-phenylindole.

To address the role of Sirt1 in modulating Aicda in vivo, we used transgenic mice expressing extra copies of Sirt1 (Sirt1super mice) (26). In Sirt1super mice, B cell Sirt1 levels were about 250 to 1000% higher than in Sirt+/+ mice, concomitant with decreased Aicda expression and decreased circulating IgG1 and IgA but normal IgM levels (Fig. 2, A and B). Sirt1super B cells stimulated in vitro by LPS plus IL-4 expressed Sirt1 225% more than wild-type (WT) B cells (Fig. 2C). The increased Sirt1 transcripts and Sirt1 protein in these Sirt1super B cells were associated with decreased Aicda transcripts and AID protein, significantly lower CSR to IgG1, IgG3, IgA, and IgE than WT B cells, but unaltered expression of Prdm1, Xbp1, and plasma cell differentiation (Fig. 2, C to E). Accordingly, enforced Sirt1 expression by a retroviral construct in normal B cells greatly reduced CSR to IgG1 (Fig. 2F). Further, selective activation of Sirt1 by SRT1720 inhibited CSR in WT B cells stimulated with LPS plus IL-4, without affecting plasma cell differentiation, cell proliferation, or viability (Fig. 2, G to I, and fig. S2A). SRT1720-induced inhibition of CSR was associated with reduced Aicda but not reduced Prdm1 or Xbp1 expression (Fig. 2J). Like SRT1720, resveratrol reduced Aicda expression and CSR in a dose-dependent fashion without altering Prdm1 expression or plasma cell differentiation (fig. S2, B and C). Thus, Sirt1 overexpression or Sirt1 activation efficiently inhibits B cell Aicda expression and CSR but not Prdm1 or Xbp1 expression or plasma cell differentiation.

(A) Ex vivo expression of Sirt1 and Aicda (qRT-PCR) in peripheral blood total B cells isolated from non-intentionally immunized Sirt1+/+ and Sirt1super mice. Data are ratios to expression in total B cells from Sirt1+/+ mice (set as 1; means SEM of values from three Sirt1+/+ mice and three Sirt1super mice). (B) Serum titers [enzyme-linked immunosorbent assay (ELISA)] of total IgM, IgG1, and IgA in non-intentionally immunized Sirt1+/+ and Sirt1super mice (means SD of six Sirt1+/+ mice and six Sirt1super mice). (C) Expression of Sirt1, Aicda, Prdm1, and Xbp1 transcripts (qRT-PCR) in Sirt1+/+ (set as 1) and Sirt1super B cells stimulated with LPS plus IL-4 for 72 hours (means SEM of three biological independent experiments, each consisting of triplicates; left) as well as immunoblotting and flow cytometry analysis of Sirt1 and AID protein levels in these cells (one of two independent experiments yielding similar results; right). (D and E) CSR (D) and plasma cell differentiation (E) in Sirt1+/+ and Sirt1super B cells (flow cytometry analysis) after stimulation with appropriate stimuli, as indicated, for 96 hours (left, one representative of three independent experiments; right, means SEM of three independent experiments). (F) CSR to IgG1 (flow cytometry analysis) in (B220+GFP+) B cells transduced by pMIG-GFP or pMIG-GFP-Sirt1 retrovirus (one of two independent experiments yielding similar results). (G to I) C57BL/6 B cells stimulated for 96 hours with LPS plus IL-4 in the presence of SRT1720 at indicated doses. Percentages of IgG1+ B cells and B220lowCD138+ plasma cells (G), cell proliferation [carboxyfluorescein diacetate succinimidyl ester (CFSE)labeled cells] (H), and viability [7-aminoactinomycin Dnegative (7-AAD)] (I) (flow cytometry analysis). Data are means SEM of three independent experiments (G) or one representative of three independent experiments (H and I). (J) Aicda, Prdm1, and Xbp1 transcript levels (qRT-PCR analysis) in purified B cells treated with nil or SRT1720 at the indicated doses and stimulated for 72 hours with LPS plus IL-4. Data are ratios to the expression in B cells treated with nil (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (K) Sirt1 transcript levels (qRT-PCR analysis) in AicdacreSirt1+/+ and AicdacreSirt1fl/fl B cells stimulated with LPS plus IL-4 for 0 and 72 hours. Data are ratios to the expression in unstimulated (0 hours) AicdacreSirt1+/+ B cells (set as 1; SEM of three biological independent experiments, each consisting of triplicates). (L) CSR to different Ig isotypes (flow cytometry analysis) in AidcacreSirt1+/+ and AicdacreSirt1fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means SEM (right) of three independent experiments. (M) Expression of indicated genes (qRT-PCR analysis) in AidcacreSirt1+/+ and AicdacreSirt1fl/fl B cells stimulated for 72 hours with LPS plus IL-4 or LPS plus TGF- and anti-Ig mAb-dextran (I-C and I-C). Data are ratios to the expression in AicdacreSirt1+/+ B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (N) AID protein levels in AicdacreSirt1+/+ and AicdacreSirt1fl/fl B cells stimulated with LPS plus IL-4 for 72 hours as analyzed by intracellular staining and flow cytometry. Data are from one of two independent experiments yielding similar results. (O) Plasma cell differentiation (flow cytometry analysis) in AidcacreSirt1+/+ and AicdacreSirt1fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means SEM (right) of three biological independent experiments. (P) Proliferation of spleen B220+ B cells labeled with CFSE and stimulated with LPS plus IL-4 for 72 and 96 hours (top) and B cell viability (7-AAD, bottom). Data are one representative of three independent experiments yielding similar results. *P < 0.05 and **P < 0.01, unpaired two-tailed Students t test.

To further define the B cellintrinsic role of Sirt1 in modulating AID expression and CSR/SHM, we cross-bred transgenic Aicdacre mice (5) with Sirt1fl/fl mice (27) to construct AicdacreSirt1fl/fl mice. In these mice, which carry a normal complement of Aicda gene together with a cre gene under control of an extra Aicda promoter/enhancer within a bacterial artificial chromosome (BAC) transgene, cre recombinase expression leading to Sirt1 deletion occurred only in B cells activated to transcribe Aicda-cre. AicdacreSirt1fl/fl mice had an intact Sirt1 locus throughout embryogenesis and were born at an expected Mendelian ratio. They were indistinguishable from their AicdacreSirt1+/+ and AicdacreSirt1+/fl littermates in size, fertility, and organ morphology during development and maturation.

In vitro, AicdacreSirt1fl/fl B cells further reduced Sirt1 expression upon exposure to Aicda- and CSR-inducing stimuli, as compared to their AicdacreSirt1+/+ counterparts (Fig. 2K). Among AicdacreSirt1fl/fl B cells stimulated to undergo CSR to IgG3 (by LPS), to IgG1 and IgE (LPS plus IL-4), or to IgA [LPS plus transforming growth factor (TGF-), IL-5, IL-4, and anti- mAb/dex], the proportion of switched IgG3+, IgG1+, IgA+, or IgE+ B cells was 50 to over 400% greater than among similarly stimulated AicdacreSirt1+/+ B cells, as confirmed by elevated postrecombination I-C1, I-C, and I-C transcripts (Fig. 2, L and M). Further, it was associated with increased expression of Aicda and AID protein, but not Prdm1, Xbp1, or germline I-C, I1-C1, I-C, and I-C transcripts, as measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) after a 72-hour culture (Fig. 2, M and N), indicating a role of Sirt1 in regulating AID but not Blimp1 expression. Plasma cell differentiation of AicdacreSirt1fl/fl B cells, as measured by proportion of CD19lowCD138+ cells, was comparable to that of AicdacreSirt1+/+ cells and so were B cell proliferation and survival (Fig. 2, O and P). Thus, ablation of Sirt1 in B cells increases Aicda expression and CSR without altering Prdm1 expression or plasma cell differentiation.

AicdacreSirt1fl/fl mice injected with NP16-CGG, a conjugated hapten that preferentially induces T-dependent NP-specific IgG1 antibodies, mounted a significantly greater NP4-specific IgG1 response than their AicdacreSirt1+/+ littermates (Fig. 3, A and B). NP16-CGGinjected AicdacreSirt1fl/fl mice also showed increased antibody-forming cells (AFCs) secreting NP4-specific IgG1 in the spleen and bone marrow (Fig. 3, C and D). In these mice, gut IgA-producing cells were also increased in small intestine lamina propria, Peyers patches, and mesenteric lymph nodes (Fig. 3E). Consistent with the augmented NP4-specific IgG1 response, somatic mutations in rearranged V186.2DJH-C1 transcripts [V186.2, ImMunoGeneTics information system (IMGT) gene name V1-72, encodes the IgH variable region of NP-binding antibodies) were increased by almost 100% in AicdacreSirt1fl/fl mice as compared to their AicdacreSirt1+/+ littermates (analysis of more than 10,000 Illumina MiSeq sequences) (Fig. 3F and fig. S3, A and B).

(A and B) Serum titers of total IgM, IgG1, and IgA (A) and high-affinity NP4-binding IgG1 (B) (ELISA; RU, relative units) in AicdacreSirt1+/+ and AicdacreSirt1fl/fl mouse littermates immunized with NP16-CGG at days 0 and 21 at different time points, as indicated (n = 7 mice in each group). Dotted lines link paired littermates. (C and D) AFCs secreting IgM and IgG1 or NP4-binding IgG1 (ELISPOTs) in the spleen and bone marrow in AicdacreSirt1+/+ and AicdacreSirt1fl/fl mouse littermates euthanized 28 days after the first NP16-CGG injection. Data are one representative of three independent experiments yielding similar results (C) or means SEM of three independent experiments (D). (E) Fluorescence microscopy analysis of IgA-producing cells in different gut tissues, as indicated, in AicdacreSirt1+/+ and AicdacreSirt1fl/fl mouse littermates AicdacreSirt1fl/fl mice (one representative of three independent experiments yielding similar results). Scale bars, 50 m. (F) Overall frequency (change/base) and distribution (pie charts) of point mutations in the V186.2 region of V186.2DJH-C1 complementary DNA (cDNA; pooled data from two mouse pairs) in AicdacreSirt1+/+ and AicdacreSirt1fl/fl mice injected (intraperitoneally) with NP16-CGG at days 0 and 21 and euthanized at day 28. Also depicted by histograms are frequencies of mutations in the framework (FR) and complementarity-determining (CDR) regions (right). P values were calculated by 2 test. (G and H) Analysis of class-switched IgMIgDIgG+ B cells, NP5-binding IgG1+ B cells, and NP5-binding CD38+IgG1+ memory B cells (flow cytometry) in spleen (G) and quantification of these cells in the NP16-CGG immunized mice (H). (I) Flow cytometry analysis of B220lowCD138+ plasmablasts/plasma cells in the spleen and bone marrow. (J) Quantification of proportion of B220lowCD138+ plasmablasts/plasma cells among total spleen and bone marrow cells. (K) Viable (7-AAD) B cells in the spleen (flow cytometry) of the immunized mice. (L and M) Flow cytometry analysis of proliferating (incorporating BrdU and BrdU+) B cells (L) and proportions of T (CD3+) cells and B (B220+) cells (M). (N) Spleen germinal center (B220+GL7+CD95+) B cells in AicdacreSirt1+/+ and AicdacreSirt1fl/fl mice as analyzed by flow cytometry 10 days after NP-CGG injection. (O) Quantification of the proportion and of B220+GL-7+CD95+ germinal center B cells among total spleen B cells, as analyzed by FACS (left), and total numbers of B220+GL-7+CD95+ germinal center B cells in each spleen (right). (P) Germinal center structure in the spleen (fluorescence microscopy). Data in (G), (I), (K) to (N), and (P) are one representative of three independent experiments yielding similar results. Scale bar, 100 m. (H), (J), and (O) are means SEM of three or four biological independent experiments. *P < 0.05, **P < 0.01, paired two-tailed Students t test.

In AicdacreSirt1fl/fl mice injected with NP16-CGG, class-switched IgMIgDIgG1+ B cells were increased by almost 55% over their AicdacreSirt1+/+ littermates (Fig. 3G). Among these B cells, the proportion of specific NP5-binding B cells was almost twice that in AicdacreSirt1+/+ littermates (19.0% versus 9.9%). In AicdacreSirt1fl/fl mice, 73.5% of these NP5-binding IgG+ B cells were CD38+ memory B cells, compared to only 30.4% in AicdacreSirt1+/+ mice. In addition, AicdacreSirt1fl/fl mice displayed a more than 320% increase in total spleen NP5-binding IgG1+ B cells, of which the proportion of CD38+ memory B cells was more than 570% greater than in AicdacreSirt1+/+ littermates (Fig. 3H). By contrast, the proportion of the spleen and bone marrow CD138+ plasmablasts/plasma cells in AicdacreSirt1fl/fl mice was comparable to that in AicdacreSirt1+/+ mice (Fig. 3, I and J) and so were spleen size and number and size of Peyers patches. AicdacreSirt1fl/fl mice showed normal B cell viability and proliferation (Fig. 3, K and L). They also showed B (B220+) and T (CD3+) cell numbers (Fig. 3M), proportion of B220+GL7+CD95+ germinal center B cells, and germinal center structure comparable to those of AicdacreSirt1+/+ mice (Fig. 3, N to P). Thus, B cellintrinsic Sirt1 deletion leads to increased antigen-specific, class-switched, and hypermutated B cell responses and memory B cell generation, without affecting B cell number, proliferation, survival, germinal center formation, or plasma cell differentiation.

To further address the direct B cellintrinsic role of Sirt1 in modulating the antibody response, we developed the NBSGW/B mouse model by engrafting highly immunodeficient NBSGW (NOD.Cg-KitW-41J Tyr+Prkdcscid Il2rgtm1Wjl/ThomJ) mice, which lack T cells, B cells, natural killer (NK) cells, functional dendritic cells, and macrophages (28), with highly purified C57BL/6 mouse spleen B cells. We segregated 10 NBSGW/B mice into two groups, 5 mice each, and injected (intraperitoneally) 5 mice with SRT1720 and 5 mice with phosphate-buffered saline (PBS) once every 2 days. All 10 mice were injected intraperitoneally, immediately after engraftment with NP-LPS (to induce an NP-specific T-independent antibody response), and all produced NP4-binding IgM, IgG3, and IgG2b antibodies (fig. S4A). However, the NBSGW/B mice given SRT1720 showed reduced total and NP4-binding class-switched IgG3 and IgG2b and marginally increased unswitched IgM antibodies, without alteration in B cell viability and numbers (fig. S4B). This was concomitant with reduced numbers of IgG3+ and IgG2b+ B cells and normal numbers of CD138+ plasmablasts/plasma cells (fig. S4C) and underpinned by reduced Aicda, but not Prdm1 expression, and decreased postrecombination I-C3 and I-C2b transcripts (fig. S4D). In NBSGW/B mice, Aicda down-regulation by SRT1720 also resulted in a reduced mutational load in V186.2DJH-C3 and V186.2DJH-C2b transcripts encoding the IgH chain of NP-binding IgG3 and IgG2b, as assessed by enumerating point mutations in the V186.2 segment of three comparable V186.2DJH-C3 and three comparable V186.2DJH-C2b clone pairs, each pair consisting of sequences from one SRT1720-treated and one nontreated mouse. Comparable pairs were defined as B cell clones expressing V186.2DJH-C3 or V186.2DJH-C2b with IgH CDR3s being identical in length (7 to 12 amino acids), identical in the first two amino acids and the last three or four amino acids. One of the three V186.2DJH-C3 clone pairs was identical in the whole IgH CDR3 sequence (ARGYFDY). The average change/base in transcripts from mice treated with nil versus mice treated with SRT1720 was 0.48 versus 0.35, 0.44 versus 0.30, and 0.48 versus 0.28 (V186.2DJH-C3) and 0.60 versus 0.35, 0.33 versus 0.19, and 0.33 versus 0.23 (V186.2DJH-C2b) (fig. S4E). Thus, activation of Sirt1 inhibits the maturation of a class-switched and hypermutated T-independent antibody response by down-regulating Aicda expression and dampening CSR/SHM, but not Prdm1 or plasma cell differentiation, in a B cellintrinsic fashion, independent of T cells, dendritic cells, NK cells, or macrophages.

In resting B cells, which do not express AID but express abundant Sirt1 (Fig. 1), histone H3 is hypoacetylated across the Aicda locus (5). Only upon activation by LPS and IL-4, which induced profound Sirt1 down-regulation and Aicda promoter activation, was the Aicda locus histone H3 acetylation greatly increased (Fig. 4A), Sirt1 deacetylates H3Ac, including H3K9 and H3K14, to shape heterochromatin (16). The lack of Sirt1 resulted in increased histone acetylation and gene expression, indicating that in B cells expressing AID, decreased Sirt1 expression enhanced Aicda histone acetylation and transition to euchromatin. Stimulation of B cells with LPS plus IL-4 increased Aicda promoter histone acetylation (H3K9Ac/K14Ac) and induced robust Aicda expression, with no change in H3K9Ac/K14Ac of Prdm1 or Xbp1 promoter (Fig. 4A). Increased Aicda promoter histone H3 acetylation reflected a reduced recruitment of Sirt1 to this region (Fig. 4B) and was not significantly increased in activated AicdacreSirt1fl/fl B cells stimulated with LPS plus IL-4 (fig. S5A), likely due to the physiologically extensive Aicda histone hyperacetylation in B cells induced to express AID and undergoing CSR (5). Thus, LPS plus IL-4induced Sirt1 down-regulation limits Sirt1 recruitment to and deacetylation of the Aicda promoter, thereby increasing Aicda promoter histone acetylation, AID expression, and CSR.

(A and B) ChIP-qPCR analysis of acetylated H3K9/K14 in Aicda, Prdm1, and Xbp1 promoters (A) and Sirt1 recruitment to the Aicda promoter (B) of WT C57BL/6 B cells nonstimulated (Nil) or stimulated with LPS plus IL-4 for 72 hours. Data are ratios to nonstimulated B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (C to I) AicdacreSirt1+/+ and AicdacreSirt1fl/fl B cells were stimulated with LPS plus IL-4 (C to I) or CD154 plus IL-4 (G to I) for 72 hours. (C) Acetylated Dnmt1 and total Dnmt1 (left, immunoblotting; one representative of three independent experiments yielding similar results) and densitometry quantification of signals normalized to -actin levels and depicted as ratios to signals in AicdacreSirt1+/+ B cells (set as 1; means SD of three independent experiments; right). (D) Recruitment of Dnmt1 to the Aicda promoter or a Prdm1 regulatory region (ChIP-qPCR). Data are ratios to recruitment in AicdacreSirt1fl/fl B cells to AicdacreSirt1+/+ B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (E) CpG DNA methylation at the Aicda promoter and Prdm1 regulatory region, as assessed by bisulfite sequencing. Depicted is the proportion of methylated dC nucleotides within the CpG motifs (pooled data from two mice in each group, with at least 5000 sequences from each mouse; means SD). (F) DNA methylation of Aicda and Xbp1 promoters, as well as Prdm1 regulatory region (MeDIP-qPCR). Data are normalized to DNA used as input (means SEM of three biological independent experiments, each consisting of triplicates). (G and H) Immunoblotting analysis of acetylated NF-B p65 and total NF-B p65 (G) and densitometry quantification of signals normalized to -actin levels and depicted as ratios to signals in AicdacreSirt1+/+ B cells (set as 1; means SD of three independent experiments) (H). (I) Recruitment of acetylated NF-B p65 to Aicda promoter or Prdm1 regulatory region (ChIP-qPCR) in B cells. Data are ratios of recruitment in AicdacreSirt1fl/fl cells to that in AicdacreSirt1+/+ B cells (set as 1; means SEM of three biological independent experiments). (J to L) Sirt1+/+ and Sirt1super B cells were stimulated with LPS plus IL-4 for 72 hours. (J) Acetylated H3K9/K14 in the Aicda, Prdm1, or Xbp1 promoter (ChIP-qPCR). Data are ratios of determinations in Sirt1super B cells to those in Sirt1+/+ B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (K) Acetylated NF-B p65 and total NF-B p65 (immunoblotting analysis; left) and densitometry quantification of signals normalized to -actin levels and depicted as the ratio to that in Sirt1+/+ B cells (set as 1; means SD of three independent experiments; right). (L) Recruitment of acetylated NF-B p65 to Aicda, Prdm1, and Xbp1 promoters (ChIP-qPCR analysis). Data are ratios to recruitment in Sirt1+/+ B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (M) Aicda expression (qRT-PCR analysis) in AicdacreSirt1+/+ and AicdacreSirt1fl/fl B cells treated with nil or SRT1720 and stimulated with LPS plus IL-4 for 72 hours (means SEM of three biological independent experiments, each consisting of triplicates). (N to Q) WT C57BL/6 B cells were treated with nil or SRT1720 and stimulated with LPS plus IL-4 for 72 hours. (N and O) ChIP-qPCR analysis of Sirt1 recruitment to Aicda, Prdm1, and Xbp1 promoters (N) as well as acetylated H3K9/K14 of these regions (O). Data are ratios of readings in SRT1720-treated B cells to those in nil-treated B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (P) Immunoblotting of acetylated NF-B p65 and total NF-B p65 in B cells; densitometry quantification of signals normalized to -actin levels and depicted as ratios of readings in SRT1720-treated B cells to those in nil-treated B cells (set as 1; means SD of three independent experiments). (Q) Recruitment of acetylated NF-B p65 to the Aicda, Prdm1, and Xbp1 promoters (ChIP-qPCR analysis). Data are ratios of readings in SRT1720-treated B cells to those in nil-treated B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). *P < 0.05, **P < 0.01, ***P < 0.001, paired two-tailed Students t test.

In addition to histones, Sirt1 deacetylates nonhistone proteins. Sirt1 has been suggested to boost DNA methylation by enhancing Dnmt1 activity through Dnmt1 deacetylation (18). LPS plus IL-4stimulated AicdacreSirt1fl/fl B cells increased acetylated (inactive) Dnmt1, as compared to AicdacreSirt1+/+ B cells, while total Dnmt1 protein levels were unchanged (Fig. 4C). Inactive Dnmt1 resulted in reduced recruitment of this DNA methyltransferase to and reduced DNA methylation of the Aicda promoter [28 and 44% of deoxycytidine (dC) nucleotides within CpG motifs were methylated in LPS plus IL-4stimulated AicdacreSirt1fl/fl and AicdacreSirt1+/+B cells, respectively, as shown by bisulfite sequencing of genomic DNA (gDNA)] (Fig. 4D), leading to increased Aicda expression. The methylation of Prdm1 promoter and the intronic regulatory region or Xbp1 promoter [as further shown by methylated DNA immunoprecipitation (MeDIP)qPCR] in AicdacreSirt1fl/fl B cells were unchanged (Fig. 4, E and F, and fig. S5, B to D).

Another nonhistone protein deacetylated by Sirt1 is NF-B p65, as occurring in macrophages, epithelial cells, and liver cancer cells, in which Sirt1 deacetylates p65 to dampen NF-B activity (17). Given the important role of NF-B in Aicda induction, we hypothesized that deletion of Sirt1 in B cells would lead to increased NF-B p65 acetylation, resulting in enhanced NF-B activity, and therefore, Aicda expression. Upon stimulation with LPS or CD154 plus IL-4, AicdacreSirt1fl/fl B cells significantly increased acetylated-p65 (p65-Ac) as compared to their AicdacreSirt1+/+ counterparts, concomitant with unchanged total p65 protein levels (Fig. 4, G and H). In AicdacreSirt1fl/fl B cells, increased p65 acetylation resulted in increased recruitment of p65-Ac to the Aicda promoter, as shown by chromatin immunoprecipitation (ChIP) with antip65-Ac mAb (Fig. 4I); despite some increased recruitment of p65-Ac to the Prdm1 promoter in AicdacreSirt1fl/fl B cells, Prdm1 expression was not increased (Fig. 2M), likely reflecting this regions unchanged H3K9/K14 acetylation (fig. S5A) and DNA methylation (Fig. 4, E and F), as well as the down-regulation of Irf4 in these B cells (fig. S6 and S7).

Thus, in resting B cells, in addition to being recruited to the Aicda promoter where it deacetylates histones, Sirt1 deacetylates and activates Dnmt1, and deacetylates and inactivates NF-B p65, overall resulting in silencing Aicda expression. Upon B cell stimulation to undergo CSR, down-regulated Sirt1 is not available for recruitment to and deacetylation of Aicda histones nor is it available to deacetylate Dnmt1-Ac, thereby keeping it in an inactive state, or NF-B p65-Ac, thereby keeping it activated. This increased acetylation of Aicda promoter histones, decreased Dnmt1 recruitment to and methylation of the Aicda promoter DNA, and increased NF-B acetylation for an overall combined activation of Aicda expression.

In Sirt1super B cells, impaired Aicda expression and CSR were associated with decreased Aicda promoter histone acetylation (H3K9Ac/K14Ac), as determined by ChIP assays of LPS and IL-4stimulated Sirt1super B cells and WT counterparts, with no concomitant change in H3K9Ac/K14Ac of Prdm1 or Xbp1 promoters (Fig. 4J). In addition, in these stimulated Sirt1super B cells, NF-B p65 acetylation and p65-Ac recruitment to the Aicda promoter were greatly reduced (Fig. 4, K and L). Similarly, Aicda expression was reduced in WT B cells treated with the potent SRT1720 Sirt1 activator; failure of SRT1720 to alter Aicda expression in Sirt1-deficient AicdacreSirt1fl/fl B cells confirmed that this was mediated by Sirt1 (Fig. 4M). SRT1720-mediated reduction of Aicda expression and CSR in B cells was also associated with reduced histone H3K9Ac/K14Ac of the Aicda promoter, but not Prdm1 or Xbp1 promoter, as a result of an increased recruitment of Sirt1 to the Aicda promoter (Fig. 4, N and O). In addition, SRT1720-mediated Sirt1 activation also significantly reduced NF-B p65 acetylation and recruitment of p65-Ac to the Aicda promoter, but not to the Prdm1 or Xbp1 promoter, in LPS plus IL-4stimulated B cells (Fig. 4, P and Q). Thus, overexpression or activation of B cell Sirt1 leads to reduced histone Aicda promoter histone and p65 protein acetylation, resulting in decreased Sirt1 and NF-B recruitment to the Aicda promoter, decreased Aicda expression, and impaired CSR.

NAD+ is the critical Sirt1 cofactor, and NAD+ availability is a function of NAD+/NADH ratio, which is metabolically regulated by glycolysis (19). In the glucose metabolic pathway, glycolysis converts NAD+ to NADH, thereby lowering cytosolic NAD+/NADH ratio. To test that this leads to decreased Sirt1 activity and increased Aicda expression, we cultured B cells with LPS plus IL-4 in medium containing increasing doses of glucose (0 to 20 mM), yielding lower cytosolic NAD+/NADH ratios. This resulted in dose-dependent increased expression of Aicda and CSR, concomitant with normal expression of Prdm1 and plasma cell differentiation (Fig. 5A and fig. S8), and reduced Irf4 expression. While this would generally lead to decreased Prdm1 expression (29), it was counteracted by the potential Prdm1 up-regulation due to increased NF-B p65 acetylation (Fig. 5A and fig. S7), thereby resulting in an unchanged Prdm1 expression level. These increased Aicda expression and CSR were not due to increased carbon/energy provided by glucose, as shown by failure of the same concentrations of galactose (galactose shares the same molecular formula with glucose and serves as a cell energy source but does not effectively feed into glycolysis) to affect Aicda expression and CSR (Fig. 5B). By contrast, B cells cultured with increased amounts of Sirt1 cofactor NAD+ (0 to 500 M) or glucose analog 2-deoxyglucose (2-DG; 0.1 to 1.0 mM), a prototypical glycolytic pathway blocker that has been shown to increase intracellular NAD+ levels and Sirt1 activity (30), reduced Aicda expression and CSR in a dose-dependent manner, with no alteration of Prdm1 expression and plasma cell differentiation (Fig. 5, C and D, and fig. S8)glucose, galactose, or NAD+ concentrations used in these experiments did not alter B cell viability (fig. S9).

B cells were cultured in glucose-free fetal bovine serum (FBS)RPMI medium supplemented with increased concentrations of (A) glucose or (B) galactose or in complete FBS-RPMI-1640 medium supplemented with increased concentrations of (C) 2-DG or (D) NAD+ and stimulated with LPS plus IL-4. Surface expression of B220 and IgG1 was analyzed by flow cytometry after 96 hours of culture. Expression of Aicda, Prdm1, and Irf4 was analyzed by qRT-PCR after 72 hours of culture. NAD+ and NADH concentrations in B cells cultured with increased amount of glucose were also determined after 72 hours. Data are from one representative of three independent experiments yielding comparable results (left) or means SEM of three biological independent experiments, each consisting of triplicates (right). C57BL/6 (E to L) or AicdacreSirt1+/+ and AicdacreSirt1fl/fl (M and N) B cells were cultured in glucose-free FBS-RPMI medium supplied with indicated concentrations of glucose and stimulated with LPS plus IL-4 for 72 (E to M) or 96 hours (N). (E and F) ChIP-qPCR analysis of acetylated H3K9/K14 in Aicda, Prdm1, and Xbp1 promoters (E) and recruitment of Sirt1 to the Aicda promoter (F) in B cells cultured with 0 or 20 mM of glucose. Data are ratios to recruitment in nil-treated B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (G) Densitometry quantification of immunoblotting signals of acetylated Dnmt1 and total Dnmt1 after normalization to -actin levels in B cells. Data are ratios of acetylated Dnmt1 and Dnmt1 in B cells cultured with increased concentrations of glucose to B cells cultured without glucose (set as 1; means SD of three independent experiments). (H) ChIP-qPCR analysis of recruitment of Dnmt1 to the Aicda promoter in B cells cultured in different concentrations of glucose, as indicated. Data are ratios of recruitment in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (I) MeDIP-qPCR analysis of DNA methylation in the Aicda promoter of B cells cultured in different concentrations of glucose, as indicated. Data are ratios of values in B cells cultured in glucose to those in B cells cultured without glucose (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (J and K) Immunoblotting analysis of acetylated NF-B p65 and total NF-B p65 in B cells cultured in different concentrations of glucose, as indicated (J); densitometry quantification of signals expressed as ratios of signal in B cells cultured in glucose to that in B cells cultured without glucose (set as 1; means SD of three independent experiments) (K). (L) Recruitment of acetylated NF-B p65 to the Aicda promoter (ChIP-qPCR analysis) in induced B cells. Data are ratios of recruitment of acetylated NF-B p65 to Aicda promoter in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means SEM of three biological independent experiments). (M) qRT-PCR analysis of Aicda expression in AicdacreSirt1+/+ and AicdacreSirt1fl/fl B cells cultured in the different concentrations of glucose. Data are ratios of Aicda expression in AicdacreSirt1fl/fl B cells to that in AicdacreSirt1+/+ B cells (set as 1; means SEM of three biological independent experiments, each consisting of triplicates). (N) Flow cytometry analysis of CSR to IgG1 in AicdacreSirt1+/+ and AicdacreSirt1fl/fl B cells cultured in different concentrations of glucose. Data are ratios of IgG1+ AicdacreSirt1fl/fl B cells to AicdacreSirt1+/+ B cells (set as 1 in each glucose concentration; means SEM of three biological independent experiments). *P < 0.05, **P < 0.01, and ***P < 0.001, unpaired two-tailed Students t test.

To further define the role of Sirt1 in glucose-mediated up-regulation of Aicda expression and CSR, we stimulated AicdacreSirt1fl/fl and AicdacreSirt1+/+ B cells with LPS plus IL-4 and cultured them in RPMI medium containing no or increasing concentrations of glucose. In WT B cells, glucose-induced decreased NAD+/NADH ratio and Sirt1 activity resulted in reduced Sirt1 recruitment to the Aicda promoter, leading to increased promoter histone acetylation, increased acetylated (inactive) Dnmt1, reduced recruitment of Dnmt1 to the Aicda promoter, as defined by ChIP-qPCR, thereby limiting Aicda promoter DNA methylation, as defined by MeDIP-qPCR. This together with increased p65-Ac (not total p65 level) and increased recruitment of p65-Ac to the Aicda promoter led to increased Aicda expression and CSR (Fig. 5, E to L). At the highest glucose concentration (20 mM), the enhancement of Aicda expression and CSR to IgG1 in AicdacreSirt1+/+ B cells was comparable to those observed in AicdacreSirt1fl/fl B cells (Fig. 5, M and N). Thus, B cell glycolysis, which results in reduced NAD+/NADH ratio, paucity of Sirt1 cofactor NAD+, or decreased Sirt1 activity, leads to increased Aicda expression and CSR, mimicking the increased Aicda expression and CSR resulting from ablation of Sirt1.

In AicdacreSirt1fl/fl mice, deletion of Sirt1 in activated B cells led to production of class-switched anti-nuclear antigen autoantibodies (ANAs), including antidouble-stranded DNA (dsDNA), anti-histone, antiribonucleoprotein (RNP), and anti-RNA IgG (Fig. 6, A and B). In B cells isolated from female lupus MRL/Faslpr/lpr (12-week-old) and BXD2 (36-week-old) mice or patients with SLE, enhanced Aicda/AICDA expression was associated with significantly reduced Sirt1/SIRT1 expression, as compared to healthy female C57BL/6 mice or healthy humans, respectively (Fig. 6, C and D). Accordingly, Sirt1 protein was significantly decreased in MRL/Faslpr/lpr B cells, in which AID expression was elevated (Fig. 6G). Further, reduced Sirt1 expression was associated with hyperacetylated Aicda/AICDA promoter histone H3 in mouse and human lupus B cells (Fig. 6, E and F). Last, SRT1720 treatment reduced AID expression in MRL/Faslpr/lpr B cells and decreased anti-dsDNA and anti-histone IgG1 and IgG2a, but not IgM, as well as reduced IgG1/IgG2a kidney deposition and glomerular damage in MRL/Faslpr/lpr mice (Fig. 6, G to I). Thus, Sirt1 deletion in activated B cells leads to emergence of lupus autoantibodies in otherwise normal female C57BL/6 mice. In mouse and human lupus B cells, high levels of Aicda/AICDA expression occur concomitantly with decreased Sirt1/SIRT1 expression, in association with increased Aicda promoter histone acetylation. In addition, Sirt1 activation by SRT1720 reduces AID expression, autoantibody production, and autoimmunity in lupus MRL/Faslpr/lpr mice.

(A) IgG1+ and IgG2a+ ANAs in sera from 35-week-old female AicdacreSirt1+/+ and AicdacreSirt1fl/fl mice (immunofluorescence microscopy of one representative of three independent experiments). Scale bar, 50 m. (B) Anti-dsDNA, anti-histone, anti-RNP, and anti-RNA IgG titers (ELISAs) in sera from 35-week-old female AicdacreSirt1+/+ and AicdacreSirt1fl/fl mice (means SD of six AicdacreSirt1+/+ and six AicdacreSirt1fl/fl mice, each tested in triplicates). (C) Expression of Sirt1 and Aicda (qRT-PCR analysis) in spleen B cells from C57BL/6 mice and lupus-prone MRL/Faslpr/lpr and BXD2 mice. Data are relative expression to -Actin (means SD of three mice in each group). Dotted lines depict trends. (D) qRT-PCR analysis of expression of SIRT1 and AICDA in B cells from healthy humans and patients with SLE. Data are normalized to expression of -ACTIN (means SD of three healthy individuals and three patients with SLE). Dotted lines depict trends. (E) Acetylated H3K9/K14 in the Aicda, Prdm1, and Xbp1 promoters (ChIP-qPCR analysis) in B cells from C57BL/6 and MRL/Faslpr/lpr mice. Data are ratios of acetylated H3K9/K14 in MRL/Faslpr/lpr mice to that in C57BL/6 mice (set as 1; means SD of three mice in each group). (F) Acetylated H3K9/K14 in the AICDA and PRDM1 promoters in B cells from healthy human individuals (HS) and patients with SLE (ChIP-qPCR analysis). Data are ratios of acetylated H3K9/K14 in the AICDA and PRDM1 promoters of B cells from patients with SLE to that of healthy individuals (set as 1; means SD of three healthy individuals and three patients with SLE). (G) Intracellular staining of Sirt1 and AID protein levels in spleen B cells from age-matched female C57BL/6 mice, untreated MRL/Faslpr/lpr mice, and MRL/Faslpr/lpr mice treated with SRT1720 (flow cytometry analysis, one representative of three independent experiments). (H) Serum anti-dsDNA and anti-histone IgM, IgG1, and IgG2a titers (ELISAs) in female MRL/Faslpr/lpr mice treated with Nil or SRT1720 (means SD of six mice in each group). (I) Photomicrographs of kidney sections from MRL/Faslpr/lpr mice treated with Nil or SRT1720, after hematoxylin and eosin (H&E) staining (left) or fluorescence staining of mouse IgG1/IgG2a (right). One representative of three independent experiments yielding similar results. Scale bars, 100 m. *P < 0.05, **P < 0.01, unpaired two-tailed Students t test.

By introducing single-strand nicks and double-strand breaks in the Ig locus DNA, AID affects the first and critical step in the cascade of events that lead to B cell CSR/SHM. Being a potent mutator, AID is under tight regulation. Its expression is cell type specific, being confined to B lymphocytes, and B cell differentiation is stage specific, being limited to activated B cells stimulated to undergo CSR/SHM (24). AID is not expressed in nave resting B cells, resting memory B cells, or plasma cells. Its targeting is also specific, being restricted to the Ig locus. However, under nonphysiological conditions, AID can target DNA outside the Ig locus (AID off-targeting) and, as a potent mutator, can cause genome instability in lymphoid and nonlymphoid cells, thereby promoting neoplastic transformation (4, 31). Genomic damage by AID off-targeting has been well documented, emphasizing the need for tight regulation of AID expression to maintain genomic integrity (31). The mechanisms targeting AID to the Ig locus, including DNA hotspots and epigenetic marks, such as histone acetylation and phosphorylation, are relatively understood. However, what keeps AID expression in check in resting B cells and limits it to B cells activated to undergo CSR/SHM has remained unaddressed.

Here, we provide evidence that Sirt1 is central to a B cellintrinsic mechanism that, together with transcription factors and other epigenetic modulators, regulates AID expression for CSR/SHM in the maturation of the antibody response. Sirt1-mediated regulation of AID is B cell differentiation stage specific, as shown by high levels of Sirt1 in human and mouse resting B cells and Sirt1 down-regulation by stimuli that induce Aicda expression and CSR/SHM. The B cellintrinsic function of Sirt1 in the regulation of these processes was addressed in vitro in experiments using purified mouse and human nave B cells and was extended to in vivo analysis of class-switched and hypermutated T-dependent and T-independent antibody responses in AicdacreSirt1fl/fl mice and NBSGW/B mice. The specificity of such a B cell Sirt1 function was underlined by Sirt1 regulation of Aicda but not Prdm1 or Xbp1, thereby affecting CSR/SHM but not plasma cell differentiation. The importance of B cellintrinsic and differentiation stagespecific Sirt1 expression was further emphasized by the fast kinetics of Sirt1 down-regulation that preceded AID up-regulation, as well as the compelling phenotypes generated upon manipulation of B cell Sirt1 expression or activity. Sirt1 would keep Aicda expression in check in resting B cells and allows Aicda to be expressed only to initiate CSR/SHM. By negatively regulating Aicda expression, Sirt1 would function as a key safeguard of AID-mediated DNA damage.

Sirt1 has been implicated in the functions of other immune elements. Through deacetylation of histone and nonhistone proteins, Sirt1 controls the intracellular localization, stability, and activity of these proteins, making this class III HDAC involved in multiple, mainly epigenetic, functions (16). In addition to deacetylating gene promoter histones, Sirt1 modifies other epigenetic mediators, including DNA methyltransferases, acetyltransferases, and transcription factors. Sirt1 has been shown to be highly expressed in medullary thymic epithelial cells, in which it is required for the expression of Aire-dependent, tissue-restricted antigen-encoding genes and, therefore, induction of central immunological T cell tolerance (32). Sirt1 negatively regulates T cell activation and would play a role in maintaining T cell tolerance and T celldependent humoral immune responses (13). It can also function as a molecular switch in controlling regulatory T cell (Treg) and T helper cell 17 (TH17) cell differentiation (33). Stimulation of nave T cells with IL-4 plus TGF-1 decreased Sirt1 expression and induced TH9 cell differentiation (34). In humans, SIRT1 is a regulator of resting CD8+ memory T cell metabolism and activity, and it is significantly down-regulated in terminally differentiated CD8+CD28 memory T cells (35). Also, Sirt1 has been suggested to modulate cytokine production by dendritic cells and skewing the balance between proinflammatory TH1/TH17 cells and anti-inflammatory Treg cells (14, 36). Last, Sirt1 inhibits inflammatory pathways in macrophages and regulates macrophage self-renewal (37).

Sirt1 keeps Aicda transcription in check by a three-pronged mechanism (fig. S10) involving (i) deacetylation of Aicda promoter histones, (ii) deacetylation of Dnmt1, and (iii) deacetylation of the p65 component of NF-B. First, Sirt1 plays a major (negative) regulatory role in Aicda locus H3 acetylation. Deacetylation of core histones promotes chromatin condensation, making promoters inaccessible to transcription factors and suppressing gene expression. Sirt deacetylates H3K9Ac and H3K14Ac (as shown here), as well as H3K4Ac and H3K36Ac (38). Accordingly, in B cells stimulated with LPS plus IL-4, H3K9Ac and H3K14Ac were hyperacetylated, reflecting Sirt1 down-regulation and, consequently, the loss of Sirt1-mediated deacetylation at these H3 Lys residues. By contrast, Sirt1 did not affect Prdm1 locus H3 acetylation, which occurred mainly at H3K27 [our analysis of primary ChIP-Seq data GEO:GSE82144 deposited in the National Center for Biotechnology Information (39)]. In nucleosomes, this H3K27 is not targeted nor is deacetylated by Sirt1 (40). Last, while histone acetylation of the Aicda promoter was increased in C57BL/6 B cells induced to undergo CSR and decreased by enforced Sirt1 expression or Sirt1 activation, the histone acetylation status of the Aicda promoter in AicdacreSirt1fl/fl B cells was unaltered, as compared to AicdacreSirt1+/+ B cells under similar CSR induction. Likely, the deep down-regulation of Sirt1 expression in B cells undergoing CSR led to Aicda promoter histone hyperacetylation, which left virtually no room for further histone acetylation upon Sirt1 ablation. All nucleosomes of Aicda exon 1 and its vicinity, including the promoter region, have been shown to be extensively acetylated in B cells induced to express Aicda and undergoing CSR (5).

Second, upon Sirt1-mediated acetylation, activated Dnmt1 was recruited to and methylated Aicda promoter DNA, thereby silencing this gene. The Dnmt1 selectivity in being recruited to and methylating the Aicda promoter DNA contrasted with the failure of this DNA methyltransferase to be recruited and methylate Prdm1 or Xbp1 promoter DNA. This is consistent with the Prdm1 promoter showing constitutively demethylated CpG islands and, therefore, being hardly further acetylated through modulation of DNA demethylation (not shown). This would not exclude the possibility, however, that Prdm1 DNA methylation can also be mediated by additional DNA methyltransferases, such as Dnmt3a and Dnmt3b (41).

Third, deacetylation of NF-B effectively inactivates this important B cell transcription factor. NF-B together with HoxC4 is critical to Aicda promoter activation (6, 7). NF-B function is modulated by posttranscriptional modifications, such as phosphorylation, acetylation, and methylation (42). Acetylated NF-B displays enhanced transcriptional activity (43), which is reduced by Sirt1-mediated deacetylation (17). The critical role of Sirt1 in modulating NF-B acetylation and, therefore, activity was further emphasized by the reduced p65 acetylation and Aicda expression by Sirt1 overexpression in Sirt1super B cells and boost of Sirt1 activation by NAD+ or SRT1720. The increased p65Ac recruitment to the Prdm1 promoter in AicdacreSirt1fl/fl B cells was likely insufficient to up-regulate the induction of Prdm1 in light of the marginal role of Sirt1 in Prdm1 promoter H3 deacetylation, as well as the acetylation and activation of Dnmt3l (due to decreased Sirt1 activity) and the consequent Irf4 DNA methylation and down-regulation by this DNA methylase (fig. S7).

In B cells stimulated in a T-dependent or T-independent fashion, the significant drop in Sirt1 led to decreased Sirt1 recruitment to the Aicda promoter, but not Prdm1 or Xbp1 promoter, and reduced Aicda deacetylation. This together with greater acetylation of NF-B p65 promoted Aicda expression and CSR in B cells in vitro, as further facilitated by reduced Aicda promoter DNA methylation resulting from acetylation and inactivation of Dnmt1, which failed to be recruited to the Aicda promoter. These events were magnified by differentiation stagespecific Sirt1 ablation in AicdacreSirt1fl/fl B cells, which further increased Aicda expression, but did not affect Prdm1 or Xbp1. In AicdacreSirt1fl/fl mice, the same events boosted B cell Aicda expression and the generation of class-switched and somatically mutated antibody response, which included increased specific switched memory B cells but not plasma cells. Non-intentionally immunized AicdacreSirt1fl/fl mice produced a spectrum of lupus-like autoantibodies, pointing at a role of intrinsic B cell mechanisms in the development of the lupus-like autoimmune condition in constitutive Sirt1 knockout mice (15). A role of Sirt1 in regulating Aicda expression and the autoantibody response was further supported by our demonstration of correlation between down-regulated SIRT/Sirt1 and up-regulated AICDA/Aicda in human and mouse lupus B cells. Such a role would be consistent with the suggestion that SIRT1 rs375891 allele modifies lupus morbidity, with rs375891T being a risk factor for nephritis (44), and the reduced expression levels of SIRT1/Sirt1 in lupus B cells, in which AID is known to be greatly up-regulated.

The definition of the mechanism by which Sirt1 is down-regulated in B cells induced to undergo CSR/SHM is beyond the scope of these investigations. Nevertheless, it is known that Sirt1 expression is regulated at both transcriptional and posttranscriptional levels (45). Interferon- (IFN-) has been shown to repress Sirt1 transcription (46). IFN- induces transcription factor Class II Major Histocompatibility Complex Transactivator (CIITA), which is recruited to the Sirt1 promoter by a transcriptional repressor hypermethylated in cancer 1 (46). Once recruited to Sirt1, CIITA represses Sirt1 transcription, via active deacetylation of core histones surrounding the Sirt1 proximal promoter (46). At the posttranscriptional level, Sirt1 can be regulated by microRNAs or RNA-binding proteins (45). More than 16 microRNAs have been shown to modulate Sirt1 expression in different types of cells. These include miR-22, which is expressed in B cells and has been shown to be up-regulated by CD154 plus IL-4 in chronic lymphocytic leukemia B cells (45). In dendritic cells, miR-22 can be induced by TLR ligands such as LPS through NF-B (47). Thus, CIITA and select microRNAs, such as miR-22, may play a role in mediating Sirt1 down-regulation in B cells, as induced to undergo CSR/SHM.

Sirtuins have emerged as important metabolic sensors of energy status in mammalian cells (2325). Sirt1 activity is directly modulated by the cellular level of its critical cofactor NAD+, whose concentration increases in response to energy or nutrient stresses such as fasting or calorie restriction (2325). All these conditions boost Sirt1 activity (19), leading to histone deacetylation and chromatin silencing. Conversely, glycolysis, as induced by high blood sugar levels, converts cellular NAD+ to NADH, decreasing NAD+/NADH ratio and NAD+ availability, which blunts Sirt1 activity (24, 25). Accordingly, increased glucose concentration led to up-regulation of Aicda and CSR. Like intrinsic B cell overexpression of Sirt1, boosting Sirt1 function by NAD+ or the small-molecule SRT1720, a specific and potent Sirt1 activator, dampened Aicda expression and CSR/SHM in the T-independent IgG response to NP-LPS in our NSGW/B mice. This novel and powerful model has unveiled a significant B cell SHM activity in the absence of T cells or other immune cells. This SHM was likely induced by our previously described mechanism of BCR and TLR4-linked co-engagement (48).

Activation of Sirt1 by SRT1720 dampened the autoantibody response in lupus MRL/Faslpr/lpr mice. In these mice, not only did SRT1720-mediated Sirt1 activation suppress the autoantibody response but it also led to a marked amelioration of lupus immunopathology. The decreased SIRT1/Sirt1 expression shown here in human and mouse lupus B cells would provide an explanation for the hyperacetylation of the AICDA/Aicda promoter in human and mouse lupus (3, 4), as confirmed by our findings in those B cells. Our demonstration of a regulation of AID expression through Sirt1 metabolic sensor would outline a possible mechanism by which the diabetes therapeutic metformin (inhibitor of gluconeogenesis) and 2-DG (a glucose analog, which competitively inhibits glucose uptake and glycolytic flux) dampened anti-dsDNA IgG and ANA IgG in lupus B6.Sle1.Sle2.Sle3 mice (49).

Sirt1 expression has been shown to fluctuate under physiological and pathological conditions (25). Collectively, our findings identified Sirt1 as a critical B cellintrinsic epigenetic element in AID silencing in resting B cells in regulating this potent dC deaminator, and, therefore, CSR/SHM in the maturation of the antibody response. Sirt1 suppresses Aicda expression and CSR/SHM through deacetylation of both histone and nonhistone proteins, that is, deacetylation of Aicda promoter histones, NF-B p65 and Dnmt1. In both human and mouse B cells, SIRT1/Sirt1 is down-regulated in response to stimuli that induced expression of AID and CSR/SHM, as it is in B cells from humans and mice with lupus. By showing that B cell Sirt1 inactivation by reduction of NAD+ can be an inducer of AID expression, our findings add an important effector function to Sirt1 as a metabolic sensor and suggest a role of glycolysis in the regulation of AID.

C57BL/6 (C57BL/6J, stock no. 000664), MRL/Faslpr/lpr (MRL/MpJ-Faslpr/J, 000485), BXD2 (BXD2/TyJ, 000075), Sirt1fl/fl (B6.129-Sirt1tm3Fwa/DsinJ, 029603), Aicdacre [B6;FVB-Tg(Aicda-cre)1Rcas/J, 018422], Sirt1super [B6.Cg-Tg(Sirt1)ASrn/J, 024510], and NBSGW (NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ, 026622) mice were from The Jackson Laboratory (Bar Harbor, Maine). Sirt1fl/fl mice carry insertion mutations in the neomycin-resistant gene and lox sequences of the Sirt1 gene flanking exon 4 that encodes a conserved Sir2 motif (27). Sirt1super mice display abnormally increased Sirt1 expression, as a result of carrying multiple copies of a BAC transgene containing the Sirt1 locus with the endogenous promoter/enhancer regions (26). In BAC transgenic Aicdacre mice, the bacterial cre recombinase gene was introduced in lieu of Aicda exon 1 in a supplementary Aicda locus and under control of the Aicda promoter/enhancers within the BAC transgene (5). We generated AicdacreSirt1fl/fl mice by cross-breeding Aicdacre with Sirt1fl/fl mice (fig. S3). For NP-CGG immunization, AicdacreSirt1fl/fl mice and their sex-matched AicdacreSirt1+/+ littermates (8 to 12 weeks of age) were injected intraperitoneally with 100 g of NP16-CGG (average 16 molecules of 4-hydroxy-3-nitrophenyl acetyl coupled with 1 molecule of chicken -globulin; Biosearch Technologies) in 100 l of alum (Imject Alum, Pierce). Mice were given a booster intraperitoneal injection of 100 g of NP16-CGG in PBS at day 21. Serum samples were collected, and mice were euthanized for ex vivo analysis at time points indicated.

To generate NBSGW/B mice, nave B cells were purified from splenocytes of 8-week-old female C57BL/6 mice by negative selection using the EasySep Mouse B Cell Isolation Kit (STEMCELL Technologies) following the manufacturers instructions and supplemented with additional anti-CD3 mAb (clone 17A2; BioLegend), resulting in more than 99% purity. Purified B cells (3.0 107 cells per mouse in 250 l of PBS) were injected intravenously through lateral tail veins into 8-week-old female NBSGW mice. NBSGW/B mice were injected intraperitoneally with NP0.5-LPS (average 0.5 molecule of NP conjugated to 1 molecule of LPS; 50 g in PBS) at days 0 (after B cell engraftment), 2, 4, and 6. For in vivo Sirt1 activation, NP-LPSimmunized NBSGW/B mice were injected intraperitoneally with SRT1720 (0.3 mg in 200 l of PBS) or PBS every other day, starting at day 0 and until the end of the experiment. Mice were euthanized 7 days after the last NP-LPS injection. All mice were housed under pathogen-free conditions and were fed autoclaved food and deionized water. The Institutional Animal Care and Use Committee of the University of Texas Health Science Center San Antonio (UTHSCSA) approved all animal protocols.

For in vitro CSR induction, human IgD+ nave B cells were purified by negative selection from peripheral blood mononuclear cells (PBMCs) of healthy donors fresh buffy coats (San Antonio Blood and Tissue Center) using the EasySep Human Nave B Cell Enrichment Kit (19254, STEMCELL Technologies) following the manufacturers directions, resulting in more than 98% purity. Nave B cells were stimulated with CD154 (5 U/ml; obtained from membrane fragments of baculovirus-infected Sf21 insect cells) and cultured in fetal bovine serum (FBS)RPMI (RPMI-1640 supplemented with 10% FBS, 50 mM -mercaptoethanol, and antibiotic-antimycotic mixture) and recombinant human IL-21 (50 ng/ml; R&D Systems) in the presence or absence of recombinant human IL-4 (20 ng/ml; R&D Systems) for up to 120 hours. B cells were then stained with 7-aminoactinomycin D (7-AAD; BD Biosciences) and fluorochrome-conjugated mAbs specific for human CD19 (clone HIB19; BioLegend) and human IgG (clone G18-125; BD Biosciences) and then analyzed by flow cytometry using an LSR-II flow cytometer (BD Biosciences). Dead cells (7-AAD+) were excluded from analysis. All flow cytometry data were analyzed using FlowJo (Tree Star).

For SIRT1 and AICDA qRT-PCR analysis and histone acetylation ChIP assays, B cells were purified by positive selection from PBMCs of healthy donors fresh buffy coats (San Antonio Blood and Tissue Center) or peripheral blood from patients with SLE (Division of Rheumatology, Department of Medicine, Long School of Medicine, UTHSCSA) using mouse biotinanti-human CD19 mAb (clone HIB19; BioLegend) and streptavidin-coupled magnetic beads, resulting in more than 99% purity. All experiments involving human blood were approved by the Institutional Review Board of UTHSCSA.

For in vitro CSR induction, nave B cells isolated from red blood celldepleted splenocytes of 8- to 12-week-old mice were purified by negative selection of cells expressing CD43, CD4, CD8, CD11b, CD49b, CD90.2, Gr-1, or Ter-119 using the EasySep Mouse B Cell Isolation Kit (STEMCELL Technologies). B cells were resuspended in FBS-RPMI at 37C in 48-well plates and stimulated with the following reagents: LPS (1 or 5 g/ml) from Escherichia coli (055:B5, Sigma-Aldrich) for CSR to IgG3; LPS (3 g/ml) or CD154 (1 U/ml) plus IL-4 (5 ng/ml; R&D Systems) for CSR to IgG1; LPS (3 g/ml) plus IFN- (50 ng/ml) for CSR to IgG2a; LPS (3 g/ml) plus TGF- (2 ng/ml; R&D Systems), IL-4 (5 ng/ml), IL-5 (3 ng/ml; R&D Systems), and anti-Ig mAb-dextran (Fina Biosolutions) for CSR to IgA. After 96 hours, cells were analyzed for surface Ig after being stained with fluorescein isothiocyanate (FITC)labeled rat mAb to mouse IgG1 (clone A85-1), mouse IgG2a (clone R19-15), mouse IgG3 (clone R40-82), and mouse IgA (clone C10-3) or phycoerythrin (PE)labeled rat mAb to mouse B220 (clone RA3-6B2), all from BD Biosciences. Cells were analyzed by flow cytometry, with dead cells (7-AAD+) excluded from analysis. All flow cytometry data were analyzed using FlowJo (Tree Star). All the Abs and mAbs used in the above experiments are listed in table S1A. For Sirt1 and Aicda qRT-PCR analysis and histone acetylation ChIP assays, B cells were isolated from C57BL/6 or lupus-prone MRL/Faslpr/lpr and BXD2 mice by positive selection using biotinylated rat anti-mouse CD19 mAb (clone 6D5; BioLegend) and streptavidin-coupled magnetic beads, resulting in more than 99% purity.

Single-cell suspensions were prepared from mouse spleen and stained with the following antibodies and reagents in different combinations for flow cytometry analysis (LSR-II flow cytometer, BD Biosciences) of B cells (B220+), T cells (CD3+), germinal center (GL-7hiB220+) B cells and plasma cells (B220lowCD138+), antigen-specific B cells, and class-switched B cells: PEanti-B220 mAb (clone RA3-6B2; eBioscience), Pacific Blue anti-B220 mAb (clone RA3-6B2; BioLegend), FITCanti-CD3 mAb (clone 17A2; BioLegend), and biotinanti-CD138 mAb (clone 281-2; BD Biosciences) followed by FITC-streptavidin (11-4317-87, eBioscience) or PE-streptavidin (12-4317-87, eBioscience), PE-Cy7-anti-CD38 mAB (clone 90; BioLegend), PE or FITCanti-IgM mAb (clone RMM-1; BioLegend), FITCanti-IgG1 mAb (clone A85-1; BD Biosciences), allophycocyanin (APC)anti-IgG1 mAb (clone X56; BD Biosciences), FITCanti-IgG3 mAb (clone R40-82; BD Biosciences), FITCanti-IgA mAb (clone C10-3; BD Biosciences), and biotinanti-IgD mAb (clone IA6-2; BioLegend) followed by FITC-streptavidin (11-4317-87, eBioscience) or APC-streptavidin (550874, eBioscience), PE-NP4 (N-5070, Biosearch Technologies), and 7-AAD, as all previously described (6, 11).

B cell division in vitro was analyzed by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution using the CellTrace CFSE Cell Proliferation Kit (Invitrogen). Briefly, B cells were incubated for 5 min at 37C in 3 ml of PBS with 2.5 M CFSE at a density of 1 107 cells/ml and then washed in FBS-RPMI. Cells were then cultured in the presence of LPS or CD154 plus IL-4 for 3 days and then stained with PEanti-B220 mAb and 7-AAD for flow cytometry analysis. For intracellular staining, cells were reacted with an anti-CD19 mAb (clone 1D3; Tonbo) and fixable viability dye eFluor 450 (FVD 450, eBiosciences), followed by incubation with the BD Cytofix/Cytoperm buffer at 4C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were resuspended in Hanks balanced salt solution with 1% bovine serum albumin (BSA) and stored overnight at 4C. Cells were then stained with anti-Sirt1 Ab (A11267, ABclonal; labeled with APC using Mix-n-Stain Fluorescent Protein & Tandem Dye Antibody Labeling Kit, Biotium) and FITCanti-AID Ab (bs-7855R-FITC, Bioss) in Perm/Wash buffer. Dead (eFluor 450+) cells were excluded. All the Abs and mAbs used in the above experiments are listed in table S1A.

To visualize Sirt1 and AID expression in B cells, cells were spun onto glass slides (800 rpm for 5 min; Cytospin 4, Thermo Fisher Scientific) and then fixed with 3% paraformaldehyde in 250 mM Hepes (pH 7.4), stained with APC-labeled anti-Sirt1 Ab (as above) or Alexa Fluor 647anti-AID Ab (bs-7855R-A647, Bioss), and mounted in ProLong Gold Antifade Reagent with DAPI (4,6-diamidino-2-phenylindole; Invitrogen). Fluorescent images were captured using a 10 objective lens with a Zeiss Axio Imager Z1 fluorescence microscope.

To analyze IgM- and IgA-producing cells, intestinal sections were heated at 80C to adhere to glass slides, washed four times in xylene for 2 min, dehydrated twice with 100% ethanol for 1 min, twice with 95% ethanol for 1 min, and washed twice in water for 1 min. Antigens were unmasked using 2 mM EDTA in 100C for 40 min followed by a cooling step at 25C, washed three times with tris-buffered saline (TBS), and blocked using 10% BSA for 15 min. Slides were again washed three times with TBS and stained with rabbit anti-IgA Ab (PA-1-30826, Thermo Fisher Scientific) followed by Alexa Fluor 488conjugated anti-rabbit IgG (H+L) Ab F(ab)2 fragment (4414, Cell Signaling Technology) and PE-conjugated goat-anti mouse-IgM mAb (clone RMM-1; BioLegend) for 2 hours in a moist dark chamber. After washing three times with Triton X-100 (0.1%) in TBS, slides were air-dried, and coverslips were mounted with ProLong Gold Antifade Reagent using DAPI (Invitrogen). To analyze germinal center structure, 10-m spleen sections were prepared by cryostat and loaded onto positively charged slides, fixed in cold acetone and stained with PE-GL7 and FITC-B220 mAb for 1 hour at 25C in a moist chamber. Coverslips were mounted using ProLong Gold Antifade Reagent with DAPI for microscopy analysis. All the Abs and mAbs used in the above experiments are listed in table S1A.

Titers of total and NP4-binding IgM, IgG1, IgG3, IgG2b, and IgA were measured by enzyme-linked immunosorbent assay (ELISA), as described (6, 11). Anti-dsDNA, anti-histone, anti-RNP, and anti-RNA IgG1 and IgG2a titers were measured by ELISA, as described (11)titers were expressed in relative units (RU), defined as the dilution factor needed to reach 50% of binding saturation, as calculated using the Prism software (GraphPad). To detect ANAs, sera were serially diluted in PBS (from 1:20 to 1:400), incubated on ANA substrate slides (HEp-2 cellcoated slides, MBL-BION), and detected with FITCanti-IgG1 mAb (clone 85-1; BD Biosciences) and FITCanti-IgG2a mAb (clone R19-15; BD Biosciences). Images were acquired with a 40 objective on a Zeiss Axio Imager Z1 fluorescence microscope. To analyze kidney IgG deposition, kidneys from MRL/Faslpr/lpr mice were fixed in 4% formaldehyde and paraffin-embedded for hematoxylin and eosin staining. For immunofluorescence, 5-m cryostat sections were loaded onto positively charged slides, fixed in cold acetone, and stained with a mixture of FITC-labeled rat anti-mouse IgG1 or anti-mouse IgG2a mAb. Cover slips were mounted using ProLong Gold Antifade Reagent for microscopy analysis. All the Abs and mAbs used in the above experiments are listed in table S1A.

MultiScreen ELISPOT plates (MAIPS4510, Millipore) were activated with ethanol (35%), washed four times with PBS, and then coated with 100 l NP4-BSA (5 g/ml; N-5050L, Biosearch Technologies), goat anti-mouse IgG1 Ab (5 g/ml; 1071-01, Southern Biotech), or goat anti-mouse IgM Ab (5 g/ml; 1121-01, Southern Biotech) in PBS overnight at 4C. The plates were then washed six times with PBS and blocked with BSA (0.5%) in RPMI/Hepes plus l-glutamine for 1 hour at room temperature. Single-cell suspensions (250,000 or 100,000 cells/ml) from the spleen and bone marrow of immunized mice were cultured in plates at 37C for 16 hours in FBS-RPMI. The cultures were removed, and the plates were washed six times, incubated with biotin-goat anti-mouse IgG1 Ab (1070-08, Southern Biotech) or biotin-rat anti-mouse IgM mAb (1022-08, Southern Biotech) for 2 hours on a shaker at room temperature, washed six times, incubated with horseradish peroxidase (HRP)streptavidin (Santa Cruz Biotechnology) for 1 hour on a shaker at room temperature, washed 10 times, and developed using the VECTASTAIN AEC Peroxidase Substrate Kit (SK-4200, Vector Laboratories) following the manufacturers protocol. Plates were imaged and quantified using a CTL-ImmunoSpot Analyzer (Cellular Technology). All the Abs and mAbs used in the above experiments are listed in table S1A.

The mutations in V186.2 of rearranged V186.2DJH-C1, V186.2DJH-C2b, and/or V186.2DJH-C3 NP-binding IgG1, IgG3, and IgG2b were analyzed by high-throughput Illumina MiSeq amplicon sequencing. Spleen B cells were isolated for RNA extraction using the RNeasy Mini Kit (Qiagen). Residual DNA was removed from the extracted RNA with gDNA eliminator columns (Qiagen). Complementary DNA (cDNA) was synthesized from 1 to 2 g of total RNA using SuperScript III First-Strand Synthesis System (Invitrogen) and oligo-dT primer. Rearranged V186.2DJH-C1, V186.2DJH-C2b, and/or V186.2DJH-C3 cDNAs encoding the anti-NP IgG1, IgG2b, and IgG3 heavy chains were amplified using a V186.2 leaderspecific forward primer together with a reverse C1-, C3-, or C2b-specific primer (11) tagged with Illumina clustering adapters and Phusion high-fidelity DNA polymerase (New England BioLabs). PCR conditions were 98C for 10 s, 60C for 45 s, and 72C for 1 min for 30 cycles. The amplified library was tagged with barcodes for sample multiplexing, and PCR was enriched and annealed to the required Illumina clustering adapters. High-throughput 300base pair (bp) paired-end sequencing was performed by the UTHSCSA Genome Sequencing Facility using the Illumina MiSeq platform. Somatic mutations and clonotypes in rearranged V186.2(V1-72)-DJH gene encoding NP-binding IgG3 and IgG2b were analyzed using IMGT/High V-QUEST (http://imgt.org/HighV-QUEST/doc.action). The frequency of mutations in rearranged V186.2DJH-C2b and/or V186.2DJH-C3 NP-binding IgG3 and IgG2b, as analyzed by MiSeq amplicon sequencing in SIRT1720-treated and nontreated NBSGW/B mice, was compared by enumerating the point mutations in the V186.2 segment of three comparable V186.2DJH-C3 and three comparable V186.2DJH-C2b clone pairs (each pair consisting of a mouse treated with nil and one with SRT1720). Comparable pairs were defined as B cell clones expressing V186.2DJH-C3 or V186.2DJH-C2b with IgH CDR3s being identical in length (7 to 12 amino acids), identical in the first two amino acids and the last three or four amino acids. One of the three V186.2DJH-C3 clone pairs was identical in the whole IgH CDR3 sequence (ARGYFDY).

Total RNA was extracted from 2.0 to 5.0 106 pelleted B cells using the RNeasy Mini Kit (Qiagen). Residual DNA was removed from the extracted RNA with gDNA eliminator columns (Qiagen). cDNA was synthesized from 1.0 to 2.0 g of total RNA with the SuperScript III First-Strand Synthesis System (Invitrogen) using oligo-dT primer. Specific transcripts were measured by real-time qRT-PCR with appropriate primers as described (6). An Applied Biosystems QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) was used to measure SYBR Green (Bio-Rad Laboratories) incorporation with the following protocol: 95C for 15 s, 40 cycles of 94C for 10 s, 60C for 30 s, and 72C for 30 s. Data acquisition was performed during a 72C extension step. Melting curve analysis was performed from 72 to 95C. The change in cycling threshold (Ct) method was used to analyze levels of transcripts. Data were normalized to the expression level of -ACTIN/-Actin except noted otherwise (e.g., normalized to the Gapdh expression level).

B cells were lysed in Laemmli buffer. Cell extracts containing equal amounts of protein (20 g) were fractionated through 10% SDSpolyacrylamide gel electrophoresis. The fractionated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad) overnight (30 V/90 mA) at 4C. After blocking and overnight incubation at 4C with mouse anti-AID mAb (clone ZA001; Invitrogen), mouse antiNF-B p65 mAb (clone D14E12; Cell Signaling Technology), rabbit antiAcetyl-(Lys310)NF-B p65 mAb (clone D2S3J; Cell Signaling Technology), rabbit antiAcetyl-DNMT1-K1127/K1129/K1131/K1133 Ab (A5595, ABclonal), rabbit anti-Sirt1 Ab (A11267, ABclonal), rabbit anti-Dnmt1 Ab (A-1700, Epigentek), or rat anti-actin mAb (clone AC-15, Sigma), the membranes were incubated with HRP-conjugated secondary Abs. After washing with TBSTween 20 (0.05%), bound HRP-conjugated mAbs or Abs were revealed using Western Lightning Plus-ECL reagents (PerkinElmer Life and Analytical Sciences). Densitometry was performed with Fiji software. All the Abs and mAbs used in the above experiments are listed in table S1.

DNA methylation was analyzed by bisulfite DNA conversion and Sanger or MiSeq sequencing. For bisulfite conversion, gDNA was treated with sodium bisulfite using the EpiTect Bisulfite Kit (Qiagen) according to the manufacturers instructions. Bisulfite-treated DNA was amplified by PCR using GoTaq Hot Start Polymerase (Promega). The primers for bisulfite sequencing PCR were designed using MethPrimer2 (www.urogene.org/cgi-bin/methprimer2/MethPrimer.cgi) and tagged with Illumina clustering adapters. PCR products were purified with a QIAquick PCR purification kit (Qiagen) and used directly for Sanger sequencing with a sequencing primer which reverse-complemented the adapter sequence. For MiSeq sequencing, the amplified library was tagged with barcodes for sample multiplexing, PCR-enriched, and annealed to the required Illumina clustering adapters. High-throughput 300-bp paired-end sequencing was performed by the UTHSCSA Genome Sequencing Facility using the Illumina MiSeq platform. DNA methylation was also analyzed by MeDIP using the MeDIP Kit (Active Motif) following the manufacturers instructions. Precipitated DNA was used as a template for qPCR analysis involving specific primers (table S1B).

ChIP assays were performed as previously described (6). B cells (1.0 107) were treated with formaldehyde (1.0%, v/v) for 10 min at 25C to cross-link chromatin. After quenching with 100 mM glycine (pH 8.0) and washing with cold PBS containing protease inhibitors (Roche), B cells were resuspended in lysis buffer [20 mM trisHCl, 200 mM NaCl, 2 mM EDTA, 0.1% (w/v) SDS, and protease inhibitors (pH 8.0)]. Chromatin was sonicated to yield DNA fragments (about 200 to 600 bp), precleared with Pierce Protein A beads (Thermo Fisher Scientific), and incubated with rabbit antiNF-B p65, antiAcetyl-(Lys310) NF-B p65 and anti-Sirt1 Abs (as above), rabbit anti-H3K9Ac/K14Ac (17-615, Millipore), rabbit anti-DNMT1 Ab (A-1700, EpiGentek), rabbit anti-Sirt1 Ab (A11267, ABclonal), mouse anti-Dnmt3l mAb (S117-9, StressMarq Biosciences), or control rabbit or mouse IgG with irrelevant specificities at 4C overnight. Immune complexes were precipitated by Protein A agarose beads, washed, and eluted [50 mM tris-HCl, 0.5% SDS, 200 mM NaCl, and proteinase K (100 g/ml) (pH 8.0)], followed by incubation at 65C for 4 hours. DNA was purified using a QIAquick PCR purification kit (Qiagen). The precipitated DNA was used as a template for qPCR analysis involving specific primers (table S1B).

Sirt1 coding region cDNA was amplified from unstimulated mouse B cells using the appropriate primers (table S1) and cloned into the pMIG retroviral expression vector. To generate the retrovirus, the pMIG vector encoding green fluorescent protein (GFP) only or the pMIG-Sirt1 vector encoding GFP and Sirt1 was used with the pCL-Eco retrovirus-packaging vector (Imgenex) to transfect human embryonic kidney293T cells by a Ca++ phosphate (ProFection Mammalian Transfection System, Promega). Viral supernatants were collected and used to transduce spleen B cells from C57BL/6 mice after a 12-hour LPS activation, as we reported (6). Transduced B cells were then stimulated with LPS plus IL-4 for 96 hours before analysis of GFP+ and/or IgG1+ B cells by flow cytometry. Dead (7-AAD+) cells were excluded from analysis.

NAD+ and NADH concentrations were measured using a fluorescence-based assay with Amplite Fluorimetric total NAD and NADH Assay Kit (Red Fluorescence, AAT Bioquest), following the manufactures instruction.

Differences in Ig titers, CSR, and RNA transcript expression were analyzed with the Students paired t test unless otherwise noted, assuming two-tailed distributions. Differences in the frequency of somatic point mutations were analyzed with 2 tests. The Excel (Microsoft) or Prism software was used for all statistical analyses and calculation of P values (*P < 0.05, **P < 0.01, and ***P < 0.001).

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We would like to thank G. Excalante for SLE blood samples, Z. Lai (UTHSCSA Genome Sequencing Facility) for high-throughput sequencing, and Y. Chen for bioinformatics analysis. We would also like to acknowledge the critical contribution of K. Gorena and M. Berton (UT Health Flow Cytometry core) for helping us on FACS analysis and cell sorting. Funding: This work was supported by NIH grants R01 AI105813, R01 AI079705, and T32 AI138944 and the Lupus Research Alliance Target Identification in Lupus Grant ALR 641363. H.G., T.S., and X.L. were supported by Xiangya Medical School, Central South University, Changsha, China, in the context of the Xiangya-UT Long School of Medicine San Antonio medical student visiting program. Author contributions: H.G., T.S., J.R.T., D.P.C., H.N.S., and X.L. performed experiments. Z.X. helped in experimental design. H.Z. designed and performed experiments, analyzed data, supervised the work, and wrote the manuscript. P.C. planned the study, designed the experiments, analyzed the data, supervised the work, and wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response - Science Advances

Heart health: A crowded, competitive, and niche market for supplements – Nutritional Outlook

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Heart health is a solid category where natural solutions have found fertile ground for the last several years. But it is also crowded and very generic. Thats why there is a lot of movement to be more specific about a heart health products benefits, as well as find new angles for innovation.

Offering a sobering view of the market, Julian Mellentin, director, New Nutrition Business, says, Heart health is arguably the most-crowded and competitive of all benefit platforms. The benefit of heart health is offered directly and indirectly by a massive array of products stretching across all categories. In heart health, whatever proposition you developno matter how good its scienceis in competition with a wealth of regular foods that consumers and the media perceive as heart healthy. These include oats, olive oil, dark chocolate, whole grains, almonds, pistachios, blueberries, and many, many others. This competitive reality has helped keep heart health brands in a niche.

Still, heart health remains one of the top reasons why overall supplement users take supplements. This reason is especially popular among supplement users aged 55+, with heart health being the third most popular reason for taking dietary supplements in this age bracket, says Nancy Weindruch, vice president, communications, Council for Responsible Nutrition (CRN; Washington, DC).

In the mass-market retail channel, the top three ingredients for heart health as of October 6, 2019, according to market researchers SPINS and IRI, were fish oil, psyllium, and CoQ10. However, the only categories of the top 10 ingredients listed in this market under cardiovascular health that posted gains compared to the previous year were psyllium (which grew 0.5%) and CoQ10 (ubiquinol; which grew 15%).

In the natural retail channel, the top three ingredients for heart health were CoQ10, fish oil, and garlic. Those posting gains in the natural channels cardiovascular category were garlic at 5%, red yeast rice at 1%, magnesium at 4%, and resveratrol at 9%.

Most top 10 ingredient categories across both channels were in the red, indicating a stagnating and/or declining market from previous years. That being the case, omega-3s and CoQ10 continue to hold the top positions in terms of market share.

Among consumers, according to CRNs 2019 Consumer Survey on Dietary Supplements, usage of omega-3/fatty acid supplements has remained steady over the past five years. Historically and currently, omega-3s are most popular in the 55+ age category, says CRNs Weindruch. Omega-3/fatty acids are in the top five most popular supplements taken by men. In fact, the number of men who take omega-3/fatty acid supplements has grown by four percentage points over the last year.

Mellentin also believes nutrients like omega-3s are among those likely to keep the heart health category afloat for years to come, among others.

Ingredients like walnuts or almonds have strong epidemiological data showing their cardio-protective effects, which helps to strengthen the naturally heart healthy beliefs around whole foods, he says. Ingredients like omega-3, beta-glucan, phytosterols, CoQ10, and lycopene dominate the heart health supplement category. It will be difficult for emerging ingredients to challenge ingredients like omega-3s, which have been studied since the 1970s.

Omega-3 Drugs Dominate Recent Headlines

The omega-3 market has had its share of ups and downs. In 2019, there was a mix of news related mostly to the publication of several high-profile studies. Indeed, REDUCE-IT1, VITAL2, and ASCEND3 were the studies that kept on giving when it came to headlines.

All published about a year ago, new findings generated from these studies continue to shine a light on omega-3s EPA and DHA, especially with regard to heart health.

Most recently, Amarins Vascepa EPA ethyl ester drug stunned the world in the REDUCE-IT1 study by showing its extraordinary ability to reduce the risk of cardiovascular (CV) events by 30%, an achievement a cholesterol medication could only dream of.

This prompted FDA in mid-December to expand label-claim approval for Vascepa for the secondary prevention of cardiovascular events, claiming that it would give patients with elevated triglycerides and other important risk factors, including heart disease, stroke, and diabetes, an adjunctive treatment option that can help decrease their risk of cardiovascular events.

However, the wave omega-3 drugs were riding abruptly crashed in January of this year when two other omega-3 drug makers, AstraZeneca and Acasti, reported the clinical trial results of Epanova (a fish-oil based EPA/DHA drug) and CaPre (a krill oilbased drug), respectively. The STRENGTH trial was halted because Epanova didnt show a likelihood of cutting LDL cholesterol when combined with a statin for patients at risk of heart disease due to high levels of LDL (or bad) cholesterol. At the same time, Acasti reported that its krill oil drug failed to sufficiently perform better than placebo in terms of reducing triglyceride levels.

In the meantime, although a full-scale direct-to-consumer marketing campaign for Vascepa is well underway, many analysts continue to question its future due to intellectual property concerns.

In light of these recent studies and reports, it is important to keep things in perspective. Fish, dietary supplements, and pharmaceutical omega-3s all have an important role to play in delivering omega-3s to support heart health. But at the end of the day, if health outcomes are the goal, then it will always come down to dose, regardless of how these omega-3s are delivered.

The Omega-3 Index: A Tool for Growth

Another aspect of heart health that will likely come into play for 2020 and well into the future is the importance of knowing ones Omega-3 Index. This is simply a measurement of the omega-3s EPA and DHA in red blood cell membranes, and it has the potential to hold more weight than measuring cholesterol as a risk factor for heart disease, according to William S. Harris, PhD, the inventor of the test.

Originally proposed more than 15 years ago by Drs. Harris and his colleague Clemens von Schacky, the Omega-3 Index has since been featured in more than 200 studies, including some of the largest in the world like the Framingham Heart Study and the Womens Health Initiative Memory study, both of which included thousands of subjects.

With a risk factor that can be easily modified (i.e., one just needs to consume more EPA and DHA to raise their Omega-3 Index), this test is particularly attractive to doctors who want to assess risk for their heart patients. Stakeholders hope that the Omega-3 Index will one day become the standard of care like a cholesterol or CRP test. Lending credence to the importance of measuring omega-3 levels was a statement4 released late last year by the International Society for the Study of Fatty Acids and Lipids (ISSFAL) relating to the importance of omega-3 blood testing in research studies.

What this basically means is that any study looking to assess the benefits of omega-3s is heavily encouraged to measure omega-3 levels before and during the study in order to better correlate health outcomes. Similarly, consumers and practitioners should establish a baseline omega-3 blood level (i.e., Omega-3 Index) before deciding where to go next when it comes to strategizing when and how to adopt supplemental heart health strategies.

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Heart health: A crowded, competitive, and niche market for supplements - Nutritional Outlook

I survived the coronavirus. It wasnt easy, but we can get through it – The Globe and Mail

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Illustration by Mary Kirkpatrick

I am a physician well on my way to recovery from COVID-19, the coronavirus, the same virus that is tearing apart families, communities, whole nations and our global economy. The fear of this viral terror is palpable.

I likely acquired the virus in the small ski town of Ischgl, Austria: not on the glorious powder-filled ski slopes but rather in the uproarious, festive, aprs-ski bars. When I began to have fevers and a mild cough a few days before flying home, I assumed I had a local flu. I remained housebound, and when flying home I wore a mask and washed my hands frequently.

By then news of Ischgl being a hotbed for coronavirus was in the media and I knew I had to get tested. I did, and then self-isolated with whatever advice and information I could organize from the Internet.

My fevers and chills and coughing had increased once I returned to Canada after four days of gradually getting better while in Europe, I got worse at home. The Public Health nurse called me confirming I had COVID-19 when I was already beginning to heal, five days after my return.

For some, COVID-19 feels like nothing. They have no symptoms, but may become healthy helpers who unwittingly become an unidentified source of transmission. For me, COVID-19 felt like a really bad flu. Night and day feverish sweats were drenching, my muscles ached, the coughing left me weary. For three days the cough was relentless although I was fortunately never truly short of breath. I lost five pounds in the first two days I was home. I had no appetite and felt nauseous.

I was also alone; my partner was still in Germany and my 15-year-old daughter was safely sequestered with her dad up the street.

At no point did I wonder about my own ability to survive what I was experiencing, but after three days of unchanged symptoms I realized how much of a physical toll this would take upon someone with much less robust health. I did plan to call 911 if I worsened, but mostly I dealt with my fear by realizing that my healthy friends and family were far more terrified of my illness than I was. Immobilized on my couch, reassuring others that I was going to be fine, I could see that I would be.

Seventeen days after symptoms began I was still in isolation but virtually symptom free. The public health department informed me that I could have gone out into the community 10 days after symptoms began if I were symptom free, while my extensive online research suggested that some people still shed the virus up to 20 days after initial symptoms. Since my province is no longer testing for recovery, even in confirmed COVID-19 cases, how can they know if I stopped shedding at five or 20 days? As the well-being of my daughter and community was at risk, I remained in isolation, erring on the side of caution.

Even as a physician, I could not access any of the medications that theoretically might decrease viral shedding. Instead, I added higher doses of vitamins D and C, zinc, quercetin, magnesium and extra antioxidants such as curcumin and resveratrol.

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What else did I do that helped? Lots and lots of sleep and rest. I only watched milder Netflix shows as I found violence and discord overwhelmed my nervous system. I drank endless fluids with sea salt to replace the electrolytes I lost in the sweats. I lived on chicken- and beef-broth soups. I made sure to eat some protein daily but oranges and green apples were all that tasted normal to me for two weeks. I drank pots of tea made from fresh lemon, ginger and honey. Then I made sure to get more rest and practised gratitude daily. Sunshine and fresh air helped immeasurably.

I know how simply lucky I am, and I have learned from my personal experience that many of us who acquire COVID-19 will be fine without accessing formal health care and by managing our symptoms at home. Where I have been most fortunate is in my community of friends and family who have dropped food and supplies at my doorstep. Many calls, texts and e-mails have kept me connected. I am grateful for the outpouring and of love and good wishes. My friend Val reminds me that, when you have a life changing experience, let it change your life. And so, I have become a COVID Ambassador: Teaching, reassuring, calming, instructing my network that this is survivable for most of us.

We can contribute to the communal good by dealing with our fears and our worries realistically, and leaving the health-care providers time, energy and limited equipment to those who are significantly short of breath with the virus or who develop secondary illnesses. Try not to judge others.

Even when I was coughing and sweating alone, praying my symptoms would not worsen, I marvelled at this amazing human body and mind, and its ability to tolerate, heal and grow strong from the challenges faced. I could have focused upon why I got sick but the greater miracle is how I got well and how we mostly stay well. I know that more lives will be tragically lost to this virus, but we also need to recognize and celebrate survival.

This global virus binds us in our humanity. When there is nothing else to do, call an elderly neighbour or someone self-isolating, buy someone some groceries, share your hoarded toilet paper, write, draw, laugh, create and talk to each other. There remains so much to be grateful for as we slow down and appreciate what we were previously too busy to notice. We are changing. We need to evolve. But most importantly we need to know we can thrive through this pandemic.

Dr. Anita Tannis lives in North Vancouver, B.C.

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I survived the coronavirus. It wasnt easy, but we can get through it - The Globe and Mail

12 Healthiest Dried Fruit Snacks Of 2020, Per Nutritionists – Women’s Health

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Jason Speakman

Dried fruit snacks are pretty much the adult version of Fruit Roll-Ups and Gushersand (shocking, I know) they're typically way healthier than those tongue-staining blue raspberry snacks of childhood.

Dried fruit can help you meet your fiber needsand it also provides a source of complex carbs, says nutritionist Jessica Cording, RD, author of The Little Book of Game-Changers.

Dietitian Keri Gans, RD, author of The Small Change Diet, agrees: Like fresh fruit, dried fruit provides plenty of vitamins and minerals." It's just, you know, dried. (Some snacks are freeze-dried, some are dried in a dehydrator machine, and others are even dried in the sun.)

But, like anything, dried fruit isnt perfect. Since dried fruit is condensed and its water is removed, you typically eat more of it and may consume many more calories than you realize, Gans says. The average portion size of dried fruit snacks is just about a quarter of a cup, which doesn't look like much because, well, it's not.

Going overboard isn't the only trap you can fall into with this stuff. Some companies add sugar and preservatives to make their dried fruit even sweeter and shelf-stable, Cording says. (People with sulfite sensitivities should look out for the preservative sulfur dioxide on ingredient lists.)

Though you can totally enjoy dried fruit snacks on their own, Cording recommends pairing them with protein and fat (like yogurt or nuts) to make them more satisfyingand to keep your blood sugar more stable.

The next time you get a hankering for the sweet fruit snacks of yesteryear, these healthy dried fruit snacks are nutritionist-approved and totally do the job (minus the blue tongue, of course).

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Best No-Sugar-Added Fruit Snack

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These apple chips are made with just one ingredient: apples. Yep,thats it. Bare alsobakes their dried fruit snacks, whichnever contain preservatives, something Cording appreciates.

Per serving: 60 calories, 0 g fat, 0 mg sodium, 14 g carbs, 2 g fiber, 11 g sugar, 0 g protein

Best Combo Dried Fruit Snack

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Bares apple and strawberry chip mix contains justapples and strawberries, which Gans likes. The delicious comboalso provides a little fiber.

Per serving: 170 calories, 0 g fat, 5 mg sodium, 42 g carbs, 6 g fiber, 30 g sugar, 1 g protein

Best Organic Option

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Peeled isnt messing around in the dried fruit department. Their gently-dried mango doesn't containpreservatives and is USDA certified organic (which Cording ishere for).

This mango snack is also gluten-free, vegan, and non-GMO Project verified.

Per 1.23 ounce serving: 110 cals, 0 g fat, 0 mg sodium, 25 g carbs, 2 g fiber, 18 g sugar, 2 g protein

Best On-The-Go Fruit Snack

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Sunsweet is practically synonymous with prunes, and theyve now packaged these little guys individually to prevent sticky fingers. Gans recommends grabbing a few and stashing them in your bag for later.

Per serving: 100 cals, 0 g fat, 0 mg sodium, 26 g carbs, 3 g fiber, 15 g sugar, 1 g protein

Best Feel-Good Snack

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Mavuno Harvests healthy dried fruit snacks are made with 100-percentfruit that's finely sliced and then rolled into bites for a nostalgic treat that Gans is a big fan of.

A cool bonus: The brand also helps support farms in one of the poorest regions on Earth. I love that the brand works with small farming cooperatives in rural Africa, Gans says.

Per serving: 171 calories, 2.25 g fat (1.85 g saturated fat), 0 mg sodium, 34.6 g carbs, 2.9 g fiber, 30.7 g sugar, 1.6 g protein

Best Fruit-Juice-Sweetened Snack

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Montmorency cherries (including the dried ones, Cording notes)are known for their health benefits, including their high antioxidant content and athletic performance-boosting abilities.

Since these cherries tend to be really tart, Eden Selecteds dried cherries are sweetened with organic apple juice concentrate.

Per serving: 130 calories, 0 g fat, 10 mg sodium, 32 g carbs, 2 g fiber, 24 g sugar (7 g added sugar), 1 g protein

Best Sun-Dried Snack

amazon.com

These cute little dates are made through an interesting process: Theyre picked, dried in the sun, steamed, and then packaged, which makes the finished product soft and surprisingly juicy.

They're also added sugar-freeand easy to tote around in theirresealable pouch, says Cording.

Per 1.25 ounce serving: 100 cals, 0 g fat, 0 mg sodium, 24 g carbs, 2 g fiber, 15 g sugar, 1 g protein

Best Sulfur-Free Snack

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Mulberries probably arent part of your regular fruit rotation, but the superfood deserves a spot in your diet.

Navitas Organics mulberries are packed with antioxidants (including resveratrol) and work well as a mildly-sweet topping for things like salads and oatmeal, Cording says.

Per serving: 110 calories, 1 g fat, 0 mg sodium, 25 g carbs, 2 g fiber, 21 g sugar, 1 g protein

Best Freeze-Dried Snack

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Freeze-drying fruit isnt necessarily better than drying it in the sun or through other methods, Cording says; its just different.

Crispy Greens freeze-dried cantaloupe has no sugar added, so you can munch on the sweetness in confidence. (Also, have you ever seen freeze-dried melon before?!)

Per serving: 35 calories, 0 g fat, 0 mg sodium, 8 g carbs, 1 g fiber, 6 g sugar, 1 g protein

Best Antioxidant Snack

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These blueberries have a totally different texture than the ones in your fridge: Thanks to the freeze-drying process they undergo, they're crunchy.

Like regular blueberries, though, these little guys are packed with antioxidants and are an awesome source of fiber.

Per serving: 80 calories, 0 g fat, 0 mg sodium, 19 g carbs, 4 g fiber, 12 g sugar, 0 g protein

Best Preservative-Free Classic Snack

amazon.com

Life Grape's vine-dried grapes are essentially raisins, but without the added sugar that comes with many varieties. They're alsonon-GMO, sulfur-free, and preservative-free.

Cording loves them on top of salads, grain dishes, breads, and baked goods.

Per serving: 80 calories, 0 g fat, 0 mg sodium, 22 g carbs, 1 g fiber, 17 g sugar, 1 g protein

Best Fiber-Packed Snack

amazon.com

These freeze-dried orange slices have a lot going for them. Theyre a phenomenal source of fiber (10 grams per serving!) and provide allof your daily recommended intake of vitamin C.

They're made withoranges and oranges alone,which is a major perk, Gans says.

Per serving: 130 calories, 0 g fat, 0 mg sodium, 12 g carbs, 10 g fiber, 12 g sugar, 0 g protein

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12 Healthiest Dried Fruit Snacks Of 2020, Per Nutritionists - Women's Health

Global USB Headsets Market Outlook 2020: Industry Trends, Analysis, Opportunities, Sales, Segmentation, Revenue and Forecast to 2026 – Cole of Duty

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The recently published research report entitled Global USB Headsets Market sheds light on critical aspects of the market like market size estimations, company and market best practices, market dynamics, market segmentation, competitive landscaping and benchmarking, opportunity analysis, economic forecasting, industry-specific technology solutions, guideline analysis, and in-depth benchmarking of vendor offerings. The report provides a clear understanding of the current and future scenarios and trends of the global USB Headsets market. The report tracks an array of important market-related aspects which can be listed as follows; the demand and supply chain, the competitive landscape, leading industries shares, profit margin, and profiles of leading companies of the global market.

This report takes into account the current and future impacts of COVID-19 on this industry and offers you an in-depth analysis of Global Trans Resveratrol Market.

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The section offers great insights such as market revenue and market share of the global USB Headsets market. The report explains a competitive edge over players competitors. Leading as well as prominent players of the global market are broadly studied on the basis of key factors. The report offers a comprehensive analysis and accurate statistics on sales by the player for the period 2015-2020. The report includes the forecasts, analysis, and discussion of important industry trends, market size, market share estimates, and profiles of the leading industry players. Company profile section of players such as Microsoft, Jabra, Sennheiser, Logitech, Plantronics, iMicro, SONY, KOSS, Sandberg, JPL, CANYON, VXi Corporation, Somic, Creative Technology,

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Global USB Headsets Market Outlook 2020: Industry Trends, Analysis, Opportunities, Sales, Segmentation, Revenue and Forecast to 2026 - Cole of Duty

Covid-19 Effect on Global Resveratrol Market-Industry Demand, Trends, and Forecast 2028 – Owned

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The Impact of COVID-19 on the Resveratrol Market Report Research Industry, 2020 report has been added to market.biz offering.

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Covid-19 Effect on Global Resveratrol Market-Industry Demand, Trends, and Forecast 2028 - Owned

Resveratrol may reverse cognitive ageing by ten years – NutraIngredients.com

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The Australian team also concludes that resveratrol consumption amongst postmenopausal women can also contribute to enhanced cognitive and cerebrovascular functions.

Our observed improvement in overall cognitive performance with resveratrol could potentially reverse cognitive ageing by up to 10 years says Dr Rachel Wong, a dementia research fellow with the National Health and Medical Research Council and the Australian Research Council (NHMRC-ARC).

We observed an improvement in overall cognitive performance which appeared to be due to improvements in processing speed and cognitive flexibility, which are critical to supporting executive function in the aging population.

Since 1990, the number of people globally living with dementia has more than doubled. In 2016, dementia mortality rates in women were almost twice that of men.

This may be partly attributable to the abrupt decline of oestrogen at menopause and the associated loss of its protective effects on cardiovascular and neural functions.

Resveratrol, found in grapes, berries and nuts, has been shown to improve endothelial vasodilator function in humans and improvements to verbal memory in older adults after six months of 200 mg of supplementation per day compared to placebo.

Further research points to doses of 250 milligrams (mg) and 500mg of resveratrol as contributing to increases of resting cerebral blood flow (CBF) in younger adults, although cognitive function was not affected.

Another study found that 75mg resveratrol, the lowest dose tested, was the most efficacious dose to acutely elicit global cerebral vasodilatation and improve performance of a sustained attention task in type 2 diabetes patients.

Along with colleagues from the University of South Australia, Dr Wong began randomising 129 volunteers to consume either two capsules containing 75mg of Evolvas Veri-te resveratrol ingredient daily or a matching placebo supplement for 12 months, followed by the alternate treatment for a further 12 months.

Results of the RESHAW (Resveratrol Supporting Healthy Aging in Women) clinical study found that compared to placebo, resveratrol improved overall cognitive performance, whilst weakening the decline in cerebrovascular responsiveness (CVR) to cognitive stimuli. The latter effect was associated with reduction of fasting blood glucose.

The evidence in this first report confirms our previous observations in both men and women that a regular low dose of resveratrol can sustain cerebrovascular function, which has helped to preserve cognitive function, particularly in the more elderly women, says Peter Howe, emeritus professor at the University of Newcastles Clinical Nutrition Research Centre.

Most importantly, it shows that these benefits are not short-lived but can be sustained with ongoing supplementation for at least 12 months.

The team, which also includes Dr Jay Jay Thaung Zaw, also from Australias University of Newcastle, think the results may be partly overseen by improvements to endothelium-dependent vasodilator function, which modulates CBF during times of demand.

We found improved resting blood flow velocities (BFV), stiffness in the cerebral vessels or pulsatility index (PI) and weakening of decline in neurovascular coupling following resveratrol supplementation, the paper discusses.

Allowing for normal ageing processes, cognitive decline and ultimately, dementia are linked to accelerated decline in resting CBF and CVR due to a decreased delivery of oxygen and nutrients in vulnerable brain regions such as the hippocampus.

Cerebral artery stiffness, marked by increased PI, is also associated with cognitive impairment and predicts the progression from mild cognitive impairment to dementia.

The study concludes that taking this evidence into account, the team think the improvements in resting BFV and PI with resveratrol highlights the ability of regular resveratrol supplementation to sustain cerebrovascular function, which may, in turn, preserve cognitive function in elderly women.

This publication from the team at the University of Newcastle highlights the benefits that Veri-te resveratrol can have on brain health, making resveratrol an essential ingredient for postmenopausal women and healthy aging,

This first peer-reviewed publication from the RESHAW clinical study provides the evidence that supplementation with Veri-te resveratrol should be a key strategy for the design and delivery of effective supplements that may attenuate the decline in brain health as women age and improve overall wellbeing post-menopause, says Clare Panchoo, VP, Health Ingredients, Evolva.

Source: Nutrients

Published online ahead of print: doi.org/10.3390/nu12030828

Sustained Cerebrovascular and Cognitive Benefits of Resveratrol in Postmenopausal Women.

Authors: Jay Jay Thaung Zaw et al.

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Resveratrol may reverse cognitive ageing by ten years - NutraIngredients.com

Eating soaked peanuts before breakfast can benefit health, help with weight loss: Know how – Times Now

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Eating soaked peanuts before breakfast can benefit health, help with weight loss: Know how  |  Photo Credit: Getty Images

New Delhi: Peanuts are a healthful food consumed widely across the world mostly as a snack item. They are rich in protein, fibre and healthy fats. Peanuts contain several essential nutrients that may offer major health benefits, including better weight loss and reduced risk of heart disease. But do you also know that eating soaked peanuts early morning can be so good for your health?

To learn more about how eating soaked peanuts can be good for health, we spoke to nutritionist Avni Kaul who reveals some significant benefits of the legume and how you can incorporate this superfood into your morning routine for optimal health.

Peanuts are high in protein, fibreand healthful fats. They also contain plenty of potassium, phosphorous, magnesium, and B vitamins along with other healthy nutrients. Besides these nutrients, peanuts contain beneficial plant compounds such as p-Coumaric acid, isoflavones, resveratrol, phytic acid, and phytosterols.

Some health benefits of eating soaked peanuts are:

Peanuts may help you lose weight or maintain weight. They contain various nutrients, including protein and insoluble dietary fibre, that make them a weight loss-friendly food.Research suggests that peanuts do not appear to contribute to weight gain despite being high in fat and calories. However, please note that there is no conclusive evidence that suggests peanuts are the best food for weight loss.

For instance, one small, 6-month study involving healthy women found that when other fat sources in a low-fat diet were replaced with peanuts, they lost about 3 kilos of weight. Similarly, another study found that healthy adults, who added 89 grams of peanuts to their daily diet for nearly 8 weeks, did not report as much weight as expected. However, these studies are all observational and, hence, the food does not guarantee weight loss.

Ideally, soaked peanuts should be consumed in the morning before breakfast. Peanuts are often associated with weight loss as they boost satiety. Peanuts are quite high in calories, so they shouldnt be eaten in excess. But eating them in moderation as part of a balanced diet may help you lose weight and improve health.

Disclaimer: Tips and suggestions mentioned in the article are for general information purpose only and should not be construed as professional medical advice. Always consult your doctor or a dietician before starting any fitness programme or making any changes to your diet.

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Global Resveratrol Market Projections , Future Opportunities Recorded for the Period until 2029 – Sound On Sound Fest

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Global Resveratrol market 2020 Report is a conceptual analysis of the market scenario. The report provides an in-depth analysis of Various geographies that govern the Market include the United States, Europe, Japan, China, Korea, India, and Middle East countries. This provides in-depth research on existing market trends. The past, present and forecast overview is presented in Resveratrol market report.

The report begins with the market overview, Resveratrol chain structure, former and current market size along with Resveratrol business opportunities in the coming years, a rise in technological innovation, supply demand and shortage, various drivers and restraining factors pull the environment. Furthermore, it depicts the production and consumption ratio of various topographical regions and the dominant market players contribution to market growth.

Ask & Request for sample report @ https://market.biz/report/global-resveratrol-market-icrw/134251/#requestforsample

The Global Resveratrol Market 2020 report Gives information on industry stats, research findings, and upcoming market Projections for development. The report delivers an in-depth analysis of the Resveratrol market, dynamics, updates, and market strategies. This study offers insightful and detailed information concerning end-users, new technologies, standardization, regulation, market projections and key Players operating in the Resveratrol Market and much more. Here the market is well broken down by the top manufacturer, end-users, and their application with their respective data. In addition, the report provides the Resveratrol statistical data in the form of graphs, tables, and pie charts format.

Top Reported Manufacturers:

DSM, Evolva, InterHealth, Maypro, Laurus Labs, JF-NATURAL, Great Forest Biomedical, Shaanxi Ciyuan Biotech, Chengdu Yazhong, Sabinsa, Changsha Huir Biological-tech, Xian Gaoyuan Bio-Chem, Xian Sinuote

The report provides the market size and forecast for the different segments and geographies for the period of 2015 to 2020

Market Key Types Segmented:

SyntheticPlant ExtractFermentation

Market Key Applications Segmented:

Dietary SupplementCosmeticFood and Beverage

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In this study, the years considered to estimate the market size of the market are as follows:

History Year: 2015-2019 | Base Year: 2019 | Estimated Year: 2020 | Forecast Year: 2020 to 2029

The examination goals of this report are:

Resveratrol Market break down and examination by worldwide limit, generation, esteem, utilization, status (2015-2020) and conjecture (2020-2029).

Focus on Resveratrol Market to examine the limit, creation, esteem, piece of the pie and improvement designs in the future.

Resveratrol Market worldwide SWOT investigation.

To describe and estimate Resveratrol Market by description, application, and locale.

To investigate Resveratrol Market key districts advertise a potential and favorable position, opportunity and test, restrictions and dangers.

To distinguish huge patterns and factors driving or hindering Resveratrol Market development.

To break down the open doors in Resveratrol Market for partners by recognizing the high development portions.

To intentionally split down each submarket concerning unique development progress and their commitment to Resveratrol Market.

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Coronavirus outbreak: Food to consume that help boost immunity to fight COVID-19 – Mid-day

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With the global spread of coronavirus at a lightning pace, it is essential to have a strong immune system to combat it effectively. As of now, there is no medical cure to this pandemic. However, individuals can still ensure critical hygiene and have a healthy food consumption pattern to keep our immune system in pink of health and to avoid infections. Fortunately, nature has given us an abundance of food that strengthens the guts and keeps one healthy.

The best way to protect oneself is to follow simple habits like sleeping well, exercising, and finding the best ways to reduce stress for a more effective immune system. Food influences our body, and hence the immune system, to respond and therefore it is important to help the immune system fight against coronavirus.

Appended below are some of the best foods that can help strengthen our immunity system:

1. Garlic

Garlic contains an important compound called Allicin which helps in fighting against germs and boosts the immune system.

Ways to consume:

Add it in your soups and curries Crush it and eat it raw

2. Ginger Ginger is a strong antioxidant that has been proven to boost the immune system. It contains many vitamins, some of which are magnesium, iron, zinc, and calcium. Ginger also helps to kill viruses that cause colds and has been said to combat chills and fever.

Ways to consume:

Add it to your coffee/tea Mix it with honey and eat it Prepare a concoction of ginger and star anise and drink it

3. Resveratrol

Resveratrol is a polyphenol compound found in certain plants and is known to have antioxidant properties. Foods like peanuts, pistachios, strawberries, cranberries, grapes etc. contain resveratrol.

Ways to consume:

Munch on the food items mentioned above

4. Load your diet with Vitamin CFoods rich in Vitamin C such as amla, peppers, broccoli, apple, kiwi, lemons, oranges etc. are found to be helpful in adding the immune system.

Ways to consume:

Prepare a fruit salad. Eat them whole and not in juice form.

5. Herbs Tulsi, oregano and rosemary are good for boosting the immune system

Ways to consume:

Eat 1-2 tulsi leaves daily Add tulsi leaves to tea or boil the water with tulsi leaves Add oregano to your salads Add rosemary oil to your dough or to the oil used for frying Inhale them

6. Zinc and SeleniumFoods rich in zinc and selenium such as almonds, cashew nuts (unsalted), sunflower seeds, pumpkin seeds are effective immunity boosters.

Ways to consume:

Nibble on them Topped cooked veggies with them

7. Probiotic and Fermented FoodsThese foods maintain and promote the growth of gut bacteria and in turn boost ones immunity.

Ways to consume:

Include dosas, idlis and dhoklas in your diet Include curd/yogurt to build good bacteria

It is indeed important to stay safe and with the help of thorough cooking. Including these food items in your diet helps boost immunity while giving high importance to nutrition. Food thus plays the biggest role in the prevention of a viral infection.

By Snehal Nanivadekar , Assistant General Manager QHSE at Elior India

Catch up on all the latest Crime, National, International and Hatke news here. Also, download the new mid-day Android and iOS apps to get latest updates

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Resveratrol supports cognitive health in aging populations, says groundbreaking RESHAW study – Nutritional Outlook

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Evolva (Reinach, Switzerland) has announced the first published findings1 from the Resveratrol Supporting Healthy Aging in Women (RESHAW) clinical study, conducted by the University of Newcastle in Australia and funded by the countrys National Health and Medical Research Council. This two-year clinical trial is the largest and longest-running trial of its kind, investigating the impact of Evolvas branded Veri-te resveratrol on cognitive performance. Interim results were announced at SupplySide West 2019.

In the double-blind, placebo-controlled crossover design, 125 post-menopausal women were assigned to either receive 75 mg of resveratrol or placebo twice daily for 12 months, then switched interventions for the next 12 months. Results showed performance on Pattern Comparison Speed tests improved in the resveratrol group, compared to placebo, which the researchers believe is due to the significant improvements in two cognitive domains, processing speed and cognitive flexibility. Both of these domains are part of executive function, requiring speed, perceptual reasoning, and accuracy to accomplish tasks. Executive function declines with age, with processing speed and cognitive flexibility being among the first cognitive changes reported in healthy older adults.

The researchers hypothesize that the cognitive improvements may be due, in part, to sustained improvement of endothelium-dependent vasodilator function, which modulates cerebral blood flow during times of demand. Subjects taking resveratrol saw significant improvements in basal blood flow velocities (systolic, mean, and diastolic), and pulsatility index, which is a measure of the stiffness in cerebral vessels, compared to placebo. Neurovascular coupling capacity also saw improvement, especially in response to tests of cognitive flexibility. This is important because cognitive decline and ultimately dementia are linked to accelerated decline in resting cerebral blood flow and cerebrovascular function due to decreased delivery of oxygen and nutrients to vulnerable parts of the brain such as the hippocampus.

This momentous publication from the team at the University of Newcastle highlights the benefits that Veri-te resveratrol can have on brain health, making resveratrol an essential ingredient for postmenopausal women and healthy aging, said Clare Panchoo, vice president, Health Ingredients, Evolva, in a press release. This first peer-reviewed publication from the RESHAW clinical study provides the evidence that supplementation with Veri-te resveratrol should be a key strategy for the design and delivery of effective supplements that may attenuate the decline in brain health as women age and improve overall wellbeing post menopause.

Our customers have been anticipating the results from RESHAW and have already initiated new product development based on this first publication; we further expect the positive outcomes to drive sales of Veri-te resveratrol globally, adds Panchoo.

References:

1. Thaung Zaw JJ et al. Sustained Cerebrovascular and Cognitive Benefits of Resveratrol in Postmenopausal Women. Nutrients, vol. 12, no. 3 (2020): 828

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Resveratrol supports cognitive health in aging populations, says groundbreaking RESHAW study - Nutritional Outlook

Errata And Addendum To Our Herman Trend Alert On The Coronavirus – NewsReleaseWire.com

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Wednesday, March 04, 2020

ERRATA AND ADDENDUM TO OUR HERMAN TREND ALERT ON THE CORONAVIRUS

I need to set the record straight on a few fronts: first, and probably most important, I have discovered that SARS and MERS were also corona viruses and thus, it would be more appropriate to call this disease that will soon be a pandemic, COVID-19. Additionally, the source which I though was snakes, was, in fact, the same as SARS---bats. Apparently, people in China eat bat meat. This winter due to high temperatures, the bats did not have the same amount of hibernation time. Therefore, their immune systems were not as strong, and they succumbed to COVID-19. When people ate the meat, they became ill with the disease. Now for the good news, according to my sources, eating bioflavonoids and dark chocolate, drinking black tea, taking 200mg of Resveratrol daily, and avoiding adult-dosages of aspirin will help you avoid contracting this dreaded disease. Finally, the mortality rate for this disease appears to have actually been between three and four percent, but when once effective treatmen tsare in place, that number will plummet. Keep your immune system strong and you will avoid COVID-19.

Joyce L. Gioia, CMC, CSP

Austin, TX

336-210-3548

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Errata And Addendum To Our Herman Trend Alert On The Coronavirus - NewsReleaseWire.com

Inflammation: The Silent Killer (and what to do about it) PART TWO – WholeFoods Magazine

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Last month we covered the basics of inflammation and the difference between acute inflammationwhen you bang your knee hard against a tableand chronic inflammation, which flies beneath the pain radar but can essentially destroy your body. I also covered a list of foods known to be high in anti-inflammatories. (To read part one, click HERE).

But supplements are also a powerful ally in the war against chronic inflammation

Many supplements have been clinically shown to lower important markers of inflammation. Heres a short guide to the most important ones.

Omega-3: The Wellness Molecule

Omega-3s are among the most anti-inflammatory substances on the planet and should be part of everyones supplement program. At this point, so much research exists to confirm this that it doesnt seem necessary to reiterate it here. Omega-3s (especially from fish) lower triglycerides, blood pressure andmost especiallyinflammation.

Omega-7: Bold New Discovery, Researched at the Cleveland Clinic

Omega-7 is a fatty acid found in cold-water fish, as well as macadamia nuts and sea buckthorn berries. It has beneficial effects on metabolic syndrome, diabetes, weight, triglycerides and insulin resistance. A study at the prestigious Cleveland Clinic found that omega-7 lowered C-Reactive Proteina test for systemic inflammationby 44%. (Barleans has a superb omega-7 product called Heart Remedy that contains the exact dose (210) of omega-7 used in the Cleveland Clinic study.)

Magnesium: The Great Relaxer

Magnesium supplements are a must for those who want to protect their heart. Magnesium lowers blood pressure, helps control blood sugar and relaxes the lining of the blood vessels. And almost all dietary surveys show that Americans arent getting nearly enough.

Magnesium dilates the arteries, thus reducing blood pressure and making it far easier for the heart to pump blood and for the blood to flow freely. Because blood pressure can contribute to inflammatory lesions in the vascular walls, its a good idea to include it in an anti-inflammatory supplement program.

Curcumin: The New Superstar of Supplements

This extract from the Indian spice turmeric has multiple benefits, not the least of which is that its highly anti-inflammatory. Scientific research has demonstrated its anti-inflammatory, anti-oxidant, anti-thrombotic, and cardiovascular-protective effects (1). It also reduces oxidized LDL cholesterol (2). Curcumin in general isnt well absorbed, so I like supplements with either C-3 or BCM-95 curcumin, as those two forms have been shown to be the most absorbable.

Resveratrol: Not just for anti-aging

Resveratrol is the ingredient in red wine thats best known for its anti-aging activity. It helps protect the arteries, improves their elasticity, inhibits blood clots, and lowers both oxidized LDL and blood pressure (3). Not a bad rsum! Its both a strong anti-oxidant and a strong anti-inflammatory, inhibiting a number of inflammatory enzymes that can contribute to heart disease. It also inhibits the ability of certain molecules to stick to the walls of the artery where they can take up residence and contribute to inflammation (4). The recommended dose is 200 mg a day of trans-resveratrol, the active component of resveratrol. Read labels carefully: Many wont tell you how much resveratrol is of the trans- variety.

MSM

MSM is methyl sulfonyl-methane, a naturally anti-inflammatory that blocks the transmission of impulses in nerve fibers that carry pain signals. Its long had the reputation of being great for joint health and the pain of arthritis. It has a high sulfur content, which makes sense when you realize that for centuries people have visited sulfur-rich hot springs for muscle aches and pains. The biochemical precursor to MSMdimethyl sulfoxidehas been studied extensively for pain and reduction of inflammation for years.

Boswellia

The herb boswellia is well-known for its ability to lower inflammation. 150 mg three times a day (or the equivalent) is recommended for two or three months. It has been used effectively in combination with ginger, turmeric, and ashwagandha, and has proved beneficial for inflammation and pain associated with osteoarthritis and rheumatoid arthritis (7). You can often find it at the store combined with curcumin.

GLA

GLA (gamma linolenic acid) is an anti-inflammatory omega-6 fatty acid (8) and the active ingredient in evening primrose oil, borage oil, and black currant oil. (The average 1,000 mg dose of evening primrose oil contains about 100 mg of GLA.) GLA seems to have a synergistic effect with the long-chain fatty acid EPA (eicosapentaenoic acid) and should be given together with omega-3 fish oil. Try to get 1,000 mg a day of GLA (in conjunction with 2-4 grams of combined EPA-DHA from fish oil).

The bottom line

Inflammation is likely to emerge as the health concern of the decade, but the good news is that you can do a great deal to combat it. Healthy, whole foods, plenty of omega-3 fats, and a minimum of sugar in your diet is a great place to start!

PS: Stress is inflammatory. As are toxic emotions. So a real anti-inflammation lifestyle needs to include some attention on those variables as well! Pay attention to your feelingsthey make a difference in both your mental and your physical health.

REFERENCES

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Inflammation: The Silent Killer (and what to do about it) PART TWO - WholeFoods Magazine

Cheers to these top vino for National Drink Wine Day – ABC News

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Happy National Drink Wine Day!

From dedicated Twitter and Facebook pages to people sharing their favorite varietal and vintage on social media, today is the day to raise a glass of wine.

A family toasts with wine in an undated stock photo.

Whether you're are looking to try a new bottle, better understand the health benefits or simply share a fun GIF, Feb. 18 is dedicated to spreading "the love" of wine.

Even celebrities like Kerry Washington, whose "Scandal" character Olivia Pope was known for her heavy pours, chimed in to celebrate the trending day.

"Wine Enthusiast" managing editor Lauren Buzzeo shared some expertise from choosing the right wine and understanding your palate to her personal favorite go-to bottle.

Wine is subjective and depends on personal tastes and experiences, but Buzzeo said for those new to wine, she would suggest starting with "fresh, fruit-forward selections with moderate acidity and low tannins, in the case of red wines."

"I generally start on the lower end of the tannic spectrum, because I find thats often what people have a hard time with initially," she explained of the bitter compounds that can leave an astringent feeling in the mouth. "Ill reach for a bright California Pinot Noir or Beaujolais, which is made from a grape called Gamay."

Wine bottles sit on a wine rack in an undated stock photo.

Buzzeo's advice is to start with what you like and fine tune based on your palate's preferences.

When it comes to white wine, Buzzeo said go for "a Spanish Albario or New World Sauvignon Blanc to start, and tune the choice from there. If youre looking for more weight, veer towards Chardonnay or Viognier."

One of the most common wine myths that Buzzeo helped dispel in honor of National Drink Wine Day is the broad generalization when people say, "I dont like Chardonnay."

"The grape is so versatile and easily influenced by both terroir and winemaker influence, that final wines from around the world run a huge spectrum," she explained. "If you think you dont like Chardonnay, instead pinpoint what about Chardonnay you dont like, because Im pretty sure theres a counterpoint to that bad experience you had and it might be amazingly delicious."

Once someone uncorks that wine they've carefully selected, the preservation game is on if you don't plan on finishing the bottle in one sitting.

Luckily, there are great gadgets on the market that Buzzeo said can help keep a bottle of wine fresh for days, weeks or even months.

"If youre constantly feeling like youre always looking for one glass and then letting the rest of the bottle go to waste before you get to enjoy more of it, invest in a Coravin," she said, "Its a fabulous system that will allow you to dispense pours from a sealed bottle for months without serious alteration to the wines profile."

She also suggested another innovative method, "trying out a wine club that specializes in single-serve pours; not only will you eliminate the possibility of any wine getting spoiled, but youll also get to try lots of different varieties from many regions to really discover a range of vinous possibilities."

Buzzeo said her go-to selection for National Drink Wine Day, would be an aged Barolo, in honor of her dad, "who introduced me to the wonderful world of wine and had a strong passion for Italian classics."

While the nationally recognized day can certainly seem "silly" at first, Buzzeo said simply, it helps raise awareness. "From Tacos to Tempranillo, who doesnt want an excuse to dive deeper and further appreciate something they either love or could stand to learn more about"

You don't need to be a sommelier to enjoy a glass of vino and various studies have shown that drinking red wine in moderation can be a healthy part of one's diet.

Dr. Melanie R. Graber, a resident with the ABC News Medical Unit, explained that people who choose to consume light to moderate amounts of alcohol may experience benefits in their health.

Red wine contains antioxidants called polyphenols, which, according to the Mayo Clinic, can "help protect the lining of blood vessels in your heart."

One polyphenol found in red wine, resveratrol, may help "prevent damage to blood vessels, reduce low-density lipoprotein (LDL) cholesterol (the "bad" cholesterol) and prevent blood clots," according to the Mayo Clinic.

A person pours red wine in an undated stock photo.

The USDA's 2015-2020 Dietary Guidelines for Americans defines moderate drinking as up to one drink per day for women and two drinks per day for men, with one drink listed as 8 to 20 grams of alcohol.

"Moderate alcohol consumption is thought to reduce risk of heart attack by increasing sensitivity to insulin, decreasing inflammation and decreasing blood clotting activity," Graber said.

While studies have been done into the benefits of red wine as it relates to reduced cardiovascular risk, the American Heart Association doesn't recommended that people who do not currently drink start drinking wine or alcohol for that sole purpose.

Various peer-reviewed medical literature on evidence-based medicine has shown that trials have had contradictory findings on coronary heart disease and it is unclear if wine is more cardio-protective than other alcohol types.

While there are some potential heart-healthy associations with drinking red wine and alcohol, drinking too much can increase the risk of liver and pancreas diseases, certain types of cancer, stroke, weight gain and other side effects.

So whether you reach for a biodynamic red from South America, chardonnay from Burgundy or a Napa Valley Cabernet, raise a glass today and enjoy responsibly.

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Cheers to these top vino for National Drink Wine Day - ABC News

The Best Serums to Brighten and Refresh Dull, Tired Skin – gearpatrol.com

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Heres the truth about serum: Its the biggest secret to having younger-looking, radiant skin.

You know all the other steps to staying healthyand showing it: wearing SPF, and applying retinol. You stay hydrated and eat healthy foods. You get lots of exercise and plenty of sleep. But, are you applying serums? They can give your skin some of the most immediate and impactful benefits.

Serum seeps into all three layers of your skin to work its magic at deeper levels than moisturizer ever could. Those moisturizing creams merely sit atop your skin. Theyre equally important, and shield skin from toxins, but they do little to reverse dark spots and smooth wrinkles. Serums, on the other hand, can correct hyperpigmentation and puffy eyes.

Serum brightens your skin complexion, and can preserve moisture levels in ways that moisturizers never could (funny, given its name). No two serums are alike in terms of ingredients and overall benefits, but almost all of them brighten your skin and smooth complexion. Here are seven of our favorite serums; apply them to clean, dried skin before moisturizers, SPFs, retinol or night creams.

If your skin suffers from redness or discoloration, then look for a corrective serum. SkinCeuticals gel serum uses thyme and cucumber extract to soothe irritation, as well as mulberry extract to even skin tone and gently hydrate the skin. If most moisturizers make your skin break out, then this is a gentle and top-notch moisturizing alternative.

Vitamin C is one of the best ingredients you can apply to skin for brighter, more even complexion. Glossiers serum is packed with it, along with magnesium, for an instant pick-me-up for tired or dull skin. Use it daily to preserve a fresh face, and to get ahead of the inevitable hangovers or early mornings.

The skin around your eyes is more sensitive than the rest of your face, and is much thinner, too hence why you quickly show signs of tiredness. (Its fluid buildup from a lack of rest, shining through your skin.) So, target the eyes with a special-blend serum that tightens and brightens at the same time. In OSEAs eye serum, hyaluronic acid preserves firmness and moisture levels, grape-seed-derived resveratrol shields this thin skin from toxins, and algae extract plumps and nourishes tired eyes (and alert eyes, if youre plenty rested already everyone benefits). It also prevents and helps reverse fine lines and other signs of aging.

Some serums are best worn during the day those that focus on hydration and pure brightening. Others, like corrective and exfoliating serums, are often best suited for bedtime so that they can sync with your bodys circadian rhythm to boost cellular turnover (and in turn guarantee that you wake up looking rested and more youthful). However, some serums cover both bases: Brickells night serum is ultra-hydrating so much so that its impact is maximized by wearing it overnight. Its loaded with plant stem cells, hyaluronic acid and vitamin C, and promises to magnify the benefits of a full nights sleep. Your friends and colleagues will notice the difference within days. (Assuming you arent pairing it with a lovely binge-drinking or chain-smoking habit.)

Theres an entire family of serums that focuses on exfoliation, by using ingredients that dissolve dead skin cells and reduce hyperpigmentation (dark spots and discoloration). Herbivores serum does so with alpha- and beta-hydroxy acids, all of which are naturally derived, despite their sciencey-sounding names. Theyre extracted from things like willow bark and sugar maple, and in turn leave skin bright, while minimizing pore size and smoothing skin texture.

Its hard to categorize Caudelies skin-brightening serum, since it uses the brands proprietary blend of ingredients (Viniferine, derived from grape vines) but they seem to deliver on their promise to brighten skin 62 times better than Vitamin C. All around the globe, this is what Caudelies spa customers pay top dollar for, and you can enjoy the same complexion-smoothing effects for less than a buck a day (assuming you just use a couple drops daily).

If your skin texture needs attention just as much as your skin tone, then look for a corrective and smoothing serum. This one from PCA Skin is pumped with peptides, and it waxes poetic on your skin to undo signs of aging (like the appearance of fine lines, wrinkles, dark spots and acne spots). It also helps skin retain moisture thanks to the inclusion of hyaluronic acid, rice bran extract, squalene, ensuring firmness and brightness.

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2019 Review: Resveratrol Market Growth Analysis and Market Sizing – The Market Journal

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This intelligence report provides a comprehensive analysis of the Global Resveratrol Market. This includes Investigation of past progress, ongoing market scenarios, and future prospects. Data True to market on the products, strategies and market share of leading companies of this particular market are mentioned. Its a 360-degree overview of the global markets competitive landscape. The report further predicts the size and valuation of the global market during the forecast period.

Some of the key players profiled in the study are:

DSM (Netherlands), Naturex (France), Evolva (Switzerland), Sabinsa (United States), InterHealth (United States), Maypro (United States), Chengdu Yazhong (China), JF-NATURAL (China), Jiangxi Hengxiang (China) and Great Forest Biomedical (China)

Increasing geriatric population is driving the market of resveratrol market. Resveratrol belongs to the stilbenoids group which contains phenol rings connected to each other. It is found mostly in the skin or seeds of grapes and berries which are used in the preparation of red wine. Resveratrol contains antioxidant which helps lowering the blood pressure. In also decreases cholesterol and increases the HDL. In addition, it protects the brain from any damage and prevents or treats different types of cancer.

Get Latest insights about acute features of the market (Free Sample Report + All Related Graphs & Charts) @ https://www.advancemarketanalytics.com/sample-report/64172-global-resveratrol-market

Market Drivers

Market Trend

Opportunities

Each segment and sub-segment is analyzed in the research report. The competitive landscape of the market has been elaborated by studying a number of factors such as the best manufacturers, prices and revenues. Global Resveratrol Market is accessible to readers in a logical, wise format. Driving and restraining factors are listed in this study report to help you understand the positive and negative aspects in front of your business.

This study mainly helps understand which market segments or Region or Country they should focus in coming years to channelize their efforts and investments to maximize growth and profitability. The report presents the market competitive landscape and a consistent in depth analysis of the major vendor/key players in the market.Furthermore, the years considered for the study are as follows:Historical year 2013-2017Base year 2018Forecast period** 2019 to 2025 [** unless otherwise stated]

**Moreover, it will also include the opportunities available in micro markets for stakeholders to invest, detailed analysis of competitive landscape and product services of key players.

Enquire for customization in Report @ https://www.advancemarketanalytics.com/enquiry-before-buy/64172-global-resveratrol-market

The Global Resveratrol segments and Market Data Break Down are illuminated below:The Study Explore the Product Types of Resveratrol Market: Natural, Artificial

Key Applications/end-users of Global Resveratrol Market: Dietary supplements, Pharmaceutical, Personal care products, Others

Region Included are: North America, Europe, Asia Pacific, Oceania, South America, Middle East & Africa

Country Level Break-Up: United States, Canada, Mexico, Brazil, Argentina, Colombia, Chile, South Africa, Nigeria, Tunisia, Morocco, Germany, United Kingdom (UK), the Netherlands, Spain, Italy, Belgium, Austria, Turkey, Russia, France, Poland, Israel, United Arab Emirates, Qatar, Saudi Arabia, China, Japan, Taiwan, South Korea, Singapore, India, Australia and New Zealand etc.

Objectives of the Study

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Strategic Points Covered in Table of Content of Global Resveratrol Market:

Chapter 1: Introduction, market driving force product Objective of Study and Research Scope the Resveratrol market

Chapter 2: Exclusive Summary the basic information of the Resveratrol Market.

Chapter 3: Displaying the Market Dynamics- Drivers, Trends and Challenges of the Resveratrol

Chapter 4: Presenting the Resveratrol Market Factor Analysis Porters Five Forces, Supply/Value Chain, PESTEL analysis, Market Entropy, Patent/Trademark Analysis.

Chapter 5: Displaying the by Type, End User and Region 2013-2018

Chapter 6: Evaluating the leading manufacturers of the Resveratrol market which consists of its Competitive Landscape, Peer Group Analysis, BCG Matrix & Company Profile

Chapter 7: To evaluate the market by segments, by countries and by manufacturers with revenue share and sales by key countries in these various regions.

Chapter 8& 9: Displaying the Appendix, Methodology and Data Source

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Key questions answered

Definitively, this report will give you an unmistakable perspective on every single reality of the market without a need to allude to some other research report or an information source. Our report will give all of you the realities about the past, present, and eventual fate of the concerned Market.

Thanks for reading this article; you can also get individual chapter wise section or region wise report version like North America, Europe or Asia.

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2019 Review: Resveratrol Market Growth Analysis and Market Sizing - The Market Journal

How Your Diet And Self-Care Habits Affect Your Skin, From A Derm – Women’s Health

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When conventional treatments arent working, its time to consider not just the magic elixirs that youre putting on your face but also whats going on in your diet and daily life. Ahead, some tips from Jeanette Jacknin, MD, a San Diegobased dermatologist who approaches treatment from a holistic perspective.

Sorry to break it to you, but everything youve heard about quitting dairy and sweets for better skin is legit, Dr. Jacknin notes. She also says to avoid fried food, soda, alcohol, and anything else youd consider junk. A Mediterranean or whole foodsbased diet, with a lot of fresh vegetables and fish like salmon, is ideal, she says. Several studies show low-glycemic-load diets improve acne.

Theres no one-size-fits-all method for calming down and cutting stress, so Dr. Jacknin works with her patients to see what gets them in their Zen zone. Is it a HIIT class with your friends? A solo hike in nature? A morning meditation sesh? Find your outlet, but know that adequate sleep, regular exercise, and speaking to a mental health professional are pretty much a 10/10 across the board.

Dr. Jacknin combines botanical actives and Rx ones, depending on severity and preference. More intense cases may require a prescription, but on milder spots, she suggests applying 5 percent tea tree oila natural antibacterialtwice a day. Also clutch? Ingredients like green tea and resveratrol (an antioxidant found in grape skin), which are known to reduce inflammation. Try Este Lauder Advanced Night Repair Intense Reset Concentrate ($80, esteelauder.com).

Este Lauder Advanced Night Repair Intense Reset Concentrate

$80.00

This article originally appeared in the March 2020 issue of Women's Health.

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How Your Diet And Self-Care Habits Affect Your Skin, From A Derm - Women's Health

Type 2 diabetes: Sprinkle this fruit on your breakfast to lower your blood sugar – Sky Statement

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Type 2 diabetes means your body either resists the effects of insulin a hormone that regulates the movement of sugar into your cells or doesnt produce enough insulin to maintain normal blood sugar levels. Uncontrolled blood sugar levels may not pose serious health risks initially, but over time, the condition can lead to life-threatening complications such as heart disease and stroke. Fortunately, making healthy lifestyle tweaks can compensate for the poor insulin production in the body, keeping the threats at bay.

Diet forms an essential part of blood sugar management and although you do not have to cut out any food groups per se, it is strongly advised to reduce your intake of certain items known to cause a spike in blood sugar.

Foods that contain high amounts of carbohydrate spell trouble for blood sugar management because carbohydrate is broken down into glucose relatively quickly and therefore has a more pronounced effect on blood sugar levels than either fat or protein.

In fact, for optimal blood sugar management, a recent study recommends eating a diet consisting of low carb options and an increased share of protein and fat.

The study, conducted by Bispebjerg Hospital in collaboration with other partners, found diet with a reduced carbohydrate content and an increased share of protein and fat improves the patients ability to regulate his or her blood sugar levels independent of weight loss.

READ MORE:Type 2 diabetes: Eating this winter spice could lower blood sugar

Type 2 diabetes: Grapes have been shown to decrease blood sugar (Image: Getty Images )

A helpful way to distinguish between high-carb and low-carb food items is to follow the Glycemic Index (GI) a relative ranking of carbohydrate in foods according to how they affect blood glucose levels.

Carbohydrates with a low GI value are more slowly digested, absorbed and metabolised and cause a lower and slower rise in blood glucose and therefore insulin levels.

The GI ranking is usually found on the front of food packets to help people identify low-carb options when shopping.

One food that ranks low on the GI index is grapes and the fruit has been shown to decrease blood sugar levels.

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In a 16-week study in 38 men, those who took 20 grams of grape extract per day experienced decreased blood sugar levels, compared to a control group.

Additionally, resveratrol a compound found in grapes has been shown to increase insulin sensitivity, which may improve your bodys ability to use glucose and hence lower blood sugar levels.

Furthermore, research reveals that resveratrol increases the number of glucose receptors on cell membranes, which may have a beneficial effect on blood sugar.

Exercise also lowers blood sugar in two key ways.

Type 2 diabetes: Losing weight without trying to is a warning sign (Image: Getty Images )

As the American Diabetes Association explained, insulin sensitivity is increased, so your muscle cells are better able to use any available insulin to take up glucose during and after activity.

In addition, when your muscles contract during activity, your cells are able to take up glucose and use it for energy whether insulin is available or not.

The NHS recommends aim for at least 2.5 hours of activity a week to lower blood sugar.

You can be active anywhere as long as what youre doing gets you out of breath.

This could be:

Type 2 diabetes: A low-carb diet with a higher share of fat and protein can lower blood sugar (Image: Getty Images )

According to the NHS, many people have type 2 diabetes without realising because symptoms do not necessarily make you feel unwell.

Symptoms include:

You should speak to your GP if you have any of the symptoms of type 2 diabetes or youre worried you may have a higher risk of getting it, advises the health body.

It added: The earlier diabetes is diagnosed and treatment started, the better. Early treatment reduces your risk of other health problems.

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Type 2 diabetes: Sprinkle this fruit on your breakfast to lower your blood sugar - Sky Statement

Food for thought: What to eat on exam day – University of Virginia The Cavalier Daily

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What you consume before a test can impact your energy and stamina, your ability to focus and your mood. Our brains use up to 20 percent of our energy every day. Heres what you should eat during finals season to keep your brain going.

Dinner: Salmon, broccoli and potatoes

Its well-known that getting enough sleep the night before an exam is important for test performance. The food you eat the night before is as well. Try this salmon, broccoli and potato dish for dinner to boost your memory, increase your nutrient-intake and sleep better the night before your big test.

Salmon is rich in omega-3 fatty acids, which are incredibly important for your body and brain health. Sixty percent of your brain is fat, and half of that fat is omega-3. These fatty acids have countless health benefits, but among them are eased anxiety, improved mental disorders and better sleep.

Broccoli is high in vitamin K, which has been linked to better brain memory. One cup of broccoli delivers more than 100 percent of the Reference Daily Intake of vitamin K.

Potatoes are a good source of energy and supply of nutrients. Although they arent directly linked to brain health, potatoes provide our bodies with glucose for energy. The brain needs a steady supply of energy to function sufficiently. Therefore, potatoes are a healthy option to give your body the energy it needs, as well as additional fiber, calcium, iron and B vitamins.

Dessert: Anything dark chocolate

You will want to avoid a sugar high before going to bed, but if you choose wisely, your dessert can actually benefit your health.

Dark chocolate contains brain boosting compounds such as flavonoids, caffeine and antioxidants. Flavonoids gather in the areas of the brain that deal with learning and memory, and have the potential to promote memory, learning and cognitive function. Try making these dark chocolate brownie-cookies for a healthy dessert. Itll give you enough sugar to satisfy your sweet tooth and boost your brain.

Breakfast: Egg, avocado and peanut butter-banana toast

As the saying goes, breakfast is the most important meal of the day. This is because breakfast resupplies our brain with glucose energy after going several hours without food. Heres why you should eat egg and avocado toast, peanut butter and banana toast or both on the morning of your final.

Eggs are rich in nutrients that boost your mood, memory and energy levels. They are a good source of choline and B vitamins. The body uses choline to create acetylcholine, a neurotransmitter that is tied to memory, mood and muscle control. B vitamins are involved in synthesizing brain chemicals, converting glucose into energy and preventing age-related mental decline. Vitamin B deficiencies are related to fatigue, memory problems, muscle weakness and more.

Avocados lower blood pressure and heighten cognitive ability. High blood pressure is linked to a decline in cognitive ability, and unsaturated fats have been shown to reduce blood pressure. Avocados are a sufficient source of healthy unsaturated fat, and therefore, eating an avocado can correlate with increased brain function.

Bananas can improve memory and brain function. They are rich in magnesium, fiber and fructose. Magnesium deficiency is related to fatigue, weakness and increased anxiety. Increasing magnesium intake will fight fatigue and reduce anxiety. The fiber in a banana slows down the breakdown and release of its sugars from fructose into the bloodstream. Thus, bananas are a key lasting source of energy.

Peanut butter and peanuts are a great source of unsaturated fats, which give you energy. They also contain resveratrol, which improves blood flow to the brain by as much as 30 percent, helping you concentrate. Peanuts also make you feel good. They contain polyphenols, which are linked to mood enhancement.

Whole grain bread contains valuable nutrients that refined grains dont. Whole grains contain all parts of the grain kernel, which holds all of the key nutrients that are removed in the refining process for the flour used in white bread. This eliminates all of the vitamins, minerals, healthy fats, protein and fiber. The bran and fiber slow the breakdown of starch, creating a longer and more steady supply of energy.

Lunch: Quinoa and veggie salad

The key to lunch is making sure you eat enough so that youre not hungry but not too much that you feel sleepy. This quinoa and veggie salad is a delicious, healthy option that will give you enough energy to finally take on your final. It makes six servings, meaning you can adjust your portion size to what works for you, and you will have leftovers for other exam days.

Quinoa, like potatoes, is a healthy starch and great source of energy. Its extremely high in protein, containing sufficient amounts of all essential amino acids. Therefore, quinoa is an excellent protein source for vegetarians and vegans.

Spinach contains nutrients that benefit memory and mental focus. One-half a cup of spinach contains five times the amount of vitamin K you need in a day. Like the broccoli you had for dinner last night, the Vitamin K in spinach is linked to better brain memory.

Corn and beans will supply you with energy and vitamins crucial to the brain and nervous system. Corn is an energy enhancer because it contains complex carbohydrates that get released more slowly than other carbohydrates. Beans are packed with B vitamins, which help make the neurotransmitters that pass signals between nerves in the brain.

If you want to add meat to this dish, add chicken or fish. Avoid turkey, as it contains L-tryptophan which produces sleep-promoting melatonin and serotonin. An addition of turmeric to this dish, measured to taste, could also be very beneficial. Its an antioxidant with the active ingredient curcumin, which has been shown to directly enter and benefit brain cells. It has also been linked to improved memory and eased depression.

Take a study break, and cook a meal! Itll relieve stress and refuel your brain. As the semester comes to a close, hopefully these dishes give you the energy you need to make it through your final endeavors.

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Food for thought: What to eat on exam day - University of Virginia The Cavalier Daily

Reducing the effects of inflammation – The Guardian

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Dr Edward Bullmore (Inflammation is the new frontier in public health, Journal, 20 January) is right in saying that inflammation seems to be everywhere. In fact, many of the diseases of ageing have been linked to the rise in three inflammatory cytokines that all of us secrete over the age of about 65. These include osteoarthritis, rheumatoid arthritis, cardiovascular disease, some cancers, macular degeneration, muscle loss leading to frailty, dementia and type 2 diabetes. Interestingly, those living into their late 90s and longer secrete an anti-inflammatory cytokine. So these individuals are able to do naturally what Bullmore wants to do with drugs.

But Id like to suggest a further way of reducing the effect of inflammatory cytokines. A wide body of published research details the anti-inflammatory effects of various foods. These include ginger and curcumin (from turmeric), green tea, omega 3 fatty acid (from oily fish and flaxseed), resveratrol (from grapes, berries, peanuts and red wine) and punicalagin (from pomegranate). These natural anti-inflammatory substances are also available as food supplements.

Bullmore mentioned that depression could also be treated by anti-inflammatory drugs that crossed the blood-brain barrier. This is interesting because there is a body of research that indicates curcumin crosses the blood-brain barrier and can be effective in depression.Dr Colin SutherlandKendal, Cumbria

I think Edward Bullmores article overstates the case for depression being linked medically to inflammation. Last year, a bout of arthritis in my right knee left me barely able to walk for a month. I was mostly confined to my room, unable to sleep because of the pain, and gave up all healthy outdoor activities. Was I depressed? You bet! Strangely, the depression lifted magically once the pain subsided and I was mobile again.John ODwyerSteeple Claydon, Buckinghamshire

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Reducing the effects of inflammation - The Guardian