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Women’s march from #NRA2DOJ July 14-15 Fairfax VA to Washinton DC

Women's March from #NRA2DOJ - July 14 & 15. Fairfax, VA to Washington, DC

This Fourth of July, we celebrated the power of our voice. This has been a year of patriotic resistance, as we collectively struggle against the attacks on our fundamental rights. We are fueled by the spirit of love and liberation that we experienced on January 21st, when millions of us came together for the largest coordinated protest in U.S. history, grounded in the nonviolent principles of the Civil Rights movement.

We continue to resist efforts to silence and intimidate us from exercising the power of our voice. When many of us called on the NRA to take action on behalf of Philando Castile — in line with their stated mission and purpose — the response was not only silence but the resurfacing of an NRA ad featuring right wing propagandists and deeply divisive, “us versus them” language, appearing to be a direct endorsement of violence against women, our families and our communities for exercising our constitutional right to protest.

In response, Women’s March co-president Tamika Mallory penned an open letter to the NRA calling for the ad to be removed and an apology to be issued to the American public for the false and inflammatory rhetoric. Instead of distancing themselves from the vitriol, the NRA responded by releasing a new video attacking Tamika and other leaders personally and doubling down on the implied call to arms. This is the kind of incendiary speech that leads to acts of hate and violence, and it is unequivocally meant to create a chilling effect on communities speaking up and using the power of our collective voice.

On July 14th & 15th, Women’s March and partners will mobilize a mass demonstration, again grounded in the principles of Kingian nonviolence, to denounce the false and intimidating rhetoric of hatred and send a clear message that our movement will proudly and bravely continue to strive for the respect of the civil and human rights of all people.

We can’t do it without you. Help us raise the funds necessary to support the marchers each step of the way. Your contribution goes towards water, food, security and other logistics to ensure an effective mobilization.

Donate to support the Women’s March from #NRA2DOJ

A week after our nation celebrated the values of liberty and justice, let’s come together to say that our movement is stronger than fear. Please spread the word about the Women’s March from #NRA2DOJ on July 14th & 15th, 2017, and let the NRA know that we are the majority, who believe in equity, justice and safety for all.

In community,
Women’s March

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p53 , resveratrol , pancreatic cancer and apoptosis

Intrigued by pancreatic cancer, infection and environmental toxins, I stumble into this topic from Wiki. One of my clients died of stomach , esophagal and pancreatic cancer. Previous to that , he had shingles and other infections. Her immune system became weaker during the last 5 years before his death. He disregarded stomach pains and takes TUMs regularly and other anti-viral med for his shingles. His autopsy revealed stomach cancer.

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The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes ¹⁰. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity.

The p53 upregulated modulator of apoptosis (PUMA) also known as Bcl-2-binding component 3 (BBC3), is a pro-apoptotic protein, member of the Bcl-2 protein family.^[3]^[4] In humans, the Bcl-2-binding component 3 protein is encoded by the BBC3 gene.^[3]^[4]

The expression of PUMA is regulated by the tumor suppressor p53. PUMA is involved in p53-dependent and -independent apoptosis induced by a variety of signals, and is regulated by transcription factors, not by post-translational modifications. After activation, PUMA interacts with antiapoptotic Bcl-2 family members, thus freeing Bax and/or Bak which are then able to signal apoptosis to the mitochondria. Following mitochondrial dysfunction, the caspase cascade is activated ultimately leading to cell death.^[5]

Structure

The PUMA protein is part of the BH3-only subgroup of Bcl-2 family proteins. This group of proteins only share sequence similarity in the BH3 domain, which is required for interactions with Bcl-2-like proteins, such as Bcl-2and Bcl-xL.^[3] Structural analysis has shown that PUMA directly binds to antiapoptotic Bcl-2 family proteins via an amphiphatic α-helical structure which is formed by the BH3 domain.^[6] The mitochondrial localization of PUMA is dictated by a hydrophobic domain on its C-terminal portion.^[7] No posttranslational modification of PUMA has been discovered yet.^[5]

Mechanism of action

Biochemical studies have shown that PUMA interacts with antiapoptotic Bcl-2 family members such as Bcl-xL, Bcl-2, Mcl-1, Bcl-w, and A1, inhibiting their interaction with the proapoptotic molecules, Bax and Bak. When the inhibition of these is lifted, they result in the translocation of Bax and activation of mitochondrial dysfunctionresulting in release of mitochondrial apoptogenic proteins cytochrome c, SMAC, and apoptosis-inducing factor(AIF) leading to caspase activation and cell death.^[3]

Because PUMA has high affinity for binding to Bcl-2 family members, another hypothesis is that PUMA directly activates Bax and/or Bak and through Bax multimerization triggers mitochondrial translocation and with it induces apoptosis.^[8]^[9] Various studies have shown though, that PUMA does not rely on direct interaction with Bax/Bak to induce apoptosis.^[10]^[11]

Regulation

Induction

The majority of PUMA induced apoptosis occurs through activation of the tumor suppressor protein p53. p53 is activated by survival signals such as glucose deprivation^[12] and increases expression levels of PUMA. This increase in PUMA levels induces apoptosis through mitochondrial dysfunction. p53, and with it PUMA, is activated due to DNA damage caused by a variety of genotoxic agents. Other agents that induce p53 dependent apoptosis are neurotoxins,^[13]^[14] proteasome inhibitors,^[15] microtubule poisons,^[16] and transcription inhibitors.^[17] PUMA apoptosis may also be induced independently of p53 activation by other stimuli, such as oncogenic stress^[18]^[19] growth factor and/or cytokine withdrawal and kinase inhibition,^[4]^[20]^[21] ER stress, altered redox status,^[22]^[23] ischemia,^[8]^[24] immunemodulation,^[25]^[26] and infection.^[5]^[27]

Degradation

PUMA levels are downregulated through the activation of caspase-3 and a protease inhibited by the serpase inhibitor N-tosyl-L-phenylalanine chloromethyl ketone, in response to signals such as the cytokine TGFβ, the death effector TRAIL or chemical drugs such as anisomycin.^[28] PUMA protein is degraded in a proteasome dependent manner and its degradation is regulated by phosphorylation at a conserved serine residue at position 10.^[29]

Role in cancer

Several studies have shown that PUMA function is affected or absent in cancer cells. Additionally, many human tumors contain p53 mutations,^[30] which results in no induction of PUMA, even after DNA damage induced through irradiation or chemotherapy drugs.^[31] Other cancers, which exhibit overexpression of antiapotptic Bcl-2family proteins, counteract and overpower PUMA-induced apoptosis.^[32] Even though PUMA function is compromised in most cancer cells, it does not appear that genetic inactivation of PUMA is a direct target of cancer.^[33]^[34]^[35] Many cancers do exhibit p53 gene mutations, making gene therapies that target this gene ^{[clarification needed]} impossible, but an alternate pathway may be to focus on therapeutic to target PUMA and induce apoptosis in cancer cells. Animal studies have shown that PUMA does play a role in tumor suppression, but lack of PUMA activity alone does not translate to spontaneous formation of malignancies.^[36]^[37]^[38]^[39]^[40]Inhibiting PUMA induced apoptosis may be an interesting target for reducing the side effects of cancer treatments, such as chemotherapy, which induce apoptosis in rapidly dividing healthy cells in addition to rapidly dividing cancer cells.^[5]

PUMA can also function as an indicator of p53 mutations. Many cancers exhibit mutations in the p53 gene, but this mutation can only be detected through extensive DNA sequencing. Studies have shown that cells with p53 mutations have significantly lower levels of PUMA, making it a good candidate for a protein marker of p53 mutations, providing a simpler method for testing for p53 mutations.^[41]

Cancer therapeutics

Therapeutic agents targeting PUMA for cancer patients are emerging. PUMA inducers target cancer or tumor cells, while PUMA inhibitors can be targeted to normal, healthy cells to help alleviate the undesired side effects of chemo and radiation therapy.^[5]

Cancer treatments

Research has shown that increased PUMA expression with or without chemotherapy or irradiation is highly toxic to cancer cells, specifically lung,^[42] head and neck,^[43]esophagus,^[44] melanoma,^[45] malignant glioma,^[46] gastric glands,^[47] breast^[48] and prostate.^[49] In addition, studies have shown that PUMA adenovirus seems to induce apoptosis more so than p53 adenovirus.^[42]^[43]^[44] This is beneficial in combating cancers that inhibit p53 activation and therefore indirectly decrease PUMA expression levels.^[5]

Resveratrol, a plant-derived stilbenoid, is currently under investigation as a cancer treatment. Resveratrol acts to inhibit and decrease expression of antiapoptotic Bcl-2family members while also increasing p53 expression. The combination of these two mechanisms leads to apoptosis via activation of PUMA, Noxa and other proapoptotic proteins, resulting in mitochondrial dysfunction

The loss of SETD8 triggers cellular senescence

The SETD8/PR-Set7 Methyltransferase Functions as a Barrier to Prevent Senescence-Associated Metabolic Remodeling

DOI: http://dx.doi.org/10.1016/j.celrep.2017.02.021

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Figure 1

The Loss of SETD8/PR-Set7 Induces Senescence

(A) Schematic representation of senescence models used in this study. SA-β-Gal staining of growing, oncogene-induced senescence (OIS), and replicative senescence (RS) cells are shown. Numbers indicate the percentage of SA-β-Gal-positive cells (each n > 300 cells). Scale bars, 100 μm.

(B) Western blot analysis of SETD8 and p16^INK4A in growing, OIS, and RS cells.

(D) EdU incorporation assay on day 3 of Ctr- or SETD8-KD cells. EdU-positive and EdU-negative cells were distinguished by measuring the fluorescence intensity of EdU-labeled DNA in each cell (each n > 1,600 cells).

(E) SA-β-Gal staining on days 3–15 of Ctr- or SETD8-KD (each n > 300 cells). Scale bars, 100 μm.

(F) SAHF on days 3–15 of Ctr- or SETD8-KD (each n > 300 cells). Scale bars, 10 μm.

(G) Western blot analysis of SETD8, p16^INK4A, and p21^CDKN1A on days 3 and 6 of Ctr- or SETD8-KD.

(H) Growth curves for DMSO-treated or the SETD8 inhibitor UNC0379-treated IMR-90 cells.

(I) SA-β-Gal staining on day 6 of DMSO or UNC0379 treatment (each n > 300 cells). Scale bars, 100 μm.

(J) SAHF on day 6 of DMSO or UNC0379 treatment (each n > 300 cells). Scale bars, 10 μm.

Values are means ± SD (n = 3). p values were calculated using the Student’s t test (^∗p < 0.05, ^∗∗p < 0.01).

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Highlights

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SETD8 protein is downregulated in senescent cells
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The loss of SETD8 triggers cellular senescence
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SETD8 represses p16^INK4A and ribosome-associated genes through H4K20 methylation
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SETD8 regulates the structure and function of nucleoli and mitochondria

Summary

Cellular senescence is an irreversible growth arrest that contributes to development, tumor suppression, and age-related conditions. Senescent cells show active metabolism compared with proliferating cells, but the underlying mechanisms remain unclear. Here we show that the SETD8/PR-Set7 methyltransferase, which catalyzes mono-methylation of histone H4 at lysine 20 (H4K20me1), suppresses nucleolar and mitochondrial activities to prevent cellular senescence. SETD8 protein was selectively downregulated in both oncogene-induced and replicative senescence. Inhibition of SETD8 alone was sufficient to trigger senescence. Under these states, the expression of genes encoding ribosomal proteins (RPs) and ribosomal RNAs as well as the cyclin-dependent kinase (CDK) inhibitor p16^INK4A was increased, with a corresponding reduction of H4K20me1 at each locus. As a result, the loss of SETD8 concurrently stimulated nucleolar function and retinoblastoma protein-mediated mitochondrial metabolism. In conclusion, our data demonstrate that SETD8 acts as a barrier to prevent cellular senescence through chromatin-mediated regulation of senescence-associated metabolic remodeling.

Introduction

Cellular senescence is induced by various cellular stresses such as oncogene expression, telomere attrition, and genome-scale perturbation of chromatin. It is characterized by irreversible cell cycle arrest, senescence-associated β-galactosidase (SA-β-Gal) activity, and the senescence-associated secretory phenotype (SASP), as well as alterations of gene expression and chromatin (Benayoun et al., 2015, Campisi and d’Adda di Fagagna, 2007, Kuilman et al., 2010). These changes in senescent cells contribute to tumor suppression, tissue repair, and developmental processes, as well as age-related deterioration of tissue functions in vivo (Baker et al., 2016, Muñoz-Espín and Serrano, 2014, van Deursen, 2014). The senescent cells also undergo metabolic remodeling as indicated by enlarged cell size and increased protein content. Various metabolic pathways, including protein synthesis and degradation, autophagy, glycolysis, and mitochondrial oxidative phosphorylation (OXPHOS), are essential for the establishment of senescence (Salama et al., 2014, Wiley and Campisi, 2016). However, it is not clear how senescent cells remodel their metabolic status in combination with other senescence-associated features.

Chromatin-modifying factors play a fundamental role in gene regulation and are involved in DNA methylation, histone modification, and the formation of higher-order chromatin structures. The epigenomic landscapes in senescent cells differ from those of proliferating cells (Chandra et al., 2012, Chicas et al., 2012, Criscione et al., 2016, Cruickshanks et al., 2013, Hirosue et al., 2012, O’Sullivan et al., 2010, Shah et al., 2013), indicating that chromatin regulators play an important role in establishing and maintaining the senescent state. Growing evidence suggests that there is a reciprocal relationship between epigenetic regulation and cellular metabolism (Gut and Verdin, 2013, Hino et al., 2012, Hino et al., 2013). Most chromatin-modifying enzymes use substrates or cofactors derived from various metabolites (such as S-adenosylmethionine for many methyltransferases), while biochemical reactions depend on coordinated expression of many enzyme-encoding genes in metabolic pathways (Desvergne et al., 2006). So far, we do not understand how epigenetic and metabolic mechanisms cooperate to establish cellular senescence.

SETD8, also known as PR-Set7 or SET8, is a nucleosome-specific methyltransferase that is responsible for mono-methylation of histone H4 lysine 20 (H4K20me1) (Nishioka et al., 2002). SETD8 is involved in various genomic functions including DNA replication, mitosis, DNA repair, and gene expression via H4K20 methylation (Beck et al., 2012, Jørgensen et al., 2013). The protein levels of SETD8 are precisely controlled through proteasomal degradation, resulting in the lowest level during the S phase and the highest level during mitosis, which ensures proper cell cycle progression. However, the precise role of SETD8 in gene expression is rather confusing. Genome-wide profiling indicated that H4K20me1 is predominantly enriched downstream of the transcription start site of actively transcribed genes (Barski et al., 2007, Beck et al., 2012). Indeed, the levels of H4K20me1 in gene bodies are positively correlated with gene expression levels during cell differentiation (Cui et al., 2009). In contrast, loss-of-function analysis of SETD8 suggested that enrichment of H4K20me1 represses transcription of target genes regardless of their basal expression levels (Congdon et al., 2010, Kalakonda et al., 2008, Karachentsev et al., 2005). In addition, depletion of either of the H4K20me1 demethylases PHF8 or LSD1n resulted in reduced gene expression and elevated H4K20me1 levels at their respective target genes (Liu et al., 2010, Qi et al., 2010, Wang et al., 2015a). The repressive function of H4K20me1 is at least partially mediated by a polycomb protein, L3MBTL1, which binds this mark and compacts nucleosomes (Kalakonda et al., 2008, Trojer et al., 2007). Thus, it is conceivable that H4K20me1 is a transcriptionally repressive mark at actively transcribed genes. These studies suggest that SETD8 plays a fundamental role in gene expression as well as cell proliferation, but the role of SETD8 in cellular senescence remains unknown.

In this report, we show that SETD8 suppresses nucleolar and mitochondrial activities to prevent cellular senescence. SETD8 is downregulated in senescent cells induced by oncogenic and replicative stresses. Consistently, depletion of SETD8 directly induced senescence in cultured human fibroblasts. In the senescent state, the H4K20me1 landscape was largely remodeled at gene loci, including genes encoding ribosomal proteins (RPs) and RNAs, as well as p16^INK4A, resulting in nucleolar and mitochondrial coactivation. Our data shed light on the evidence that SETD8 links the epigenomic dynamics to energy metabolism during cellular senescence.

Results

SETD8 Is Commonly Downregulated in Senescent Cells

To explore a specific chromatin regulator involved in cellular senescence, we used two established senescence models: oncogene-induced senescence (OIS) and replicative senescence (RS) (Figure 1A). OIS cells were prepared by expression of 4-hydroxytamoxifen (4-OHT)-inducible Ras (H-RasV12) for 6–8 days in IMR-90 ER:Ras or MRC5 ER:Ras human fibroblast cells. For RS, IMR-90 cells were repeatedly passaged for 10–12 weeks (shown by late passage). These models expressed senescence markers including SA-β-Gal and reduced incorporation of the thymidine analog 5-ethynyl-2’-deoxyuridine (EdU) (Figures 1A and S1A). We found that levels of SETD8 protein were specifically decreased in both OIS and RS cells, compared with growing cells (Figure 1B). Time-course analysis revealed that SETD8 was steeply decreased on and after day 4 during establishment of OIS (Figure S1B). qRT-PCR analysis indicated that levels of SETD8 mRNA did not significantly change in either OIS or RS cells (Figure S1C). By contrast, the OIS cells treated with the proteasome inhibitor MG-132 resulted in increased levels of SETD8 protein (Figure S1D). There were two SETD8 bands of ∼45 kDa, which could be post-translationally modified or processed. These data indicate that downregulation of SETD8 is a hallmark of the senescent state, probably due to selective proteasomal degradation.

Loss of SETD8 Induces Senescence in Human Fibroblasts

To elucidate the role of SETD8 during senescent processes, we performed specific knockdown (KD) of SETD8 in IMR-90 cells with three independent small interfering RNAs (siRNAs). Compared with the control (Ctr), the proliferation of SETD8-KD cells was reduced within 2 days of siRNA treatment (Figures 1C and 1D and S2A). Cell cycle analysis revealed that the population of cells in both the S and G2/M phases were increased in SETD8-KD cells (Figure S2B). After synchronization by double thymidine block, SETD8-KD cells showed delayed progression through the G2/M phase (Figures S2C and S2D), in agreement with the role of SETD8 in mitotic progression (Karachentsev et al., 2005). Unexpectedly, we found that SETD8-depleted cells exhibited SA-β-Gal marking and senescence-associated heterochromatic foci (SAHF), as well as enlarged cell size, beginning at KD day 3 (Figures 1E and 1F and S2E). Nearly all treated cells were SA-β-Gal positive after 9 days of siRNA treatment, and this state was maintained without additional siRNA treatment (Figure 1E and S2F). Western blot analysis revealed that the cyclin-dependent kinase inhibitors p16^INK4A and p21^CDKN1A were also increased in SETD8-KD cells (Figure 1G).

To further examine the functional implication of SETD8 in senescence, we used the selective SETD8 inhibitor UNC0379, which clearly reduced the levels of H4K20me1 in a dose-dependent manner (Figure S2G) (Ma et al., 2014). The proliferation of UNC0379-treated cells was significantly reduced (Figure 1H), while SA-β-Gal and SAHF appeared in a dose-dependent manner (Figures 1I and 1J). In another human fibroblast cell line, MRC5 ER:Ras, SETD8 inhibition also significantly increased SA-β-Gal and SAHF without oncogene activation (Figures S2H–S2K). Collectively, loss of SETD8 induces growth arrest and senescence in human fibroblasts.

SETD8 Represses RP and Senescence-Associated Genes

To investigate how SETD8 depletion induces senescence, we performed genome-wide microarray analyses of Ctr and SETD8-KD IMR-90 cells at 24 hr, as confirmed by western blot analysis (Figure S3A). Gene set enrichment analysis (GSEA) using the KEGG platform revealed that 12 gene sets were significantly upregulated by SETD8-KD (Figure S3B), while no gene sets were downregulated. These data support that SETD8 is involved in transcriptional repression of the gene sets (Congdon et al., 2010, Kalakonda et al., 2008, Karachentsev et al., 2005). Among the upregulated gene sets, the ribosome, the p53 signaling pathway, and the cytokine-cytokine receptor interaction could be associated with senescence and/or metabolic activities (Figure 2A). Notably, most of the RP gene probes as well as other highly expressed gene probes (those with a signal intensity > 20,000 in Ctr-KD cells, which was in the top 1.5% of probes tested) were concurrently upregulated by SETD8-KD (Figures 2B and 2C and S3C–S3E). We confirmed the upregulation of a set of RP genes by qRT-PCR in SETD8-KD cells (Figure 2D). Although the ratio of upregulation was relatively small, the global increase of the highly expressed RPgenes could affect the ribosome biogenesis and protein synthesis in SETD8-KD cells (as shown later). In contrast, expression of the mitochondrial RP genes was comparable between Ctr- and SETD8-KD cells (Figure S3F). We then confirmed upregulation of senescence markers such as p15^INK4B, p16^INK4A, p21^CDKN1A, and interleukin (IL)-8 on day 1 and later of SETD8-KD (Figures 2E and S3G and S3H). Taken together, loss of SETD8 upregulates many genes, including a set of the RPgenes and senescence-associated genes in human fibroblasts.

SETD8 Regulates RP and Senescence-Associated Genes via H4K20 Mono-methylation

SETD8 is capable of regulating genes on its own by adding H4K20me1. To identify genes specifically targeted by SETD8, we examined the genome-wide H4K20me1 distribution in adult human dermal fibroblasts (NHDF-Ad cells) using ENCODE chromatin immunoprecipitation sequencing (ChIP-seq) datasets. Peak analysis using the SICER algorithm identified 8,967 genomic regions as H4K20me1-enriched islands, of which 94% (8,416 of 8,967) were deposited on intragenic regions of RefSeq-annotated genes. Functional annotation clustering analysis using Database for Annotation, Visualization, and Integrated Discovery (DAVID) revealed that H4K20me1 was preferentially enriched at a set of the RP genes (p = 6.12E-15; false discovery rate [FDR] = 8.10E-14) (Figures 3A, S4, and S5A). In fact, among the 86 RP genes detected in IMR-90 cells (Figure 2C), 83% (71 of 86) were enriched for H4K20me1 and 69% (59 of 86) were also upregulated by SETD8-KD (Figure 3A). In addition, p16^INK4A and p21^CDKN1A were enriched for H4K20me1 and were upregulated by SETD8-KD (Figures 2E, 3A, and S4).

Next, we tested whether loss of SETD8 affects the levels of H4K20me1. As shown in Figure 3B, global levels of H4K20me1 were decreased 23%–40% at 24 hr after SETD8-KD without changing the cell cycle distribution (Figures S5B and S5C). Consistent with previous reports, the basal levels of H4K20me1 were low at the S phase and high at the G2/M phase, compared with the G1 phase (Jørgensen et al., 2013), but depletion of SETD8 significantly decreased H4K20me1 levels at all cell cycle stages (Figure S5D). Thus, the overall reduction in H4K20me1 levels was not due to specific cell cycle stages.

We then performed ChIP to assess the levels of H4K20me1 at RP gene loci at 24 hr after SETD8-KD. RPL5, RPS24, RPL27A, and RPS18 were chosen as representative genes upregulated by SETD8-KD (Figure 2D). Interestingly, H4K20me1 was highly enriched at the intragenic regions of the RP genes, compared with the promoter regions, and depletion of SETD8 decreased gene-body H4K20me1 levels by 17%–31% (Figures 3C and S5E). In contrast, the levels of H4K20me3 did not change at these loci. Recently, it was reported that deposition of H4K20me1 at gene bodies is associated with repression of transcriptional elongation (Wang et al., 2015a). Consistent with this, we found that the levels of H3K36me3, a transcriptional elongation mark, were increased at gene bodies of most RP genes in SETD8-KD cells (Figures 3D and S5F). In addition, p21^CDKN1A and p16^INK4A genes were also enriched for H4K20me1 at gene bodies, and loss of SETD8 decreased H4K20me1 levels at these loci, together with an increase of H3K36me3 (Figures 3E and 3F and S5G and S5H). Peak analysis of H4K20me1 in three human cell lines, HeLa-S3, H1-human embryonic stem cells (hESC), and NHDF-Ad, indicated that enrichment of H4K20me1 at RP genes is conserved among different cell types (Figures S5I and S5J). Taken together, these results suggest that SETD8 is required for H4K20me1 at RP genes, p16^INK4A and p21^CDKN1A, and that depletion of SETD8 dampens H4K20me1-associated transcriptional repression of these genes.

SETD8 Regulates Ribosomal RNA Genes and Nucleolar Function via H4K20me1

The global upregulation of RP genes and enlarged cell size in SETD8-KD cells suggest that SETD8 depletion activates ribosome biogenesis during the establishment of senescence (Figures 2 and S2E). Ribosome biogenesis is tightly associated with cell growth and protein synthesis, especially due to the coordinate synthesis of RP proteins and ribosomal RNAs (rRNAs). Several hundred copies of rRNA genes are transcribed, and abundant rRNAs are processed by specific RPs at multiple steps, resulting in rRNA-protein assembly at the nucleolus (Thomson et al., 2013). Notably, as rRNA synthesis is a critical step in ribosome biogenesis, the rate of rRNA transcription is finely tuned by various signaling pathways and epigenetic mechanisms (Grummt and Längst, 2013, Moss et al., 2007). Interestingly, we found that rRNA gene bodies were modestly enriched for H4K20me1, and loss of SETD8 reduced 41%–62% of H4K20me1 levels, while H4K20me3 was rather stable at these loci (Figure 4A). In addition, the observed decrease of H4K20me1 coincided with increased deposition of H3K36me3 at gene bodies (Figure 4B), suggesting that SETD8 is involved in epigenetic regulation of rRNA genes. Next, we assessed the rate of rRNA transcription by measuring incorporation of the 5-ethynyl uridine (EU) in nucleoli coimmunostained with nucleophosmin (B23). Consistent with ChIP results, the rate of rRNA transcription was increased by SETD8-KD (Figure 4C). In agreement with the activation of ribosome biogenesis, the protein synthesis augmented in SETD8-KD cells (Figures S5K and S5L). Previously, it was reported that aberrant activation of rRNA genes evokes nucleolar stress, together with enlarged nucleolar morphology (Nishimura et al., 2015). In agreement with this, we found that SETD8-KD cells had enlarged nucleolar morphology, where the number of nucleoli per cell was decreased while the total area of nucleolus per cell was increased (Figures 4D and 4E). Collectively, our data suggest that SETD8 is responsible for H4K20me1 deposition at rRNA gene loci, and that loss of SETD8 upregulates rRNA transcription and induces nucleolar stress during senescence.

Loss of SETD8 Activates Mitochondrial OXPHOS via Retinoblastoma

Given that ribosome biogenesis is a major consumer of energy in cells (Warner, 1999) and that loss of SETD8 coordinately upregulates RPs and rRNAs, we speculated that depletion of SETD8 remodels the balance of energy metabolism for senescent state. In fact, several lines of evidence suggest that senescent cells exhibit higher mitochondrial activity than proliferating cells (Dörr et al., 2013, Hutter et al., 2004, Kaplon et al., 2013, Quijano et al., 2012, Takebayashi et al., 2015). Interestingly, we found that SETD8-KD cells increased mitochondrial oxygen consumption rate (OCR) at both the basal and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)-uncoupled maximal states (Figure 5A). In contrast, glycolytic activity, as indicated by the extracellular acidification rate (ECAR), did not largely change under the assay conditions (Figure S6A). Therefore, the ratio of basal OCR/ECAR increased by SETD8-KD (Figure 5B). In addition, depletion of SETD8 increased mitochondrial mass and membrane potential, as determined by fluorescent JC-1 staining (Figure 5C). Further, intracellular levels of reactive oxygen species (ROS) were increased approximately 2-fold in SETD8-KD cells (Figure 5D). These increases in mitochondrial activities were observed on day 3 of SETD8-KD when senescence markers were expressed only to a small extent (Figures 1E and 1F and S6B–S6F), suggesting that persistent upregulation of OXPHOS activity would contribute to establishment of senescence.

We and other groups previously reported that retinoblastoma (RB) protein plays a critical role in mitochondrial activity (Moiseeva et al., 2009, Nicolay et al., 2015, Takebayashi et al., 2015), as well as in cellular senescence (Chicas et al., 2010, Helman et al., 2016, Narita et al., 2003). Of note, the loss of SETD8 upregulated p16^INK4A and p21^CDKN1A (Figures 1G, 2E, 3E, and S5G), which could activate RB by inhibiting the cyclin-dependent kinases. To clarify whether RB is involved in mitochondrial activation in SETD8-KD cells, we performed simultaneous knockdown of RB and SETD8 (Figures S6G and S6H). Interestingly, depletion of RB reduced the S-phase population observed in SETD8-KD cells and abolished the activation of mitochondrial respiration (Figures 5E and 5F and S6I and S6J). Moreover, increases in the mitochondrial membrane potential and ROS accumulation in SETD8-KD cells were partially abolished by RB-KD (Figures 5G and 5H and S6K). These observations support the metabolic role of RB during establishment of senescence (Takebayashi et al., 2015). Further, depletion of RB completely abolished SA-β-Gal and SAHF formation in SETD8-KD cells, while the expression of p16^INK4A and p21^CDKN1A was consistently increased (Figures 5I and 5J and S6L). As was the case of RB, KD of p16^INK4A also blocked mitochondrial activation and senescent features in SETD8-KD cells as well as in OIS cells (Figures S6M–S6S). Together, these data show that loss of SETD8 promotes mitochondrial OXPHOS activity and ROS accumulation, at least in part through RB function, contributing to senescence.

The Senescent State Remodels the H4K20me1 Landscape at SETD8 Target Genes

Our primary observations showed that SETD8 protein was downregulated during the establishment of senescence (Figure 1B). To investigate whether SETD8 target genes acquire altered H4K20me1 during senescence, we performed expression microarray analyses of OIS, RS, and SETD8-KD cells. Commonly upregulated gene sets included ribosome, lysosome, cancer, and p53 signaling, which are each closely associated with senescence (Figure 6A). Interestingly, a set of RP genes were consistently upregulated in both OIS and RS (late passage), as well as in SETD8-KD cells (Figures 6B and S7A). Subsequent ChIP analysis revealed that H4K20me1 at RP gene bodies was decreased in both OIS and RS cells, while the levels of H3K36me3 were partially increased at these loci (Figures 6C and 6D and S7B and S7C). Further, H4K20me1 decreased at rRNA gene loci in OIS cells, together with upregulation of rRNA transcription and enlarged nucleolar morphology (Figures 6E and 6F and S7D and S7E).

Under the downregulation of SETD8, we observed that global amount of H4K20me1 was decreased in OIS cells (Figure S7F). It was previously reported that the global distribution of H4K20me1 is dynamically regulated during cell differentiation, where this mark is gained at upregulated genes and lost at repressed genes (Cui et al., 2009). Thus, the status of H4K20me1 is likely to be regulated in cell- and gene-specific manners. We found that the levels of H4K20me1 in the gene bodies of HMGA2 and IL8 were increased in OIS cells, where both gene expression and H3K36me3 levels were increased (Figure S7G and S7H). On the contrary, as was observed at the RP and rRNA genes, H4K20me1 decreased at p16^INK4A gene in OIS cells, even though its expression was increased (Figures 6G and 6H and S7I). Collectively, our data suggest that SETD8 plays an essential role in remodeling the H4K20me1 landscape at RP, rRNA, and p16^INK4A gene loci in senescent cells.

Discussion

This study demonstrates that the SETD8 methyltransferase plays a pivotal role in senescence-associated metabolic remodeling, which is characterized by nucleolar and mitochondrial coactivation (Figures 4, 5, and 7A). Under OIS and RS conditions, SETD8 protein was downregulated, resulting in decreased H4K20me1 at target gene loci such as the RP, rRNA, and p16^INK4A genes. As SETD8 exerts transcriptional repression via H4K20me1 (Congdon et al., 2010, Kalakonda et al., 2008, Karachentsev et al., 2005), the decrease in H4K20me1 levels led to transcriptional derepression of these genes (Figure 7B). Further, loss of SETD8 function using siRNAs or a specific inhibitor UNC0379 caused senescent phenotypes in proliferating fibroblasts, suggesting that SETD8 is essential for normal cell growth and that loss of SETD8 is sufficient to establish cellular senescence.

Based on the following two findings, we conclude that SETD8 is involved in the conversion of energy metabolism in cellular senescence. First, the activation of nucleolar function resulting in increased ribosome biogenesis is one feature of senescent cells. For example, abnormal activation of rRNA transcription, a critical process of ribosome biogenesis, is observed in OIS cells (Nishimura et al., 2015). Some oncogenes as well as tumor suppressors are involved in regulation of rRNAgenes (Moss et al., 2007). Our findings demonstrate that SETD8 regulates rRNAgenes by the establishment of H4K20me1 (Figure 4). This was corroborated by an observed loss of H4K20me1 at RP genes and their consequent upregulation in the absence of SETD8 (Figures 2 and 3). Therefore, our data suggest that SETD8 depletion upregulates ribosome biogenesis through coordinate activation of both rRNA and RP genes. Notably, a previous report suggested that aberrant activation of rRNA genes evokes nucleolar stress, which promotes p53/p21-dependent senescence (Nishimura et al., 2015). Moreover, translational control of protein synthesis is a key step in rapid production of SASP factors that accelerate senescence (Dörr et al., 2013, Herranz et al., 2015, Laberge et al., 2015). Thus, loss of SETD8 may promote senescence through activation of nucleolar function, owing to upregulation of rRNA and RP genes.

Second, increased mitochondrial OXPHOS activity is another feature of the senescent state (Dörr et al., 2013, Hutter et al., 2004, Kaplon et al., 2013, Quijano et al., 2012, Takebayashi et al., 2015). Recently, we and other groups reported that RB plays a critical role in mitochondrial activation (Franco et al., 2016, Moiseeva et al., 2009, Takebayashi et al., 2015). In agreement with this, we found that loss of SETD8 increased OXPHOS activity in an RB-dependent manner (Figure 5). It is well known that RB function is enhanced by p16^INK4A and p21^CDKN1A via inhibition of cyclin-dependent kinases (Campisi and d’Adda di Fagagna, 2007). Interestingly, SETD8 depletion upregulates p16^INK4A and p21^CDKN1A via decreased H4K20me1 deposition at their gene loci (Figures 1G, 2E, 3E, and S5G). Thus, RB is activated in SETD8-depleted cells, which in turn activates OXPHOS metabolism. As reported earlier, abnormal activation of mitochondrial OXPHOS leads to ROS accumulation, which promotes the DNA damage response and senescence (Correia-Melo et al., 2016). Indeed, we detected the presence of DNA damages, as indicated by γH2AX in SETD8-KD cells (Figure S7J). Thus, it is likely that loss of SETD8 accelerates senescence at least in part through mitochondrial OXPHOS activation, by upregulation of p16^INK4A and/or p21^CDKN1A.

It would be of great interest whether overexpression of SETD8 could prevent or suppress senescence. As previously reported (Abbas et al., 2010, Hartlerode et al., 2012, Sims and Rice, 2008), we found that overexpression of SETD8 rather decreased H4K20me1 levels in both IMR-90 and HeLa cells (Figures S7K–S7M). In addition, the transient expression of SETD8 did not affect the mitochondrial activation and the cell cycle arrest in OIS and RS cells (Figures S7N–S7Q). Mechanistically, it is important to understand how SETD8 and H4K20me1 become downregulated during senescence. SETD8 protein is decreased via proteasome degradation rather than transcriptional control under stress conditions (Figure S1). Previous studies revealed that SETD8 is a substrate of the E3 ubiquitin ligases CRL4^Cdt2 and SCF^β-TRCP, which mediate degradation of SETD8 during the DNA damage response (Beck et al., 2012, Wang et al., 2015b). Similarly, another ubiquitin ligase, APC^Cdh1, is also involved in SETD8 degradation (Beck et al., 2012). This ligase is activated via ROS-mediated DNA damage in OIS cells and is also involved in degradation of the H3K9 methyltransferases G9a and GLP (Takahashi et al., 2012). As senescent cells are subjected to persistent DNA damage caused by telomere attrition, replication stress, and/or ROS accumulation (Kuilman et al., 2010), these ligases may be cooperatively involved in SETD8 degradation and accelerate senescence.

It also remains unresolved how H4K20me1 is removed from specific gene loci after SETD8 inhibition. It has been reported that PHF8 (PHD finger protein 8) demethylates H4K20me1 as well as H3K9me1/2 at the promoters of target genes, and consequently activates gene expression (Liu et al., 2010, Qi et al., 2010). Interestingly, PHF8 acts on the rRNA gene loci at both promoter and coding regions and upregulates rRNA transcription, although it has not been determined whether its demethylase activity against H4K20me1 is implicated in this process (Feng et al., 2010). Alternatively, H4K20 is known to be specifically mono-methylated at the G2/M phase when SETD8 levels are high, and the majority of H4K20me1 modifications are subsequently di-/tri-methylated during the M/G1 phases (Jørgensen et al., 2013). In fact, the levels of H4K20me1 were stable for at least 100 days in SETD8-null non-dividing hepatocytes of mice, whereas H4K20me1 became lost once these cells divided, with DNA damage and ROS accumulation (Nikolaou et al., 2015). Thus, there are three possibilities for reducing H4K20me1 in the absence of SETD8: active removal by demethylases, active conversion from the mono- to di-/tri-methylated form by methyltransferases, or passive demethylation through cell division.

Several lines of evidence suggest that metabolic interventions could prevent senescence and the aging process. For example, experimental reduction of ribosome biogenesis or protein translation delayed replicative senescence in human fibroblasts and even extended lifespan in some organisms, including mice (Hofmann et al., 2015, MacInnes, 2016, Takauji et al., 2016). Similarly, reduction of ROS accumulation also attenuated senescent phenotypes under oncogene and replication stresses, prevented stem cell aging, and increased longevity in mice (García-Prat et al., 2016, Lu and Finkel, 2008, Sun et al., 2016). Further, caloric restriction in aged mice reduced the population of senescent cells in the small intestine and liver (Wang et al., 2010). Therefore, understanding the molecular basis of senescence-associated metabolic remodeling and the epigenomic features in cellular senescence might explain why the senescent state presents such hypermetabolic phenotypes, leading to the development of novel treatments in senescence- and age-related disorders.

Author Contributions

H.T., S.T., A.S., and M.N. designed and conducted the experiments, together with support from T.I., Y.N., N.S., and S.H. H.T., S.H., and M.N. prepared the manuscript.

————-

The p53 tumor suppressor protein is regulated by multiple post-translational modifications, including lysine methylation. We previously found that monomethylation of p53 at lysine 382 (p53K382me1) by the protein lysine methyltransferase (PKMT) SET8/PR-Set7 represses p53 transactivation of target genes. However, the molecular mechanism linking p53K382 monomethylation to repression is not known. Here we show in biochemical and crystallographic studies the preferential recognition of p53K382me1 by the triple malignant brain tumor (MBT) repeats of the chromatin compaction factor L3MBTL1. We demonstrate that SET8-mediated methylation of p53 at Lys-382 promotes the interaction between L3MBTL1 and p53 in cells, and the chromatin occupancy of L3MBTL1 at p53 target promoters.

In the absence of DNA damage, L3MBTL1 interacts with p53K382me1 and p53-target genes are repressed, whereas depletion of L3MBTL1 results in a p53-dependent increase in p21 and PUMA transcript levels. Activation of p53 by DNA damage is coupled to a decrease in p53K382me1 levels, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from p53-target promoters. Together, we identify L3MBTL1 as the second known methyl-p53 effector protein, and provide a molecular explanation for the mechanism by which p53K382me1 is transduced to regulate p53 activity.

We demonstrate that SET8-mediated methylation of p53 at Lys-382 promotes the interaction between L3MBTL1 and p53 in cells, and the chromatin occupancy of L3MBTL1 at p53 target promoters.

In the absence of DNA damage, L3MBTL1 interacts with p53K382me1 and p53-target genes are repressed, whereas depletion of L3MBTL1 results in a p53-dependent increase in p21 and PUMA transcript levels.

Activation of p53 by DNA damage is coupled to a decrease in p53K382me1 levels, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from p53-target promoters. Together, we identify L3MBTL1 as the second known methyl-p53 effector protein, and provide a molecular explanation for the mechanism by which p53K382me1 is transduced to regulate p53 activity.

https://www.jove.com/visualize/abstract/20870725/the-mbt-repeats-l3mbtl1-link-set8-mediated-p53-methylation-at-lysine

Crain’s SF News

YOUR NEWS FOR FRIDAY, JULY 07, 2017

Qualcomm settles in for a long patent battle with Apple

Just when you thought the mobile patent wars might be behind us, Qualcomm and Apple seem determined to keep them going. Qualcomm raised the stakes in its patent battle with Apple Thursday afternoon, suing it for patent infringement and asking the U.S. International Trade Commission to bar the sale of iPhones with anything other than a Qualcomm wireless modem chip from entering the country.(GeekWire)

San Francisco’s Hunters Point gets free gigabit internet

A San Francisco-based internet service provider is providing free gigabit-speed internet access at more than 200 low-income, federally subsidized housing units in Hunters Point. Monkeybrains partnered with the San Francisco Housing Development Corp., a nonprofit that owns the units, to provide high-speed fiber internet to help close the digital divide.(Silicon Beat)

Alphabet spins off geothermal energy unit as ‘Dandelion’

One of Alphabet Inc.’s X-Lab moonshot efforts that is seeking to deliver cutting edge geothermal energy technology is following in the path of Waymo before it and spinning off as its own company. Called Dandelion Inc., the company says it can affordably drill and install geothermal systems to heat and cool individual homes, a radical departure from existing geothermal energy technology. (SiliconANGLE)

OnboardIQ raises $9.1M to speed up recruiting

Recruiting poses difficulties for every organization, but it’s a particularly big challenge in service industries where companies must often hire hundreds of employees per year due to high turnover. OnboardIQ Inc., a startup based in San Francisco, has armed itself with $9.1 million in funding to tackle the issue. (SiliconANGLE)

Tesla loses crown as most valuable U.S. automaker

On the eve of Tesla’s expected completion of the first Model 3 – its entry-level, mass-market electric car – the company lost its crown as the most valuable U.S. automaker, a position it had held for the past three months. Tesla shares ended the trading day down 5.58 percent Thursday, continuing a drop that has plagued the company this week as concerns about its production capabilities continue to grow. (Washington Post)

Ad buyers give TV a pass for massaging Nielsen data

TV was called out for messing with metrics Thursday. But in an ironic twist, media buyers are largely shrugging where they’ve taken great umbrage at digital platforms misreporting their own data. Networks have been called out for tampering with their Nielsen ratings with a little sleight of numbers, perhaps abusing a fairly common practice, according to a Wall Street Journal report. (Ad Age)

Instagram debuts photo, video replies on stories

Social media giant Instagram has announced a new feature that will allow users to reply to their friend’s stories with a photo, video, or Boomerang. The photo sharing app introduced the feature via its blog today, as an update to stories. Update your app to version 10.28 to use the new add-on, but look for the new feature to expand on the growing platform and increase the medium’s engagement even further from Snapchat’s grasp.(Hypebeast)

High speed caused Amtrak derailment along Washington coast

An engineer was driving an Amtrak passenger train too fast in Washington state on Sunday and caused it to derail as it headed north, officials said Thursday. Amtrak spokeswoman Vernae Graham said an investigation found that the train failed to slow down to the 40 mph speed limit while approaching the Chambers Bay drawbridge, about 45 miles south of Seattle.(Fox Business)

Should the auto industry rethink flagship stores?

Some high-profile flagships are being rethought, not abandoned. They are becoming places to experience the brand – something that can’t be done online – rather than as a spot to learn about the brand’s products. In other cases, the rethinking is leading to multiple showcases, rather than one must-go location (Automotive News)

The next big title in media agencies: Chief AI officer?

Any time an explosive new technology takes hold, agencies have to navigate how it fits into their business. While some wait until it takes a deeper hold, others, like New York-based Crossmedia, are bullish. The independent media agency just hired a new executive director of cognitive solutions, who will head up the agency’s work in that area — covering everything from client projects that use AI like chatbots or Alexa skills to other areas of cognitive solutions. (Ad Age)

MORE NEWS

IAB adds viewability specification for in-app ads (Ad Age)

Facebook, Twitter said to seek World Cup clips from Fox (Ad Age)

To prove futility of age discrimination, startup hires only seniors (Tech.Co)

SoundCloud faces the music; cuts 40 percent of staff (Ad Age)

Uber suspends unlicensed service in Finland until next year (Reuters)

TRENDING:

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QVC

$2.58B Digital Health Funding

81 digital health funding deals from Q2 2017

By MobiHealthNews

StartUp Health’s recent funding report placed Q2 2017 as one of the biggest quarters of all time, and MobiHealthNews’s own data seems to back up that claim. During the quarter, we tracked 81 deals, totaling $2.58 billion. Read on for the full list of companies that raised equity from investors over the last three months, and click the links to check out the full story. We’ve ranked them in order from largest to smallest, with undisclosed rounds at the end.

Outcome Health — $500 million. Outcome Health, a maker of waiting room screens and tablets for patient education and pharmaceutical marketing, raised at least $500 million in first round funding, lending the company a valuation of around $5 billion. Goldman Sachs Investment Partners, Alphabet’s growth equity investment fund CapitalG, Leerink Transformation Partners, Pritzker Group Venture Capital, and Balyasny Asset Management are a few of the many investors in the round, along with some undisclosed strategic investors that include health systems and other stakeholders.

Peloton — $325 million. Peloton, the New York City-based technology company that has seen impressive success combining exercise bikes with tablets to create virtual cycling classes, raised $325 million in late stage funding. The investment brought the company’s total funding to nearly $445 million and brought its valuation to $1.25 billion. Wellington Management, Fidelity Investments, Kleiner Perkins, and True Ventures led the round, with additional contributions from Comcast NBCUniversal, GGV Capital, Balyasny, and QuestMark.

Modernizing Medicine — $231 million. Modernizing Medicine, which makes a mobile-based EHR for specialists, raised $231 million from global private equity firm Warburg Pincus. The company has raised funding a number of times before, so this raise took its total funding to $318 million.

Bright Health — $160 million. Bright Health, the Minnesota-based health insurance startup that boasts “a smarter, more connected” experience, raised a new round of $160 million. Greenspring Associates led the round, with additional participation from new investors Greycroft Partners, Redpoint Ventures and Cross Creek Advisors and existing investors New Enterprise Associates (NEA), Bessemer Venture Partners, and Flare Capital Partners. The raise followed an $80 million round from Bright Health in April of last year, bringing the company’s total funding to $240 million.

Patient Point — $140 million. PatientPoint, the Cincinnati, Ohio-based company that makes education and engagement platforms for patients and providers, raised $140 million in new funding from Searchlight Capital Partners and Silver Point Capital. The company – which provides content through a variety of delivery methods including digital waiting rooms and interactive touchscreens in exam rooms – plans to use the latest funding to expand its already considerably large digital footprint.

Clover Health — $130 million. Clover Health, an insurance startup with a patient engagement bent, raised $130 million in a round led by existing investor Greenoaks Capital Management. New investors Google Ventures, Palm Drive Capital, and Western Technology Investment and existing investors Sequoia Capital and First Round Capital also contributed.

Blink Health — $90 million. New York City-based startup Blink Health, which makes an app and online tool to help consumers find low prices on medications, raised $90 million in Series B funding in a round led by 8VC. Previously, the company raised $75 million – also led by 8VC – bringing the company’s total funding to date at $165 million.

Sharecare — $85 million. Atlanta, Georgia-based Sharecare, a wellness and patient engagement company known for its serial acquisitions, received an investment from Summit Partners that brings the company’s total funding to $300 million. Given the company’s previous funding, Crunchbase pegged the May funding raise at $85 million. Sharecare will use the latest capital to expand their workforce and invest in new strategies to continue growing.

Outset Medical — $76.5 million. Outset Medical, a startup focused on innovating the practice of kidney dialysis, raised $76.5 million in a series C round led by T. Rowe Price Associates. In addition to new investor T. Rowe Price Associates, existing investors Fidelity Management & Research Company, Partner Fund Management LP, Warburg Pincus, Perceptive Advisors and The Vertical Group also participated. This round of funding brings the company’s total funds raised to $185.5 million.

ClassPass — $70 million. New York-based ClassPass, a virtual fitness membership network, raised $70 million in Series C funding in a round led by Temasek. Existing investors Acequia Capital, CRV, General Catalyst, GV, M13 and Thrive also contributed to the funding, which ClassPass will use to expand operations and develop new products.

babylon — $60 million. UK-based health chatbot company babylon, which raised $25 million in early 2016, raised an additional $60 million in April. While the company didn’t disclose the investors, the round reportedly included Sawiris, an Egyptian billionaire business family, NNS holdings, Vostok New Ventures, and existing backers Kinnevik.

Omada Health — $50 million. San Francisco-based Omada Health raised $50 million in new funding, led by commercial partner Cigna. Civilization Ventures and Sanofi Genzyme BioVentures also contributed to the round. Omada last raised money in September 2015, when it took in $48 million. This latest round brings the company’s total funding to $127.5 million.

Glooko — $35 million. Diabetes data management company Glooko raised $35 million in new funding. The company already had strategic investments from Samsung and Medtronic; in this new round the Mayo Clinic and insulin pump maker Insulet join those ranks. Toronto-based Georgian Partners led the round, with participation from existing investors Canaan Partners and Social Capital, in addition to the aforementioned health and tech stakeholders.

VitalConnect — $33 million. San Jose, California-based VitalConnect, a maker of wearable sensors for remote monitoring, raised $33 million in Series C financing in a round led by MVM Life Science Partners and Baxter International. The company, which last raised money in June 2016, plans to use the funding to move forward with commercializing its remote monitoring platform and medical-grade wearable sensor system.

Medrio — $30 million. Clinical trial software company Medrio raised $30 million in new funding from Questa Capital Management. This is the first ever institutional funding round for the 12-year-old company, and will be used to support growth as well as the development of new software programs.

PolicyGenius — $30 million. PolicyGenius, which makes a virtual insurance marketplace platform, raised $30 million in Series C funding in a round led by Norwest Venture Partners, bringing the company’s total funding to $52 million. The company, which works with payers to establish a quoting engine that allows shoppers to do side-by-side comparisons of policies, started its business looking at life insurance, then expanded to health, renter’s, disability and pet insurance.

Science 37 — $29 million. Remote clinical trial company Science 37 raised $29 million in a round led by Glynn Capital Management with participation from Google’s investment arm GV. One more new investor, AmGen Ventures, contributed, as did existing investors Lux Capital, Redmile Group, dRx Capital, and Sanofi Ventures. The round brings the company’s total funding to $67 million.

Amino — $25 million. San Francisco-based Amino, which offers an online healthcare marketplace platform, raised $25 million in Series C funding. Highland Capital Management led the round, with Accel, Aspect Ventures, CRV, Northwestern Mutual Future Ventures, Pilot Wall Group, and others contributing. Previously, the company raised a little over $19 million, and this new investment brings Amino’s total funding to date to nearly $45 million.

ZappRx — $25 million. Digital specialty prescribing platform ZappRx raised $25 million in Series B funding in a round led by Qiming US Healthcare Fund. GV, formerly Google Ventures, and SR One also contributed funding. The added capital will be used to expand ZappRx’s workforce as the company explores greater adoption from providers and other partners.

TwoPoreGuys — $24.5 million. Santa Cruz, California-based Two Pore Guys, which is developing a novel method of diagnostic testing via a device that digitally detects molecules, raised $24.5 million in a round led by Khosla Ventures. Two Pore Guys (aka 2PG) will use the funding to scale up the manufacturing of its hand-held device.

Kry — $22.6 million. Stockholm, Sweden-based Kry, purveyors of an app-based telemedicine service, closed on almost $22.6 million in Series A funding led by Silicon Valley venture firm Accel Partners. Index Ventures, Creandum and Project A also returned to contribute to financing. The company received $6.8 million in seed funding August 2016, and the latest funding brings Kry’s total capital to date to $29.4 million.

Capsule — $20 million. Pharmacy startup Capsule, which hand delivers drugs in New York City, raised $20 million in a round led by Thrive Capital, with contribution from Sound Ventures and Virgin Group. The company is working to establish itself apart from other such virtual pharmacies like Blink Health and PillPack, as well as the behemoth pharmacy benefits manager Express Scripts.

Art Medical — $20 million. Israel and Palo Alto-based Art Medical, a developer of smart intubation devices for use in intensive care, raised $20 million in a round led by Advanced Medical Technologies. Founded in 2009, Art Medical previously raised $7 million from a combination of funding from the Israel Innovation Authority and Bill Gates’ Grand Challenge. In the eight years since founding, the company has been working on a suite of connected, sensor-laden tubes to continuously monitor for complications and collect data of patients in the ICU.

Trice Medical — $19.3 million. Trice Medical, which has developed a tiny, needle-based camera to triage joint injuries rather than doing so with a traditional MRI, raised $19.3 million in Series C funding to take the device’s reach further into the US market. Safeguard Scientificis, HealthQuest Capital, BioStar Ventures and Smith & Nephew contributed, bringing Trice’s total funding to date at $40.9 million.

DayTwo — $17 million. DayTwo, an Israeli startup focused on delivering insights to consumers based on their gut microbiome, raised $12 million from an impressive suite of investors including Johnson & Johnson Innovation and the Mayo Clinic. Seventure Partners’ Health for Life Capital Fund also contributed along with private investors including cofounder Marius Nacht.

DocPlanner — $16.8 million. DocPlanner, a Polish appointment booking software company, raised $16.8 million (15 million euro) in new funding from Enern Investments, Target Global, and One Peak Partners. Existing investors Enern and Target Global led the round. This brings the company’s total funding to $51 million (46 million euros).

ChartSpan — $16 million. Greenville, South Carolina-based ChartSpan Medical Technologies, which makes digital care coordination tools, raised $16 million in a round led by Cypress Growth Capital. ChartSpan’s platform runs the gamut of practice management software, mobile patient engagement and records management tools. The turn-key service is available to health systems and ambulatory practices throughout the country, enabling them to provide out-of-office healthcare services to their patients.

RapidSOS — $14 million. RapidSOS, a 4-year-old startup with roots at Harvard and MIT, raised $14 million to bring 911 and other first responder networks into the smartphone age. Highland Capital Partners led the round, with participation from A3 Ventures, The Westly Group, Two Sigma Ventures, Motorola Solutions Venture Capital and Responder Ventures. Former FCC Chairs Tom Wheeler, Julius Genachowski, and Dennis Patrick also contributed along with other undisclosed individuals.

Mindstrong Health — $14 million. Mindstrong Health, the mental health startup that former NIMH director Dr. Thomas Insel left Verily for last month, raised $14 million. Foresite Capital and ARCH Venture Partners led the round, with additional participation from Optum Ventures, Berggruen Holdings, and the One Mind Brain Health Impact Fund.

Drchrono — $12 million. Mobile EHR drchrono raised $12 million in new funding in a round led by Runa Capital. Maxfield Capital, Eric Dunn (CEO of Quicken Inc. and Intuit’s first CTO), and FundersClub also participated in the round. The company is raising money in order to be more competitive with larger EHRs and also has plans to build more iOS apps and continue its work with Apple as a mobility partner.

ClearDATA — $12 million. Austin, Texas-based ClearDATA, a cloud services provider that exclusively serves the healthcare and life sciences market, raised $12 million from a stable of existing investors including Merck Global Health Innovation Fund, Norwest Venture Partners, Excel Venture Management, Heritage Group, HLM Venture Partners, and Flare Capital Partners. This round brings ClearDATA’s total funding to $55 million.

Aira — $12 million. San Diego-based Aira raised $12 million to continue developing its smart glasses that combine AI and remote human agents to help blind and low-vision people navigate the world. JAZZ Venture Partners and Arboretum Ventures led the round and existing investors Lux Capital, ARCH Venture Partners and Felicis Ventures participated. The National Federation of the Blind joined the round as a strategic investor. The round brings the company’s total funding to about $15 million.

Doc Halo — $11 million. Cincinnati, Ohio-based clinical communications provider Doc Halo raised $11 million in a series A round led by Bane Capital Ventures. Cincinnati-based Refinery Ventures also contributed to the round. Doc Halo has been operating for seven years in the clinical comms space, but this was its first outside funding round; cofounders Dr. Jose Barreau and Dr. Amit Gupta, both former practicing physicians, self-funded the company initially with $5 million of their own money.

Lemonaid Health — $11 million. San Francisco-based Lemonaid Health, which offers a simple, text-based telemedicine app, raised $11 million in Series A funding. Novartis Venture Fund and Hikma Ventures led the round, with participation from Correlation Ventures, Adaptive Healthcare Fund, Vega Ventures and 415 Ventures. The company last received funding in 2015, and the latest investment brings Lemonaid’s total capital to date to around $20 million.

Evidation Health — $10 million. San Mateo, California-based Evidation Health, which was launched last year by GE Ventures and Stanford Healthcare, raised $10 million from Sanofi-Genzyme BioVentures. GE Ventures and B Capital were return investors, and both firms participated in the efficacy-focused company’s $3.4 million round in October 2016. The latest investment brings the company’s total funding to at least $31 million.

Figure 1 — $10 million. Clinical image and knowledge-sharing app Figure 1, which is sometimes described as an Instagram for doctors, raised $10 million in funding in a round led by Kensington Capital Partners, with additional support from Samsung NEXT Ventures, John Hancock/Manulife Financial, Hedgewood, and WTI. Existing investors Union Square Ventures, Rho Canada Ventures, and Version One Ventures also participated.

Your.MD — $10 million. London-based medical chatbot company Your.MD raised $10 million in a round led by Orkla Ventures, the venture arm of consumer goods company Orkla. Existing investor Smedvig Capital also participated in the round. Your.MD last raised funds in 2015; this round brings the company’s total funding to $17.3 million.

Xealth — $8.5 million. Xealth, a spin-out from Providence St. Joseph Health’s digital innovation group that helps doctors to prescribe digital health apps, raised $8.5 million in a round led by DFJ Venture Partners. Other investors in the round included hospital systems Providence St. Joseph Health, University of Pittsburgh Medical Center (UPMC), Hennepin Healthcare System, and Froedtert Health.

HealthLoop – $8.4 million. Mountain View, California-based HealthLoop, which offers a platform to connect patients with their care team between visits, has raised $8.4 million from iCarbonX DigitalLife Alliance, NextEquity, Lafayette General Hospital through its Health Innovation Fund, Canvas Ventures, and Summation Health Ventures. HealthLoop has been quietly getting into hospitals around the country over the last few years including Cleveland Clinic and Kaiser Permanente-Southern California. Last summer HealthLoop announced a deployment at Memorial Sloan Kettering Cancer Center’s Josie Robertson Surgery Center.

Artemis Health – $8.3 million. The Salt Lake City, Utah-based maker of data analytics tools for employee benefit managers, raised $8.3 million in a round led by Maverick Ventures. In addition to the funding, the company added two new members to its board: Ashok Subramanian, former co-founder and CEO of Liazon and the head of the Group Exchange business at Willis Towers Watson, and Ambar Bhattacharyya, managing director at Maverick Ventures.

Simple Contacts – $8 million. Simple Contacts, a New York City-based ocular telemedicine company, raised $8 million in its second round of funding. The round was led by Goodwater Capital with participation from existing investors Justin Kan, Notation Capital, and Autonomous Ventures. The company plans to use the new funding to expand its product and marketing teams, to both improve the product and bring it into more markets.

Conversa – $8 million. San Francisco-based Conversa, makers of a healthcare conversation platform, raised $8 million in Series A funding to expand its product offerings and build up its clinical conversation library. Northwell Ventures, the funding arm of New York healthcare system Northwell Health, led the round, with contribution from Epic Ventures and Healthgrades.

Viz – $7.5 million. San Francisco-based Viz looking to apply artificial intelligence to medical imaging, and raised $7.5 million in first-round funding. The funding round was led by DHVC and Alphabet Chairman Eric Schmidt’s Innovation Endeavors. AME Cloud Ventures and Susa Ventures also contributed. Viz is initially focusing on stroke, but intends to expand its capability to a number of health conditions where diagnostic imagery is used and time is of the essence.

AiDoc – $7M. Flush with a new $7 million in funding led by Tel Aviv VC firm TLV Partners, AiDoc is working to take its artificial intelligence-powered medical imaging tool into clinics to help radiologists work through their case load faster. The company has built deep learning algorithms to analyze imaging and clinical data quickly, enabling it to scan for visual abnormalities in medical scans.

CirrusMD – $7 million. Denver-based telemedicine startup CirrusMD, which focuses on a text-first approach to virtual visits for large health systems and payers, closed on $7 million in Series A funding led by Colorado Impact Fund. Since the company’s founding five years ago, CirrusMD lists 1.3 million patients with access to the platform, which represents an 850 percent growth since last year. With the latest funding, the company plans to expand sales, marketing, customer support resources and further innovate on their platform and data analytics.

Digital Pharmacist – $6.5 million. Austin-based Digital Pharmacist, a company that offers prescription management apps, has raised $6.5 million in new funding in a round led by Activate Venture Partners and LiveOak Venture Partners. The company, which was formed in January from a merger of RxWiki and Telemanager, has raised $13.7 million to date.

Solv – $6.25 million. San Francisco-based Solv Health, which is developing an app that allows people to find and book same-day doctor’s appointments at urgent care clinics, has raised $6.25 million in Series A funding. The investment round was led by Benchmark Capital with participation from Theresia Gouw of Aspect Ventures and Malay Gandhi of Ensemble Labs (Gandhi is also the former CEO of seed fund Rock Health). The new funding will be used to expand testing to more users around the state, and Benchmark Capital’s General Partner Bill Gurley will join Solv’s board of directors.

LogicStream Health – $6 million. The Minneapolis company whose software uses EHR data to help hospitals maximize workflow efficiency raised $6 million in a new round of funding led by Noro-Moseley Partners. Some existing investors also contributed. LogicStream will pour the new funds into product development as well as expanding sales and marketing teams to meet what the company says is a growing demand for the software.

Upfront Healthcare Services – $5.6 million. Chicago-based Upfront Healthcare Services – which offers a data analytics platform to help large physician practices ascertain how best to schedule and allocate resources –raised $5.6 million in Series A funding. The round was led by Nashville Capital Network, with contribution from Echo Health Ventures, Hyde Park Venture Partners and Martin Ventures.

Vericred – $5.5 million. Healthcare data company Vericred, which offers a platform with provider-network dataset, raised $5.5 million in a round led by FCA Venture Partners. The company, whose centralized data platform allows individuals and business to enroll, understand and use health insurance and employee benefits, has raised nearly $10 million to date.

Spry Health – $5.5 million. Palo Alto-based Spry Health raised $5.5 million in funding led by Grove Ventures and Stanford StartXFund to commercialize Loop, a wearable wristband and remote patient monitoring platform for people with chronic illnesses.

Sansoro Health – $5.2 million. Sansoro Health, a company that connects digital health technologies with electronic health records, raised $5.2 million. Bain Capital Ventures led the round. Combined with last year’s $1.2 million raise from Healthy Ventures and Treehouse Ventures, the company’s total funding is now at $6.4 million. The company has been around for about two years and in that time has deployed its platform at 20 healthcare organizations. The new funding will be used to expand sales and marketing operations.

Savonix – $5.1 million. San Francisco-based Savonix, maker of an app for neurocognitive assessment, raised $5.1 million in a new round of funding. DigiTx Partners led the round with additional contribution from ReThink Impact. Savonix will use the new funding to expand into new international markets including China and Japan. The company also plans to develop additional partnerships and to hire more data scientists and product developers. Dr. David Kim, CEO at DigiTx Partners, and Heidi Patel, Partner at ReThink Impact, will join Savonix’s Board of Directors.

FitnessGenes – $5 million. London-based FitnessGenes, which creates personalized fitness and nutrition plans for individuals based on a the results of an at-home DNA testing kit, raised $5 million. Sino-German High Tech Fund (SGHF), an investment fund comprised of China’s Donghai Securities and Germany’s Grunderfonds, led the round. The company – which was founded by doctors from the University of Oxford and Birmingham in 2013 and also operates under the name MuscleGenes – is working to add another $5 million in funding to close out the Series A and plans to use the capital to advance their platform and product offering as well as expand operations in North America and Europe.

Doc+ – $5 million. Doc+, a Russian digital health company that combines telemedicine with digital-enabled house calls, raised $5 million from two existing investors: Yandex, an internet services company, and Baring Vostok venture fund. This is the second round of funding and brings Doc+’s total funding to $10.5 million. The company’s primary business is digital-enabled house calls, similar to US companies like Heal and Pager. The visits are currently available in Moscow and Saint Petersburg, and the company has plans to add a third Russian city soon.

Mpirica – $4.6 million. Seattle-based healthcare quality transparency company Mpirica has raised $4.6 million in a round led by equity crowdfunding platform OurCrowd. With its team of data scientists and physicians, Mpirica uses claims data to rate surgeons based on its own proprietary methods. Three-digit quality scores, modeled after FICO credit scores, are available online for 864 different surgical procedures.The company plans to use the funding to support a national rollout to Medicare Advantage customers.

Murj – $4.5 million. Santa Cruz, California-based Murj, developer of a digital data collection platform for implantable cardiac devices, has officially emerged from stealth mode. Backed by $4.5 million in venture funding, the company wants to streamline workflow for clinicians grappling with increasingly more data from heart-monitoring devices, both remotely and in the office.

PreparedHealth – $4 million. Chicago-based Prepared Health, which makes a care coordination platform focused on aging-in-place populations, raised $4 million in seed funding in a round led by Chicago Ventures. Pritzker Group Venture Capital, Beverly Capital and Meridian Street Capital also contributed. The company will use the new funding to invest in R&D and enter new markets.

Saranas – $ 4 million. Houston, Texas-based medical device company Saranas raised $4M to support continued development on their sensor-enabled bleeding detection device intended for use during cardiac procedures. The device is still in the product testing phase, and Saranas plans to submit for FDA clearance later this year.

Cocoon Cam – $4 million. Silicon Valley-based Cocoon Cam raised $4 million in Series A led by Happiness Ventures. Previously, the company raised a little more than $1 million, and will use the latest investment continue developing, marketing and selling their smart video camera-based baby monitor.

Gixo – $3.7 million. Backed by $3.7 million in funding led by Greylock Partners, San Francisco-based fitness app company Gixo officially launched its subscription-based live workout app to Android and iOS in May. Cowboy Ventures and xSeed Capital also contributed funding to the round.

Devicare – $3.3 million. Barcelona, Spain-based Devicare, which is developing remote patient monitoring devices for people with chronic conditions, raised nearly $3.3 million (3 million euros) in seed funding. Investment holding company Emesa Coporacion Empresarial and several family and corporate partners contributed to the round. The company has raised $4.9 million (4.5 million euros) since its founding in 2012.Devicare will use the funding to launch clinical studies for two of their main products in Spain.

Lybrate – $3 million. Indian startup Lybrate, which helps patients in its home country connect to doctors – both in person and by telemedicine – raised another $3 million in a round with undisclosed investors. The company now has $15 million in investment funding. As TechCrunch points out, Lybrate is a direct competitor with Practo, which has raised $180 million, more than 10 times as much.

ReThink Medical – $3 million. San Francisco-based ReThink Medical raised $3 million to develop a medical wearable to predict and prevent heart failure. The round was led by Emergent Medical with additional funding from Norwich Ventures and Launch Capital. The company is also operating with a previously-awarded NIH grant as well as a strategic partnership with Japanese medical device company Terumo Corporation, which includes an investment.

TuringSense – $3 million. TuringSense, which is developing interactive wearables for sports and physical therapy, raised $3 million in a new round of funding. The company also raised $3 million in its first round two years ago. The newest round was led by VC firm and returning investor Ideosource, with participation from new investors The Core Group and Fenox Venture Capital.

Catalia Health – $2.5 million. Catalia Health, which makes an AI-powered patient engagement robot called Mabu, raised $2.5 million in a new round led by Khosla Ventures. Additional investments came from new investors NewGen Capital and Macnica Ventures and existing investors Q Venture Partners Limited, InnoLinks Ventures, Abstract by Flight.VC, DeNA and Lucky Capital. This brings the company’s total funding to $3.75 million.

DBS System – $2.5 million. This Swiss company that makes a line of portable blood tests called HemaXis raised $2.5 million led by investiere. (The funding was in Swiss francs, but the values of the currencies are currently approximately equal). The company’s method, called microsampling, requires considerably less blood than existing blood tests. The funds raised are going to the development of a second-generation device called HemaXis DP, which will be able to passively separate plasma from serum without the need for centrifugation or filtration.

Simple Habit – $2.5 million. San Francisco-based meditation app Simple Habit raised $2.5 million in a new round of funding that includes investors New Enterprise Associates (NEA), Fabrice Grinda’s FJ Labs, Foundation Capital and the founders of Dropbox and Gusto. This is the first round of funding for the company, whose app launched on the iOS App Store in April 2016.

Scorpio Labs – $2.5 million. Israeli equity crowdfunding platform OurCrowd invested $2.5 million in digital microscopy and diagnostics company Scopio Labs, which is the 23rd health technology company to receive funding from investor.

BioDirection – $2 million. Medical device company BioDirection raised $2 million in interim funding to develop their point-of-care products intended for diagnoses and management of concussions and other traumatic brain injuries. Their first product, called the Tbit System, uses the company’s nanotechnology biosensor to detect and measure protein biomarkers that the brain releases immediately after head trauma. With the portable system, BioDirection aims to make earlier diagnosis and treatment without the need for CT scans.

EverlyWell – $2 million. Austin, Texas-based EverlyWell, maker of a digital platform that lets consumers to order lab tests online, provide samples and receive results, raised another $2 million in a round led by Next Gen Venture Partners. Jack Novak, SoGal Ventures, Full Tilt Capital and other unnamed venture capital firms contributed. Last year, EverlyWell raised $2.5 million, and also had $500,000 in seed funding, which brings the company’s total funding to date to $5 million. EverlyWell plans to use the latest funding to hire more leadership positions, explore new partnerships and improve their product features.

eVisit – $2 million. Arizona-based telemedicine platform provider eVisit raised $2 million from Kickstart Seed Fund, Arizona Founders Fund and angel investor Jeremy Andrus. Previously, the company raised $1 million in July 2016. The latest funding will be used to scale eVisit’s sales, marketing and support teams as they expand.

NowRx – $2 million. Mountain View, California-based NowRx, an app-based pharmacy delivery company, raised $2 million in seed funding. The startup, which has been in operation a little over a year, plans to use the capital to expand their customer base.

Osso VR – $2 million. Osso VR, a startup looking to disrupt the surgical device training space with virtual reality, raised $2 million in seed funding in a round led by Signalfire, with participation from Anorak Ventures.

Thriva – $1.9 million. London-based mail order consumer blood testing startup Thriva raised $1.9 million (1.5 million pounds) in a round of seed funding. Individual investors include Zoopla CEO Alex Chesterman and TransferWise CEO Taavet Hinrikus and funds investing include Seedcamp, 500 Startups and the London Co-Investment Fund (LCIF).

Spring – $1.5 million. New York City-based Spring, which offers clinical decision support for providers around behavioral health, raised $1.5 million in seed funding. William K. Warren Foundation, a philanthropic organization associated with the St. Francis Health System, and serial entrepreneur Kevin Ryan (founder of Gilt and Business Insider) led the round. Additional contributions came from RRE Ventures, North Sound Ventures, Saddlefire Ventures, and Rough Draft Ventures. Spring is commercializing technology that was originally developed at the Yale School of Psychiatry, which has been clinically validated in studies published in JAMA Psychiatry and Lancet Psychiatry.

BrainCheck – $1.5 million. Houston-based BrainCheck, developer of a mobile neurocognitive assessment platform, raised $1.5 million to expand sales, distribution channels, and research opportunities for its product. True Wealth Ventures led the latest round, which, together with the company’s previous raise of $3 million, brings BrainCheck’s total to $4.5 million.

HelloMD – $1 million. HelloMD, a service that uses telemedicine to connect people with doctors willing and able to prescribe medical marijuana, raised just over a million dollars via equity crowdfunding on SeedInvest. The raise includes $200,000 from SeedInvest itself via the platform’s “Selections Fund”. At the time of the announcement, the round was not complete, so the company could end up raising significantly more.

HealthReveal – $500 thousand. Healthcare technology company HealthReveal, which equips payers and providers with data analytics and remote monitoring to ensure patients receive guideline-directed medical care, added another $500,000 to its Series A funding with an investment from Northwell Ventures. Along with the $10.8 million HealthReveal raised a few months ago(led by GE Ventures, Greycroft Partners and Flare Capital Partners), the new funding brings the company’s total funding to date at $11.3 million.

Impathiq – $350 thousand. Winston-Salem, North Carolina-based Impathiq, which makes a data informatics platform that allows hospitals and health systems to integrate new apps and programs into their electronic medical records in accordance with interoperability standards, has announced it raised $350,000 in seed funding and has saved hospitals over $3 million in the year since its launch.

NarrativeDx – undisclosed. Austin, Texas-based NarrativeDx raised an undisclosed Series A round, led by LiveOak Venture Partners, Cultivation Capital and HealthX Ventures. The company uses AI and natural language processing to analyze patient feedback and return suggestions on how a healthcare organization can improve the patient experience.

Washington Post: A linguist studied Trump for two years: ‘He speaks like everybody else,’ not a politician

Putin denies election hacking after Trump pressed him, Tillerson says

Secretary of State Rex Tillerson, who attended the lengthy meeting, said it did not focus on the United States moving to punish Russia for the allegations that it hacked and leaked information that would help Trump win the election. Instead, Tillerson said, the two presidents discussed “how do we move forward from what may be simply an intractable disagreement at this point” regarding the election-hacking issue.

By Abby Phillip, David Filipov and Damian Paletta • Read more »

The Fix: Trump’s past comments make it difficult to believe that he confronted Putin with much vigor

The Russian foreign minister said President Trump accepted the explanation that Russia didn’t hack the U.S. elections. He also said Trump talked about how people in the United States were “exaggerating” the situation.

By Aaron Blake • Read more »

U.S., Russia agree to collaborate on cease-fire in Syria

The deal would mark the first joint operation between Washington and Moscow since they began backing opposing sides in Syria’s civil war that began more than six years ago.

By Karen DeYoung • Read more »

At G-20, world aligns against a range of Trump policies

The growing international isolation of the United States under President Trump was starkly apparent as the leaders of major global economies mounted a near-united opposition front against Washington on issues ranging from climate to free trade.

By Michael Birnbaum and Damian Paletta • Read more »

The Fix: This is one of Trump’s most bizarre tweets yet — for 3 reasons

Trump’s tweet about John Podesta not turning over the DNC servers is downright confusing.

By Aaron Blake • Read more »

Merkel appears to roll her eyes at Putin. The Internet goes bonkers.

Cameras captured the German chancellor interacting with the Russian president.

By Thomas Johnson • Read more »

FBI investigated complaints that Bobby Knight groped women at U.S. spy agency

Four female employees accused the Hall of Fame basketball coach of touching them inappropriately before and after his speech at the NGA. Knight denied wrongdoing, and prosecutors decided not to bring charges, his attorney said.

By Craig Whitlock • Read more »

Perspective

After a reporter in a sleeveless dress was turned away, it’s time to acknowledge that no one knows what’s ‘appropriate’ on the Hill

Traditions involving dress code apply to lawmakers and their staff, as well as journalists — but apparently not to first ladies or first daughters.

By Robin Givhan • Read more »

Cruz faces off with a ‘dirty’ liberal and seems to relish debating health care with critics

The senator from Texas supports a deeper rollback of the Affordable Care Act than most of his Republican colleagues. He’s also up for reelection next year — and has calculated that staying true to his conservative base is his best path to victory.

By Sean Sullivan • Read more »

KKK marchers say they will be armed Saturday at Virginia rally

The group is protesting the Charlottesville city council’s decision to remove a statue of Confederate Gen. Robert E. Lee from a public park and rename that park.

By Joe Heim • Read more »

Illegal immigrant charged with murder in stabbing deaths of her 4 children and their father in Georgia

Immigration authorities said it is unclear when or where the suspect, who is a Mexican national, entered the United States.

By Lindsey Bever and J. Freedom du Lac • Read more »

The Fix | Analysis

A linguist studied Trump for two years: ‘He speaks like everybody else,’ not a politician

“We’re used to hearing somebody speak who sounds much more educated, much smarter, much more refined than your everyday American,” a Georgetown University academic said.

By Bastien Inzaurralde • Read more »

Analysis

The Trump economy looks a lot like the Obama economy so far

More people working is certainly good news. But if the president suggests that this month’s jobs report is uniquely good news, take it with a grain of salt.

By Philip Bump • Read more »

World Chocolate day

Happy Chocolates day! Chocolates are number one gift sought after by lovers and women. Men who eats chocolates are calmer than those who do not (this one is my opinion).

Here are some nutrients and benefits of chocolates (may be attributed to the magnesium content):

Protection from Disease-Causing Free Radicals.
Potential Cancer Prevention.
Improved Heart Health.
Good for Overall Cholesterol Profile.
Better Cognitive Function.
Blood Pressure and Blood Sugar Aid.
Antioxidant-Rich Superfood

World Chocolate Day – Wikipedia

https://en.wikipedia.org/wiki/World_Chocolate_Day

World Chocolate Day, sometimes referred to as International Chocolate Day, is an observance that occurs globally every year on July 7. Celebration of the day …

Chocolate Day – 7th Jul, 2017 | Days Of The Year

https://www.daysoftheyear.com/days/chocolate-day/

There are a number of dates throughout the year promoting variations of local, national or international Chocolate Day – our research leads us to believe that…

World Chocolate Day: 15 things all chocolate lovers know to be true …

metro.co.uk › Lifestyle › Food

23 hours ago – Today is World Chocolate Day, and you can freely celebrate your love of all things chocolate. It is safe to say that chocolate is one of your major …

Today is World Chocolate Day! Let’s celebrate with some facts – News …

http://www.news5cleveland.com/…/world-chocolate-day-is-july-7-lets-celebrate-with-some-…

10 hours ago – There are actually four times a year people in the U.S. give a giant shout-out tochocolate.

World Chocolate Day: The 10 best chocolate shops in Denver …

http://www.thedenverchannel.com/…/world-chocolate-day-the-10-best-chocolate-shops-in-…

5 hours ago – DENVER – Friday is World Chocolate Day, and we couldn’t be more excited! The addictive sweet treat is ubiquitous in the Denver7 newsroom …

World Chocolate Day 2017: 6 Reasons Why Chocolate Is Good For …

http://www.india.com/…/world-chocolate-day-2017-6-reasons-why-chocolate-is-good-for-y…

12 hours ago – On World Chocolate Day 2017, here are all the good reasons for you to start having more chocolate. – World Chocolate Day 2017: 6 Reasons …

When is World Chocolate Day in 2017? – When-Is.com

http://www.when-is.com/world-chocolate-day-2017.asp

World Chocolate Day in 2017 is on Friday, the 7th of July (7/7/2017). World Chocolate Day(sometimes called International Chocolate Day) occurs globally every …

Sweet celebration: It’s World Chocolate Day | National | mysuncoast.com

http://www.mysuncoast.com/…world-chocolate-day/article_31eaa4f8-6318-11e7-8d54-8b7…

9 hours ago – Either way, it’s just one day dedicated to honoring the sweet stuff. In the United States, we observe International Chocolate Day on Sept.

Aphrodisiac herbs in Adevi Aphrodisiac Chocolates – ADEVI …

http://www.adevichocolates.com/aphrodisiac-herbs.html

Herbal Aphrodisiacs are the nature’s remedies to the sexual health. May be the word “herbs” sounds innocent, but don’t underestimate their potency- herbal …

Is chocolate an aphrodisiac? | HowStuffWorks

science.howstuffworks.com › Science › Innovation › Edible Innovations

Point to one person who hasn’t bought a box of chocolates in desperation for their boyfriend or girlfriend, and we’ll show you a liar. Why is this go-to gift for …

The Claim: Chocolate Is an Aphrodisiac – The New York Times

http://www.nytimes.com/2006/07/18/health/18real.html

Jul 18, 2006 – Nowadays, scientists ascribe the aphrodisiac qualities of chocolate, if any, to two chemicals it contains. One, tryptophan, is a building block of …

Herbal Infused Aphrodisiac Love Chocolates | BEDSPICE

http://www.bedspice.eu/en/thestory/

Fairtrade organic love chocolate fused with wildcrafted herbs that works as a libido … The best naturalaphrodisiac for both men and women by BEDSPICE!

Tobacco, smokers and health education

Disgust but not health anxiety graphic warning labels reduce motivated attention in smokers: a study of P300 and late positive potential responses

Justinn R Cochran, MSc; Robert R Kydd, PhD; John MJ Lee; Natalie Walker, PhD; Nathan S Consedine, PhD

The role of nicotine dependence in e-cigarettes’ potential for smoking reduction

Arielle S. Selya, PhD ; Lisa Dierker, PhD; Jennifer S. Rose, PhD; Donald Hedeker, PhD; Robin J. Mermelstein, PhD

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CDC – Fact Sheet – Health Effects of Cigarette Smoking – Smoking …

https://www.cdc.gov/tobacco/data_statistics/fact_sheets/…cig_smoking/index.htm

May 15, 2017 – Cigarette smoking harms nearly every organ of the body, causes many diseases, and reduces the health of smokers in general.^1,2. Quitting …

Health Risks of Smoking Tobacco – American Cancer Society

https://www.cancer.org/cancer/cancer…/tobacco…/health-risks-of-smoking-tobacco.ht…

Nov 12, 2015 – Smoking not only causes cancer. It can damage nearly every organ in the body. Learn more about the health effects of smoking tobacco here.

Burden of Tobacco Use in the U.S. | Data and Statistics | Campaign …

https://www.cdc.gov/tobacco/campaign/tips/…/cigarette-smoking-in-united-states.html

Jump to Current Cigarette Smoking Among Specific Populations—United States – Factors that may affect smoking prevalence include sacred tobacco’s …

10 of the Worst Diseases Smoking Causes | American Lung Association

http://www.lung.org › … › State of Tobacco Control › By the Numbers

More people die from lung cancer than any other type of cancer. Cigarette smoking is the number one risk factor for lung cancer; it’s responsible for 87 percent of …

Why Quit Tobacco – Reasons to Stop Smoking – Partnership For A …

http://www.tobaccofreemaine.org/quit_tobacco/index.php

There are compelling reasons to quit using tobacco or help a loved one stop smoking. Experience health benefits, financial benefits, and look and feel better …

Cigarette Smoking Effects, Statistics & How to Quit Smoking

http://www.emedicinehealth.com › … › lung disease & respiratory health az list

Oct 21, 2016 – Information on the health dangers of cigarette smoking, and smoking cessation help.Cigarette smoking can lead to cancers, emphysema, …

WHO | Tobacco

http://www.who.int/mediacentre/factsheets/fs339/en/

Tobacco fact sheet from WHO providing key facts and information on surveillance, second-hand smoke, quitting, picture warnings, ad bans, taxes, WHO …

WHO | Prevalence of tobacco smoking

http://www.who.int/gho/tobacco/use/en/

Far more males than females smoked tobacco. Although it is declining worldwide and in many countries, the prevalence of tobacco smoking appears to be …

Tobacco smoking and all-cause mortality in a large Australian cohort …

bmcmedicine.biomedcentral.com/articles/10.1186/s12916-015-0281-z

Feb 24, 2015 – Prevalence of current tobacco smoking among Australian adults, 1945–2013. Data are from Scollo MM and Winstanley MH (1945–2010) [9], …

What is tobacco smoke? Sociocultural dimensions of the association …

https://www.ncbi.nlm.nih.gov/pubmed/20550508

by A Leone – ‎2010 – ‎Cited by 27 – ‎Related articles

The definition of smoking as the inhalation of the smoke of burned tobacco that may occur occasionally or habitually as a consequence of a physical addiction to …

Opoid pain relievers like Morphine, Codeine etc.
Anti-cholinergic agents like Atropine, Trihexiphenidyl
Antispasmodics like dicyclomine
Tricyclic antidepressants like amytriptyline
Calcium channel blockers used in arrhythmias and high blood pressure such as verapamil
Anti-Parkinsonian drugs – Parkinson’s disease itself may cause constipation and the drugs used for this condition including Levodopa cause constipation as well
Sympathomimetics like ephedrine and terbutaline. Terbutaline is commonly used on bronchial asthma
Antipsychotics like clozapine, thioridazine, chlorpromazine used for psychiatric disorders
Diuretics for heart failure like furosemide
High blood pressure lowering agents like methyldopa, clonidine, propranolol etc.
Antihistamines like diphenhydramine
Antacids especially calcium and aluminium containing
Calcium supplements
Iron supplements
Antidiarrheal agents (loperamide, attapulgite)
Anticonvulsants e.g. phenytoin, clonazepam
Pain relievers or NSAIDs (Non steroidal anti-inflammatory drugs) like ibuprofen, aspirin etc.
Miscellaneous compounds including Octreotide, polystyrene resins, cholestyramine (for lowering high blood cholesterol) and oral contraceptives
————

Foods to eat when constipated

baked beans
black-eyed peas
garbanzo beans
lima beans
pinto beans, or
kidney beans
kiwi
plums
pears
sweet potatoes with skin

Job rate under 4 USA Presidents

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