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Monocytes can be differentiated into macrophages in vivo and these cells play an important role in innate and adaptive immune responses. To reveal the global gene transcription change that occurs during monocyte to macrophage differentiation, we performed genome-wide RNA sequencing and analyses in human primary monocytes and monocyte-derived macrophages. We show that 1208 genes (with >twofold differences) were differentially expressed in macrophages compared with monocytes, including 800 upregulated and 408 downregulated genes. Gene ontology, pathway, and protein–protein interaction analyses indicated that the upregulated genes were related to macrophage functions in phagocytosis, metabolic processes, and cell cycle. The majority of downregulated genes comprised genes involved in the inflammatory response and locomotion. Genes encoding transcription regulatory factors, such as FOXO1, RUNX3, NF-κB1, and C/EBP δ, were highly expressed in monocytes and appeared to function in significant transcriptional repression, resulting in slight metabolic activity. Our transcriptome comparison between human monocytes and monocyte-derived macrophages using RNA sequencing revealed novel molecules and pathways associated with the differentiation process. These molecules and pathways may represent candidate targets involved in the pathophysiology of these important immune cells.
Monocytes and macrophages, which belong to the mononuclear phagocyte system, define innate immune-mediated processes that include clearance of cellular debris and microbial infections, control of cancer progression, and secretion of immunoregulatory bioactive factors including interferons, interleukins, growth factors, and chemokines (DeNardo et al., 2010; Serbina et al., 2008). Commitment to the mononuclear phagocyte lineage is determined at the stage of macrophage and dendritic cell progenitor (MDP), at which point erythroid, megakaryocyte, lymphoid, and granulocyte fates have been precluded. Cellular cloning and transplantation studies have shown that MDPs give rise to monocytes and common dendritic cell (DC) progenitors (Auffray et al., 2009; Fogg et al., 2006). Blood-circulating monocytes can migrate from blood vessel to most body tissues and become resident macrophages. Although monocytes and macrophages are known to be highly heterogeneous in phenotype, tissue distribution, and function (Taylor et al., 2003), the dynamic molecular, biochemical, and cellular events that underlie monocyte differentiation remain unclear.
In recent years, research efforts have focused on biochemical and genetic changes that control monocyte differentiation. Previous studies in mice have demonstrated the critical functions of the macrophage colony-stimulating factor (M-CSF) receptor and its two known ligands M-CSF and IL34 (Lin et al., 2008) during monocyte to macrophage differentiation since the M-CSF deficient mice or its receptor deficient mice have dramatic reduction of F4/80 cell density (Dai et al., 2002). Other cytokines and chemokines, such as GM-CSF, FLT3, CXCL12, and CCL18, have also been shown to control macrophage development and homeostasis (Brasel et al., 1996; Sanchez-Martin et al., 2011; Schraufstatter et al., 2012; Wiktor-Jedrzejczak et al., 1994). Genome-wide gene expression profiles associated with monocyte to macrophage differentiation have also been investigated. Serial analysis of gene expression (SAGE) (Hashimoto et al., 1999; Suzuki et al., 2000) and microarray (Li et al., 2007; Martinez et al., 2006; Rodriguez-Prados et al., 2010) data allow the study of thousands of genes that are clustered into different pathways during monocyte to macrophage differentiation or activation. However, these approaches do not offer precise gene sequencing information and lack direct quantification of gene expression.
Next-generation deep sequencing technology has been proven to be a powerful tool for transcriptome analysis (Balakrishnan et al., 2012). This direct sequencing methodology enables the simultaneous sequencing of millions of different DNA molecules without requiring prior annotation (Cloonan and Grimmond, 2008; Morozova and Marra, 2008). Sequencing-based methods generate absolute gene expression measurements and show many advantages over previous microarray-based assays. For example, sequencing-based methods generate an unbiased view of the transcriptome, are not limited by predictions of expressed transcripts used to determine array content, and are more sensitive in detecting low-abundant transcripts and small changes in gene expression (t'Hoen et al., 2008; Wilhelm et al., 2008).
In the present study, to understand the molecular mechanisms involved in monocyte to macrophage differentiation better, we employed Illumina mRNA sequencing (RNA-seq) to perform transcriptome comparative analysis between monocytes and monocyte-derived macrophages for the first time. cDNAs generated from both monocyte and macrophage mRNAs were subjected to deep sequencing and gene expression profiles between monocytes and macrophages were analyzed. Our findings provide a comprehensive view of the changes in the monocyte transcriptome following M-CSF-stimulated differentiation into macrophages. The results can improve our understanding of gene regulation during monocyte hematopoietic differentiation on a genome-wide scale.
Monocytes were purified from freshly collected, leukocyte-rich buffy coats obtained from a healthy blood donor (American Red Cross Blood Service, Columbus, OH, USA) as previously described (Dong et al., 2007). Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over a Ficoll-Paque gradient. Primary monocytes were then isolated from PBMCs using Histopaque-Percoll gradient centrifugation. Cells were cultured in RPMI medium with 10% fetal bovine serum. To generate monocyte-derived macrophages, isolated monocytes were cultured in the presence of M-CSF (50 ng/mL; PeproTech) for 7 days. Total RNAs were extracted from freshly isolated monocytes or differentiated macrophages using a Qiagen RNeasy kit.
cDNA libraries were prepared according to the Illumina TruSeq RNA Sample Preparation Guide for TruSeq RNA Sample Prep Kit (FC-122-1001). Briefly, poly-A-containing mRNA molecules were purified from 4 μg of total RNA using poly-T oligo-attached magnetic beads, then fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA polymerase I and RNaseH. These cDNA fragments were then subjected to an end-repair process using a combination of T4 DNA polymerase, Escherichia coli DNA Pol I large fragment (Klenow polymerase), and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3′ to 5′ exo minus) and dATP to yield a protruding 3-'A' base for ligation of Illumina adapters, which have a single 'T' base overhang at the 3′ end. These products were purified and enriched by 15 cycles of polymerase chain reaction (PCR) to create the final cDNA library. Agencourt AMPure XP magnetic beads by Beckman Coulter were used at each step of the library-making process to purify the desired fragments. The final purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on HiSeq 2000, following the manufacturing protocols.
The reads obtained by RNA-seq were compiled using Bowtie 2 read aligner software. Reads were aligned to the human mRNA reference sequence database (NCBI, ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/human.rna.fna.gz). Only uniquely mapped reads with less than two mismatches were used. The number of each read was further normalized to reads per kilobase of exon model per million mapped reads (RPKM); thus, the values were considered the final expression levels for each gene (Mortazavi et al., 2008).
We used the web-based GO analysis tool, GOrilla (http://cbl-gorilla.cs.technion.ac.il) to map all differentially expressed genes (DEGs) to terms in the GO database, searching for significantly enriched GO terms in DEGs compared with the genomic background. Our analysis was limited to categories with a P-value <0.001. Within the significant category, the enrichment factor was given by (b / n) / (B / N), where b is the number of DEGs within the particular category, B is the total number of genes within the same category, n is the number of DEGs in the gene reference database list, and N is the total number of genes in the gene reference database list.
Genes involved in different steps of a common pathway tend to overlap in their expression profiles. As such, pathway-based analysis can provide insights into the biological functions of genes. Pathways were constructed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and Path-Net was used to identify differential networks and core regulators involved in monocyte to macrophage differentiation (Yi et al., 2006).
Direct binding between different proteins can also reveal shared functionality. The online data analysis tool Ingenuity Pathway Analysis (IPA) version 7.1 (Ingenuity® Systems, www.ingenuity.com) was used to construct a network between the top 10 up- or downregulated genes and their mediators. Transcriptional-Net, a network map of differentially expressed transcription regulators, was constructed using Expression2kinase (X2K) software (http://www.maayanlab.net/X2K). Transcription factors likely to regulate gene expression were identified by X2K and transcriptional networks were built according to protein interaction databases, including BIND (http://www.isc.org/software/bind), BioGrid (http://thebiogrid.org/), and HPRD (http://www.hprd.org/).
Total RNA from monocytes and monocyte-derived macrophages was used for qPCR analyses. Reverse transcription with random hexamer primers was performed with the reverse transcription system (TaKaRa, Japan). Gene-specific primers were designed based on the gene sequences using Primer Premier software. qPCR assays were performed using SYBR® Premix Ex TaqTM (TaKaRa) with an Eppendorf RealPlex4 instrument under the following two-step conditions: denaturation at 95 °C for 5 s and annealing and extension at 60 °C for 40 s for a total of 40 cycles. Hypoxanthine phosphoribosyl transferase 1 (HPRT1) was used as an endogenous reference to assess the relative levels of mRNA transcripts. The relative gene expression changes were measured using the −ΔΔCt method.
Isolated monocytes were over 90% pure, as determined by surface marker CD14 staining, and did not acquire the morphological appearance of differentiated macrophages at day 0. When monocytes were cultured with M-CSF, these monocytes strongly adhered to the plates and displayed morphology characteristic of differentiated macrophages in vitro at day 7. These cells were enlarged with rough edges and distinct from small round, suspending monocytes.
To understand the transcriptional events associated with M-CSF-dependent monocyte to macrophage differentiation further, we performed high-throughput RNA-seq for two poly-A purified RNA samples using an Illumina Genome Analyzer. Table 1 shows that approximately 244 million and 146 million reads were obtained after eliminating low-quality reads, resulting in 188 million and 93 million reads from macrophages and monocytes, respectively. Of these high-quality reads, 188 million (77%) and 93 million (63%) reads were aligned to the human mRNA reference sequences and 26,266 and 25,870 transcripts were measured in primary macrophages and monocytes, respectively. RPKM was used to represent the expression level of each transcript (Mortazavi et al., 2008). Among the transcripts obtained, 1369 transcripts and their variants with P-values <0.01 and estimated absolute log2-fold change >1 were annotated to 1208 DEGs (Supplementary data 1, 2, and 3).
The gene expression profiles derived from RNA-seq were also compared with the profiles generated from microarray analysis (Martinez et al., 2006). The RNAs of these two groups were obtained from both human primary monocytes and M-CSF-stimulated macrophages. Despite technological differences and sample variations, the correlations between these two groups were not low, with R-values of 0.66 and 0.67, for macrophages and monocytes, respectively (Fig. 1A). It suggested that our results were in accordance with the microarray data, as indicated by the significant number of genes determined in both techniques. We also observed that several genes with low expression levels could be detected by RNA-seq.
The top 10 upregulated and downregulated genes following monocyte to macrophage differentiation are shown in Fig. 1B. The list is headed by selenoprotein P (SEPP1), followed by complement component 1q subcomponent C (C1QC), triggering receptor expressed on myeloid cells 2 (TREM2), hepcidin antimicrobial peptide (HAMP), and spleen tyrosine kinase (SYK). The most downregulated mRNAs following differentiation included DNM2, IL-8, CCR2, and CCR7. The IPA web-based tool was used to identify known interactions between these most up-or downregulated genes and their interplayers. As shown in Fig. 2, the most significant interactions between these genes were related to inflammation, as demonstrated by TNF, IFN-γ, and IL-6 in the middle of the map. Downward of the map, the interactions between APOE and PPARG, two important proteins involved in lipid metabolism, were also evident. These results indicate that during monocyte to macrophage differentiation, significant mRNA expression changes in cell function are related to inflammation and lipid metabolism.
To characterize the functional consequences of gene expression changes associated with monocyte to macrophage differentiation, we performed GO enrichment analysis of DEGs based on the GO database. A total of 747 of the 800 upregulated genes and 372 of the 408 downregulated genes were obtained from 1208 DEGs after data mining, reflecting functional molecular changes during differentiation. The biological process of GOs in obtained results was the most robust; thus, we focused on biological processes defined at level 3 with a P-value <0.001. As shown in Table 2, upregulated and downregulated genes shared common GOs including responses to chemical stimulus and regulation of biological quality of GO analysis but appeared to have distinct gene expression patterns. The upregulated genes were mostly involved in metabolic processes, cell cycle, cell division, oxidation–reduction process, and plasma lipoprotein particle clearance. Of these, macrophage scavenger receptor 1 (MSR1), apolipoprotein C-I (APOC1), apolipoprotein E (APOE), scavenger receptor class B member 1 (SCARB1), and CD36 were the genes most enriched in the plasma lipoprotein particle clearance category. By contrast, downregulated genes were characterized with functions of regulation of biological process, locomotion, and cellular component movement. Genes involved in locomotion and cellular component movement numbered 42 and 34, respectively. The majority of the genes in these two categories encoded chemokines and their receptors, such as CCR2, CCR7, CXCR4, CXCR3, CCL5, CCL24, CXCL2, CXCL3, CXCL5, CXCL10, CXCL11, and XCL2. Other enrichment GOs for inflammatory response, apoptosis, oxygen response, and endocytosis were also analyzed. Among these GOs, 33 genes including ALOX5, NF-κB1, DEFB1, and IL-8 functioned in inflammatory response and19genes including NR4A2,VEGFA,SOD2, and CYP1A1 were responsible for oxygen response. Our results suggest that monocytes gradually lose their abilities for cell trafficking and migration upon differentiation into macrophages but simultaneously gain cell metabolic activity and undergo proliferation, and phagocytosis.
To characterize the functional consequences of gene expression changes associated with monocyte differentiation, we performed pathway analysis of DEGs based on KEGG database using the two-sided Fisher exact test. Significant signaling pathways included hematopoietic cell lineage, steroid biosynthesis, cell cycle, lysosome, peroxisome proliferator-activated receptor (PPAR) signaling pathway, cell adhesion molecules, chemokine signaling pathway, nucleotide oligomerization domain (NOD)-like receptor signaling pathway, and cytokine–cytokine receptor interaction.
The majority of DEGs annotated in the lysosome pathway were upregulated, except Lamp-3 (also known as CD63), one of the components of late endosomal/lysosomal membranes. As shown in Fig. 3, Path-Net was used to determine the interaction network of 24 significant pathways (P < 0.05) of macrophages in comparison with those of monocytes. Path-Net was built according to interactions between KEGG database pathways to permit systematic identification of interactions between significant pathways (Yi et al., 2006). The interactions between different pathways were illustrated by their common proteins, as indicated by the lines connecting to pathway labels in Fig. 3. Significant pathway interactions in Path-Net were identified among hematopoietic lineage, cell adhesion molecular, NOD-like receptor signaling, graft-versus-host disease, viral myocarditis, prion disease, and type I diabetes mellitus pathways with interactions ≥8 on the map. A small network was also obviously associated with metabolic process, including pyruvate metabolism, glycerolipid metabolism, propanoate metabolism, butanoate metabolism, tryptophan metabolism, and arginine and proline metabolism.
To identify the regulatory mechanism upstream of genome-wide differences in gene expression, X2K software was applied to identify the transcription regulators among DEGs (Supplementary data 4). The Z-score represents the ability of a gene to mediate, and Degree represents the gene–gene interaction number. The interaction network between these regulators, the so-called transcriptional-Net, was constructed using the BIND, BioGrid, and HPRD protein interaction databases within the software. Key genes with Degree of ≥30 included AR, EP300, SP1, CREBBP, HDAC1, HDAC3, SMAD3, and VDR (Fig. 4). AR, a member of the nuclear receptor superfamily, becomes activated upon androgen binding and translocates into the nucleus to modulate the expression of its target genes. Besides its central function in the growth and development of the normal prostate gland and in the proliferation and progression of prostate cancers, a recent study showed that monocyte/macrophage AR can modulate the inflammatory response by enhancing TNF production (Lai et al., 2009). Both EP300 and CREBBP have histone acetyltransferase activity and participate in the ESR1/SP pathway, with functions in regulating lipid metabolism (Li et al., 2001). HDAC1 and HDAC3, two histone deacetylases, are responsible for the deacetylation of lysine residues on the N-terminal part of core histones and several other non-histone substrates, resulting in transcriptional repression for transcriptional regulation, cell cycle progression, and developmental events (Hayashi et al., 2010). Our results suggest that during monocyte to macrophage differentiation, genome-wide gene expression is largely affected by chromatin epigenetic modification via histone acetylation or deacetylation.
Monocytes and macrophages contribute directly to the immune defense against microbial pathogens. To validate several antiviral DEGs identified by RNA-seq, we selected six genes suspected to interfere with HIV-1 replication during differentiation for qPCR confirmation (Dyer et al., 2008). These genes included interferon-stimulated gene ISG15, two genes encoding tripartite motif proteins (TRIM22 and TRIM47), and other three proteins (TUBA, NUP98, and APOBEC3A). The HPRT1 product was used as an internal control. Primers for qPCR are listed in Supplementary data 5. The mRNA expression of the six genes determined by qPCR was consistent with the changing trends of gene expression from RNA-seq analysis (Fig. 5), supporting the reliability of RNA-seq results. For example, we detected more TRIM22 (9.9-fold), ISG15 (6.2-fold), and NUP98 (3.5-fold) in monocytes compared with those in macrophages using qPCR, whereas RNA-seq showed that there were 6.5-fold, 10.5-fold, and 9.6-fold more for TRIM22, ISG15, and NUP98 in monocytes, respectively. These results indicate that the susceptibility of HIV-1 to monocyte/macrophage is due to changes in the expression of several antiviral proteins during differentiation.
Monocytes originating from bone marrow hematopoietic progenitors are capable of differentiating into morphologically and functionally heterogeneous effector cells. These biological processes are linked to specific sets of cellular functions regulated as a result of cell differentiation. M-CSF is an important factor for monocyte survival and differentiation. However, changes in signal transduction and the subsequent gene expression profile are not well characterized. In this study, we determined how RNA-seq-based high-resolution transcriptome data could be used to understand the biology of monocyte to macrophage differentiation via M-CSF stimulation further.
In vitro monocytes adhere to plastic surfaces and undergo spontaneous differentiation even without exogenous M-CSF (Kaplan and Gaudernack, 1982). We obtained monocyte-derived cells with morphologies resembling those of macrophages cultured with M-CSF. M-CSF-stimulated macrophages reportedly maintain the inactive phenotype (M0), which can be further differentiated into classically activated (M1) or alternatively activated (M2) cells, depending on the stimulus (Dai et al., 2002; Gordon and Martinez, 2010). In our study, expressions of a certain subset of macrophage activation-induced cyto-kine genes (IL-8, IL-1β, and IL-15), M1-specific genes (Nos2 and IL12b), and M2-specific gene (Arg1) were constantly expressed at low levels or dramatically decreased with M-CSF stimulation, except the M2-specific gene CD163, which showed an increase of over 10-fold in expression (Supplementary data 1 and 2). Since only 8% transcriptional profile variance exists in the shift from M0 to M2 (Martinez et al., 2006), the cells we generated were inactive M0 macrophages. Furthermore, the interaction network of most up/downregulated proteins in Fig. 2 showed significant association with inflammation, as evidenced by the TNF-α, IFN-γ, and IL-6 expression profiles. The expressions of inflammatory cytokine gene IL-1β and pro-inflammatory cytokine genes (IL-6, IL-12, and IL-15) were very low, supporting the inactivation of M-CSF-stimulated macrophages with blunt inflammatory responses. Previous studies exploring the global gene profile change that occurs during human monocyte to macrophage differentiation have been limited to SAGE or commercially available microarrays (Hashimoto et al., 1999; Lehtonen et al., 2007). The major advantages of high-throughput RNA-seq are significantly improved detection accuracy, ability to identify alternative splicing without probe dependency, and de novo analysis of novel transcripts and long non-coding RNAs. Total reads were mapped to 25,870 and 26,266 transcript sequences in the human reference database for monocytes and macrophages and differentially expressed transcripts were identified according to P-value and absolute log2 RPKM change. The correlations between RNA-seq and microarray data were fairly well, ranging from 0.66 to 0.67 as previously reported (Marioni et al., 2008). While our comparison features several limitations, including technique difference, bioinformatic algorithms, standardization, and sample variance, it is consistent with a previous study because a large proportion of the genes were determined by two methods (Martinez et al., 2006). Furthermore, qPCR validation of RNA-seq confirmed the reliability of our RNA-seq. RNA-seq is able to detect a wide range of gene expressions, as exemplified by the significant enrichment of the solute carrier family in this study, which controls the uptake and efflux of crucial compounds such as sugars, amino acids, nucleotides, inorganic ions, and drugs. This family regulates transcription through DNA-binding proteins and metal response elements, enzyme activity, including metallo-proteases, superoxide dismutase, inducible NO synthase, endosomal fusion, and metabolism. We observed 35 of DEGs for solute carriers measured by RNA-seq.
Our results reveal that locomotion-related gene change is associated with monocyte to macrophage differentiation (Table 2). Most DEGs encoding chemokines CCL5, CCL24, CXCL2, CXCL10, CXCL3, CXCL5, and CXCL11 and their receptors CCR2, CCR7, CXCR4, and CXCR3, except CCL2 and CCL13, were downregulated. CCR2 expression on the surface of inflammatory monocytes and binding with CC chemokines MCP-1(CCL2), MCP-3(CCL7), and MCP-4(CCL13) can direct monocyte recruitment to inflamed tissue sites (Franci et al., 1995). This effect accounts for why monocytes tend to migrate to inflammatory sites and be differentiated into tissue-specific macrophages. With decreased CCR2, monocytes lose their ability for trafficking but retain the ability to recruit immune cells because of the expressions of CCL2 and CCL13. The chemokine expression profile determined in our study is also different from that of polarized macrophages (Martinez et al., 2006), where macrophage activation is associated with upregulated chemokines, which contribute to their pathophysiology.
The phagocytic capacity of macrophages is central to their function. This capacity principally involves the clearance of cellular debris during physiological homeostasis and the removal of exogenous particles, including microorganisms. We observed that genes encoding phagocytosis receptors, such as MSR1, mannose receptor (MR), Fcγ receptors (FcγR), and CD36, were upregulated, enabling macrophages to uptake apoptotic cells and foreign pathogens (Anderson et al., 1990; Febbraio et al., 2001). Subunits of vacuolar type proton ATPase (ATP6V1H, ATP6V0E2, and ATP6V0D2) and acid hydrolase cathepsins (CTSK, CTSF, CTSL, CTSK, CTSW, and CTSC), which are required for the maintenance of the acidic milieu, markedly increased, suggesting greater degradation capacity in the phagosomes of differentiated monocytes.
Of the biological processes identified among the upregulated genes during monocyte differentiation, metabolic processes are among the most prominent (Table 2). These processes include not only lipid, steroid, and fatty acid metabolic pathways, as previously described (Liu et al., 2008; Martinez et al., 2006), but also amino acid and alcohol metabolic pathways. Proteins important in lipoprotein homeostasis, such as APOE and APOC1, are significantly induced in macrophages, consistent with another study that employed SAGE and utilized short sequence tag oligonucleotides and M-CSF (Hashimoto et al., 1999). Receptors important for the binding of modified lipoproteins, such as MSR1, SCARB1-3, and LDL-related protein, were all highly expressed. Genes including lipase A (LIPA), which promotes chylomicron formation and atherosclerosis (Kodvawala et al., 2005), FABP3, which regulates cholesterol export (Hansson et al., 2003), and FADS1, a member of the fatty acid desaturases, were also upregulated in macrophages. Protein network and Path-Net analysis revealed that the PPARG (PPARγ) pathway, particularly PPARγ and acyl-CoA cholesterol acyltransferase 1 (ACAT1), was significantly affected during differentiation (Figs. 2 and and3).3). PPARγ is known to induce lipid uptake and storage (Quinn et al., 1987) and activate CD36 expression (Nagy et al., 1998), thereby promoting intracellular cholesterol accumulation and foam cell formation. ACAT1 is an intracellular enzyme that biosynthesizes cholesteryl esters from long-chain fatty acyl-CoA and cholesterol in various tissues. CYP27A1 is a molecule responsible for producing 27-hydroxy-cholesterol for the alternative bile acid synthesis pathway. ACAT1 and CYP27A1 were both upregulated in the PPARγ pathway, in accordance with a previous study (Quinn et al., 1987). Therefore, the PPARγ pathway significantly contributes to the robust activity of lipid metabolism during macrophage differentiation.
Compared with macrophages, monocytes lack active gene expression for metabolic processes. However, we detected a number of transcription factors including FOXO1, RUNX3, NF-κB1, CCAAT/enhancer binding protein δ (C/EBP δ), FOSB, and nuclear receptor family NR1H3, which were all significantly expressed in monocytes (Supplementary data 4). For example, FOXO1, a forkhead box class-O transcription factor was recently shown to be an inhibitor of lipogenic gene expression (Deng et al., 2012). Both RUNX1and RUNX3, which function by recruiting co-repressors and histone deacetylases, are involved in transcriptional repression and gene silencing during T-cell development (Durst and Hiebert, 2004). NF-κB1 is capable of inhibiting transcription via histone deacetylase HDAC1 (Zhong et al., 2002). Thus, these genes contribute to transcriptional repression in monocytes, resulting in slight metabolic processes. Transcriptional-Net, which was used to dissect the regulatory mechanismofgenome-wide geneexpression, indicated thatglobal gene expression is also controlled by histone acetylation or deacetylation during chromatin epigenetic modification in monocyte to macrophage differentiation (Fig. 4). The detailed mechanism of modulation of general cellular metabolic activities during macrophage differentiation remains unclear.
In agreement with earlier studies, our data indicate that a number of cell cycle-related genes, including cyclin proteins, cyclin-dependent kinase (CDK), and cyclin-CDK inhibitors, are significantly changed to drive the cell cycle (Table 2). Although human mononuclear phagocytes, unlike mouse macrophages, are generally considered terminally differentiated non-proliferating cells, reports of human macrophage proliferation have been published (Cheung and Hamilton, 1992). In this study, we found that DNA checkpoint-related genes DNA-PK, MAD2L, and CHEK2 were upregulated in macrophages, indicating the subtle quality control of cell proliferation. We also observed that STMN1 and RSK2, two key components in ERK-1/2 signaling pathway, were upregulated. M-CSF triggers ERK-1/2 activation and inhibition of this pathway results in arrested bone marrow macrophages at the G0/G1 phase (Valledor et al., 1999). Finally, we observed that C/EBP δ expression decreased, the over-expression of which can result in G0/G1 proliferative arrest and a moderate increase in apoptosis in K562 cells (Gery et al., 2005). Thus, human monocyte-derived macrophages appear to favor proliferation rather than remaining in the G0 quiescent state.
Monocytes and macrophages serve as sentinels involved the eradication of various pathogens. It has been well known that monocytes are an important HIV-1 reservoir and potential contributors to viral latency (Ellery et al., 2007; Lambotte et al., 2000). Despite the expression of HIV-1 receptors, undifferentiated monocytes are resistant to HIV-1 infection in vitro, whereas monocyte-derived macrophages and DCs are permissive for productive HIV-1 infection. Therefore, the identification of the restricting factors during monocyte differentiation would be interesting. Our results confirm that several proteins with antiviral functions are highly expressed in monocytes and downregulated with macrophage differentiation (Fig. 5), including TRIM22, TRIM47, ISG15, TUBA, APOBEC3A, and nuclear pore complex protein NUP98. However, we did not detect an expression change in SAMHD1, which was recently identified as an HIV-1 restriction factor in myeloid cells (Goldstone et al., 2011). The correlations between the expression of these factors and HIV-1 infection during monocyte differentiation must be further characterized.
In conclusion, we showed that critical gene expression profiles change during monocyte to macrophage differentiation, including those genes involved in inflammation, phagocytosis, metabolism, cell cycle, and transcriptional regulation. Downregulated transcriptional repression during monocyte differentiation can result in increased metabolism and activated cell cycles, which promote macrophage differentiation. Transcriptome comparison of the genes selectively expressed in human blood monocytes and M-CSF-induced macrophages will provide useful information in defining the ontogeny, development, and function of cells in monocyte and macrophage lineages.
This work is supported by the China NSFC grants 81101257 and 31270977; the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT); the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); and in part by the NIH grant AI098524 (L.W.) from the United States. The authors thank Ms. Heather Hoy for the help of sample preparation, Dr. Hongyan An and Dr. Ming Yi for their excellent assistance during data analysis and Dr. Renjing Liu for the scientific editing on the manuscript.
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.gene.2013.02.015.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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SourceYourInterior.com releases e-Auction Phase
Today, SourceYour announced the release of the e-Auction Phase on SourceYourInterior.com.
Bastiaan Brouns, creator of SourceYour and SourceYourInterior.com said: "In the e-Auction Phase, the platform shows its real power. Full data integrity is guaranteed; data which has been checked and approved is transferred one-to-one from the Information and Qualification Phases. In addition, our e-Auction Phase can be executed on six different criteria with full transparency to the bidders".
The new e-Auction Phase is a unique tool to get the best long-term value for money. It has the following setup:
In addition to standard items, such as communication options, product specification, date and time of the e-Auction, options for extension and type of contract, a customer selects the weight he attaches to the different auction elements: product cost, transport, delivery and installation cost, price of services, guarantee period, continuity of supply period and terms of payment.
Customers can establish their own level of transparency by deciding whether they want the bidders to know the names of other bidders and of the customers' incumbent suppliers. They can weigh the different e-Auction elements, ascertain when the contract will be awarded and decide if the contract will be automatically awarded to the winner (and if not, what following steps will be taken).
This involves the execution of the e-Auction at the specified date and time. Tracking the e-Auction is made easy for all participants: the dashboard is built specifically for each e-Auction and is derived from the customized set-up by the customer.
Bastiaan Brouns explained: "In general, e-auctions only focus on the price of the product and are reputed to reduce the cost by an average 18%. At SourceYour, we believe you get what you pay for – we believe the focus should be on value and total cost of ownership. Buying a product is the easy part, the challenge is to own it at the lowest cost during its lifetime."
See the demos on the e-Auction Phase and the other phases on: http://sourceyourinterior.com/demos
About SourceYour and SourceYourInterior.com
SourceYour and SourceYourInterior.com are initiated by SY Services BV in Amsterdam, The Netherlands.
Aimed at Facilities & Furniture, the aim of SourceYour is to make purchasing processes sustainable.
spend less money | reduce your waste | improve your decision
Rethinking the way things are done and supporting education to create sustainable improvement are core values of SourceYour. Everybody deserves a chance to know better.
SourceYour | so you know better
Blog: http://duotrainin.wpengine.com
Facebook: http://www.facebook.com/sourceyour
YouTube: http://youtube.com/soyouknowbetter
For feedback or more information, please contact:
E firstname.lastname@example.com


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Friday, March 09, 2012
Erin go braugh (or "the Dude abides")
Here is the one photo I managed to get for you...after a lengthy round of cursing, I eventually sent it to Nate's phone, where I then emailed it to myself. All for you. You're welcome.
This is not even the best photo in my opinion, but it was the one that screamed Boston. The best has to be either the shamrock-shaped rice krispy treats, which are of course covered in disgusting green flecked icing, or the leprechaun beards and fuzzy green hats. Did I mention these were for sale in the grocery store?
Needless to say, St. Patrick's Day is enthusiastically celebrated in Boston culture, and you don't need to be Irish to join in the fun. The title of my post includes my three words for the day, which I shared with Jenni from the Blog. My word choice has to do with two separate recent events. First, I was looking at the cupcakes and noticed a little shamrock reading: Erin go braugh. I figured it was something like "Go Ireland!" or "Good luck!" in Gaelic (it turns out it is the former, except more like "Ireland Forever"), and I think it's interesting from an anthropological perspective that so many people have retained knowledge of the meaning of this phrase that it can be found scrawled on cupcakes and t-shirts all over Beantown.
The second of the titular events has to do with a little bear Charlie has become fond of lately.
It is pure coincidence that he's decked out in St. Paddy's shamrock green, but it does make me chuckle that she wants to bring him everywhere, as if she somehow intuitively understands the culture here and is an active participant. The funniest part of this little guy is that she inexplicably calls him Doo. Not Doo, acutally, but "DOOOOO!" And when this is screamed enthusiastically, it sounds a lot like "DUDE!"...which is of course what Vivi and I now call him. Or El Duderino, if you're not into the whole brevity thing.
Do you have plans to turn regular grub green with food coloring? This is a holiday I can let pass with little fanfare, but I wouldn't mind if someone made me these Irish car bomb cupcakes. I consider that an unlikely prospect though, so this is how I intend to celebrate St. Paddy's:
Do you celebrate St. Paddy's? What are your plans?
4 comments:
So...um...I've never really participated in St. Patrick's day. I considered running in a St. Patrick's day dash a few times but it never happened. Usually I grab something with a little green on it to wear at the last minute to stave off comments. Now that my kiddo is getting older I'm realizing that it's assumed we'll participate in the holiday. Since I mainly associate the holiday with wearing green and getting drunk (sorry if that offends anyone) I'm not sure where to start with a toddler. I'm not a fan of food coloring so I won't be dying anything green. I suppose we'll be focusing on naturally green foods. I saw a rainbow fruit platter on pinterest that may be in our future. Otherwise, I'll just keep trying to reconcile the historical origins of the holiday with the current celebration that seems to focus on cultural misinterpretations and stereotypes. That said, I know a lot of people who have a lot of fun with the holiday and love it...that's probably way more than you really wanted to know about my feelings on this topic. I'll stop now before people start judging and hating me : )
- The Lone Home Ranger said...
Ha! I love it. Nate and I are both partly Irish, so we share a fondness for all things associated with Ireland (except perhaps the Lord of the Dance); however, I do think it's rather silly that people dye food green. I'm with you on being icked out by green food coloring. I love the natural food rainbow idea! My favorite St. Patty's ever was when we were living in Madison, WI and took my best friend to Chicago for the parade. We got pictures by the green river. Maybe I'll go dig them up and put one up here. It was so amazing and fun!
- sarah saad said...
اهم شركات نقل العفش والاثاث بالدمام والخبر والجبيل اولقطيف والاحساء والرياض وجدة ومكة المدينة المنورة والخرج والطائف وخميس مشيط وبجدة افضل شركة نقل عفش بجدة نعرضها مجموعة الفا لنقل العفش بمكة والخرج والقصيم والطائف وتبوك وخميس مشيط ونجران وجيزان وبريدة والمدينة المنورة وينبع افضل شركات نقل الاثاث بالجبيل والطائف وخميس مشيط وبريدة وعنيزو وابها ونجران المدينة وينبع تبوك والقصيم الخرج حفر الباطن والظهران
شركة نقل عفش بجدة
شركة نقل عفش بالمدينة المنورة
شركة نقل عفش بالرياض
شركة نقل عفش بالدمام
شركة نقل عفش بالطائف
شركة نقل عفش بمكة
- sarah saad said...
شركة تنظيف خزانات بجدة الجوهرة من افضل شركات تنظيف الخزانات بجدة حيث ان تنظيف خزانات بجدة يحتاج الى مهارة فى كيفية غسيل وتنظيف الخزانات الكبيرة والصغيرة بجدة على ايدى متخصصين فى تنظيف الخزانات بجدة
شركة تنظيف خزانات بجدة
اهم شركات كشف تسربات المياه بالدمام كذلك معرض اهم شركة مكافحة حشرات بالدمام والخبر والجبيل والخبر والاحساء والقطيف كذكل شركة تنظيف خزانات بجدة وتنظيف بجدة ومكافحة الحشرات بالخبر وكشف تسربات المياه بالجبيل والقطيف والخبر والدمام
شركة مكافحة حشرات بالدمام
شركة كشف تسربات المياه بالدمام


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It's been one hell of a week for Nasty Gal founder Sophia Amoruso.
At 32 years old, Amoruso ranks higher than Beyoncé on Forbes' Richest Self-Made Women list and is fast becoming a household name thanks to her internationally best-selling book, #GIRLBOSS.
She's currently on a whirlwind tour of Australia, speaking at Business Chicks events in Brisbane, Melbourne and Sydney, where audience members payed $169 a head to hear "the good, bad and ugly bits" of Amoruso's journey through the e-commerce world. However, yesterday news broke that Nasty Gal is filing for bankruptcy.
The timing is impeccable. No one could have predicted this, not even Amoruso. "I didn't expect this to happen while I'm here," she told the audience at this morning's Sydney event. "Leading up to a choice like this that a board has to make to fulfil its judiciary responsibilities, there are so many factors - and options on the table."
"Many were considered... and ultimately what was crazy is that filing a Chapter 11... it's actually the most responsible thing to do right now, which is just like a bonkers thing to actually consider."
"As it stands, what happened yesterday actually allows us to continue operating and to provide some relief and a moment of pause as we get out of things, like a 500,000 square-foot lease in Kentucky."
Amoruso implied that things took a turn for the worst when Nasty Gal took on investors. "I think we should run our own businesses. Nasty Gal was profitable until we took investor money. When women are in control of their own businesses, they can scale and grow thoughtfully."
"For my first business... I got really far," she continued, fighting back tears. The crowd cheered Amoruso's honest admission – which you would expect from an event that champions women supporting women.
At this point, it appears that the future of Nasty Gal is up in the air.


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AN UNLICENSED and drunk driver is in custody after driving the wrong way down a one-way street in the city, sparking a police chase early this morning.
Police on Hindley St saw the white Holden Commodore driving the wrong way down Bank St about 1.30am.
Officers attempted to pull over the car on South Rd at Croydon a short time later, but the driver refused to stop.
Another police patrol saw the car on Matthews Ave at Seaton, where the driver abandoned the vehicle and attempted to run away.
The 31-year old man, of no fixed address, was arrested soon after and allegedly recorded a blood-alcohol level of 0.015.
Police charged the man with offences including engaging in a police pursuit, dangerous driving and drink and unlicensed driving.
He was refused police bail and is expected to appear in Adelaide Magistrates Court later today.
The incident was one of three car chases in Adelaide overnight, including one in which a member of the public was injured.
Originally published as Wrong-way driver leads police on chase


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Picture: The Killers perform at the Tennants Vital 07 festival in Belfast's Ormeau Park Belfast, Ireland - 22.08.07
The Killers frontman Brandon Flowers has finally given the reason behind his hatred of THE BRAVERY - he is jealous of them.
The MR BRIGHTSIDE singer has been involved in a long-running spat with the New York electro rockers, originally slamming them for copying his style.
But he now admits he simply becomes angry when he hears his rivals are doing well.
He says, "I've never actually said anything bad about anyone who didn't deserve it, but occasionally it's brought on by jealousy.
"When I hear a good song it really does p*ss me off."07/06/2005 17:26


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The Blaze Nitrile Glove satisfies a variety of different purposes and features textured fingertips for a surer grip. The high visibility orange makes these gloves ideal for hazardous work while the extra-long cuff protects more arms. These non-sterile, ambidextrous gloves are approved for medical use. High visibility orange color makes this glove perfect for safety and EMS applications: paramedics, firefighters, EMT's and other first responders.
- Hi-visibility orange color
- Textured fingertips
- Extra-long cuff (10.5 in.)
- Approved for medical use
- Non-sterile/ambidextrous
- For single use only
- Power-free, 5 mil
- 200-count


text:
The Randall RD412D Diavlo 4x12 Straight Guitar Speaker Cabinet was designed to match perfectly with high-gain amps like the Randall Diavlo series heads. With four 12-inch Randall-designed speakers, and an angled baffle front loaded design with tuned triple porting, it's clear the RD412D cab is designed to move some serious air! With a power rating of 320 watts and a heavy duty steel grill, the Randall RD412D is the perfect choice for traveling guitarists. Casters included.
What started as a simple string and pedal sales catalog has grown exponentially over the past thirty years. The early days were a time where catalogs didn't have much of a presence in the industry, but what began as a college dorm room operation grew rapidly. In 1986 we moved to a full product offering and 64-page catalog, which over the years has grown to 162 pages. Join the AMS family and get your free catalog now!
Copyright © 1996-2017 American Musical Supply, Inc. All rights reserved. American Musical Supply reserves the right to correct pricing and/or product specifications in the event of typographical errors found in our print catalog or on our website. If a product is listed at an incorrect price due to such errors or because of inaccurate information received from a supplier, American Musical Supply shall have the right to refuse or cancel any orders placed, whether or not the order has been confirmed and your credit card charged. If your credit card has already been charged for the purchase and your order is canceled, American Musical Supply will issue a credit to your credit card account in the amount of the charge.


text: Not sure what i've done to my phone, it's not bricked, but I can't get it to go back to the default settings. Each time I do a factory reset, it still has the tether app installed and other things as well. How do I take it back to the original, or at least somewhat updated original firmware? Please provide a specific link. I went to the samsung firmwares website and had no idea how to navigate that stupid thing.


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Places Covered: Uttarkashi, Yamunotri, Gangotri, Haridwar, Rishikesh, Badrinath, Pipalkoti, Kedarnath, Rampur
Char Dham yatra Package Itinerary
Day1: Delhi
Departure to Haridwar by Bus. Over Night Journey
Day2: Haridwar – Rishikesh – Barkot
Arrive at Haridwar check-in the hotel and relax for some time. Later proceed to Barkot. Enroute visit Rishikesh where you visit Shivananda Ashram, Ram jhoola, Swarg Ashram, Geeta Ashram, Lakshman Jhoola. After exploring these places continue to Barkot. On arrival check in the hotel. Overnight stay at the hotel.
Day3: Barkot – Yamunotri – Barkot
After Breakfast proceeds to Hanumanchatti then take a jeep to Janakichatti from where you can start trek to Yamunotri temple. After visiting Yamunotri temple return back to Barkot for the overnight stay.
Day4: Barkot – Uttarkashi
After Breakfast morning is at leisure. Later in Afternoon pack your bag and start drive to Uttarkashi. On arrival at Uttarkashi check in the hotel. Overnight stay at the hotel.
Day5: Uttarkashi – Gangotri – Uttarkashi
After Breakfast proceeds to Gangotri. On arrival take a dip in the holy Ganges. After performing pooja return back to Uttarkashi. Overnight stay at the hotel.
Day6: Uttarkashi – Srinagar
After Breakfast packs your bag and checks out the hotel then proceed to Srinagar. On arrival check in the hotel and relax for some time. Overnight stay at the hotel.
Day7: Srinagar – Gupta Kashi – Rampur
In Morning check out the hotel and proceed to Rampur. On the way, you visit Rudraprayag the confluence point of 2 Holy rivers – Alaknanda & Mandakini and then continue drive to Rampur. On arrival check in the hotel and relax for some time. Overnight stay at the hotel.
Day8: Rampur – Kedarnath – Rampur
Early in the morning proceed to Sonprayag then to Gaurikund and later proceed to Kedarnath by trek, poni or helicopter. Arrive at Kedarnath visit temple for darshan. Later return back to Rampur for the overnight stay.
Day9: Rampur – Badrinath
After Breakfast check out the hotel and drive to Badrinath. On arrival check in the hotel and relax for some time. Overnight stay at the hotel.
Day10: Badrinath – Pipalkoti
Early in the morning visit Badrinath Temple for darshan and then visit Mana village, the last village before the Indo – Tibetan border. In afternoon start drives to Pipalkoti. On arrival check in the hotel for the overnight stay.
Day11: Pipalkoti – Haridwar
After Breakfast at hotel check out the hotel and proceed to Haridwar. Later in evening visit Hari-Ki-Pauri to attend aarti. After aarti back to the hotel to board a bus to Delhi.
Day12: Delhi
Arrive at Delhi and proceed to your onwards journey.
Package Price
Apr to Oct (Ac)
Adult on Twin Sharing
Rs 22,900
Rs 21,900
Adult on Triple Sharing
Rs 22,400
Rs 20,900
Child(5-11)Without Bed
Rs 9,400
Rs 18,400
Single Adult In a Room
Rs 25,900
Rs 24,900
Note: Govt. Service Tax @9.00% applicable.
Tour Requirements
- For Adults – Photo identification in the form of Voters ID / Passport / Aadhar Card / Driving Licence / Ration card & Carry 4 passport sized photographs.
- For students – Photo identification in the form of Passport / Aadhar Card / PAN card Carry original as well as 2 photocopies of school or college Photo Identity card, Carry 4 passport sized photographs.
- For Infants – Carry 2 passport sized photographs
For Booking And More Details Visit www.southerntravelsindia.com
Southern Travels Pvt. Ltd.
18/2, Arya Samaj Road,
Karol Bagh,
New Delhi-110005
Email -: firstname.lastname@example.com
Phone-: +91-11-43532800 (100 Lines),
43532806, 43532807, 28753466


text:
What's on
Food Events and Training
Find out about food festivals, markets, talks, film screenings, community feasts and training.
Shape Up in the Kitchen
Learn to cook nutritious and healthy meals to take control of your weight.
Shape Up
Free weight management programmes for you and your family.
Bespoke Courses
Our staff can lead workshops and training on a range of food topics
Eat Well Workshops
Our nutrition team delivers free sessions to community groups or those working with communities across the city.


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Overcoming the related challenges of child labour and the lack of decent work opportunities for youth will be critical to Rwanda's progress towards the Millennium Development Goals. The effects of child labour and poor youth employment outcomes are well-documented: both can lead to social vulnerability and societal marginalisation, and both can permanently impair productive potential and therefore influence lifetime patterns of employment and pay.
This Quick Note provides an overview of the World Bank report "Striving for Better Jobs: the Challenge of Informality in the Middle East and Africa". It looks at the hopes of Arab youth during the spring revolution and the current economic situation by focusing on informal employment at a human development angle.
This note presents and analyzes the main design features of a variety of non-publicly provided Active Labor Market Programs in Arab-Mediterranean Countries, with a specific focus on programs targeted at youth. Programs from nine countries are included in the inventory: Morocco, Algeria, Tunisia, Egypt, Lebanon, Syria, Jordan, West Bank and Gaza, and Yemen.


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Goodland Republic, Sept. 1, 1911
Died: Aug. 31, 1911
Striken at Fair Grounds and Was Taken to His Home in North
Goodland---Funeral Sunday.
J. H. Bardwell of this city, suffered a stroke of apoplexy at the fair grounds at about 4:30 p.m.. Thursday. He was taken at once to his nearby home in north Goodland and medical aid was summoned. Dr. Carmichael came and pronounced it a case of apoplexy. Everything possible was done to recussitate him but without avail, and he sank away and died about 9:30, five hours after the stroke.
The members of the family, living in Cheyenne county, and elsewhere, were notified, and the funeral was held from the house at the Congregational church at 11 o'clock Sunday forenoon, and burial was made in the city cemetery. Rev. J. R. Thomas of the Methodist church preached the funeral sermon and officiated at the interment; the Woman's Relief Corps and the G. A. R. post, of which he was a member, attended in a body, and read the ritual of their orders at the grave. The floral offerings were profuse and beautiful and blooming flowers, evergreens, and the flag, were laid upon the casket in token of regard, sorrow, sympathy and eternal hope.
Comrade Bardwell was quartermaster
of the W. S. Robinson Post, and he was
also a member and official of the Methodist church. Rev. Thomas took for his text words
from Hebrews 11:14-16.
"For they that say such
things declare plainly that they seek a country. And truly,
if they had been mindful of that country from whence they came out. they might have had
opportunity to have returned. But highly, now they desire a better country, that is. an
heavenly; wherefore God is not ashamed to be called their God; for he hath prepared for
them a city."
The sermon was appropriate to the occasion.
There was a large attendance at the funeral. The immediate relatives of the deceased present at the funeral were: Mrs. J. H. Bardwell. the widow, and her brother, Rev. James F. Johnson of Luray. Kan.; Alvin Bardwell of Loveland. Colo.; Helen F. Bardwell of Goodland; James L. Bardwell. Walter J. Bardwell. and George I. Bardwell all of Cheyenne county. Kan.; and Earl M. Bardwell of Rivenna. Neb. One daughter. Mrs. Lillie Bardwell Biggs, of Huntington. Ark., was not here at the funeral. All the other living children and several grandchildren were present at the services. Mr. Bardwell. the deceased, has a brother, M. L. Bardwell. residing at Gill. Colo., but was not here at the funeral.
Joseph H. Bardwell was born near Buckville. Ohio, in 1844. In boyhood his parents moved to Michigan and later to Illinois. February 10, 1862. he enlisted in "I" company, First Illinois Light Artillery, and after serving two years he "veteranized." re-enlisted for three more years or the close of the war. He was discharged in Chicago; July 26, 1865, after having served three years and nearly six months. He was united in marriage to Sarah A. Johnson, at Springfield, 111.. August 19, 1869. From this union eight children were born, all living but one son, Harry C. Bardwell, who died in Nashville, Mo., in 1887. The wife and mother, five sons and two daughters, still survive the deceased and deeply mourn his loss. The family moved from Illinois to Missouri, and then to Kansas, where they have since resided. The deceased, when about 25 years of age, was converted and joined the Methodist church, of which religious body he has since been a faithful and consistent member. In the language of the great "Apostle to the Gentiles," he has fought a good fight, he has finished his course, he has kept the faith; and henceforth there is laid up for him a crown of righteousness, which the Lord, the righteous judge, will give him in that day."
"Servant of God, well done! Thy glorious warefare's (sic) past; The battle's fought, the race is won. And thou are crowned at last."
The resident of Mr. Bardwell and family has been quite brief, something like a year or so, in Goodland; but in this short time our people, who have come to know him, have learned to respect him, and those quite intimately acquainted, to esteem him highly. His mild, quiet, unobstrusive (sic) manner has impressed many and prepared the way for lasting friendships. But he has passed to the calm and rest of the "heavenly country."
Card of Thanks
We desire to thank our many kind friends for the assistance, kindness, and sympathy, also for the beautiful floral offerings, during the recent death of our beloved Husband and father. We wish to make special mention of the service rendered by the W. R. C. and G. A. R.
Mrs. Annie Bardwell and Family.


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AJ Help Desk Software - Customer Support and Ticketing Software
AJ Help Desk - Our web based Help Desk Software offers easy handling tools that satisfy clients or customers. Powerful PHP based Help Desk Software caters you to operate in facile manner and support all your business activities. The AJ Help desk software support product is a must-have for any new or existing on line business. Even if the majority of your business is off-line, many people prefer on line product and service support. Customers need their questions answered quickly and personnel need a way to effectively manage their work. Help desk software is an economic , easy-to-use and useful tool for the growth of your business. It helps customers who can simply submit an inquiry to the appropriate department and expect a timely response. With the use of this product the customer and technical support staff are better able to manage troubleshooting.Visit publisher site: AJ Help Desk Software - Customer Support and Ticketing Software
Listing Details
- Version:
- 1.0
- Filed in:
Scripts / PHP / Scripts & Programs / Customer Support / Help Desk
- Submitted on:
- Last Updated:
- Mar 4, 2011
- Publisher:
- AJ Square Inc Other listings by this publisher
License & Pricing Information
- License Type:
- Commercial
- Price:
- Additional Info:
- Free Demo Available for AJ Help Desk Software.
User ReviewsBe the first to review this listing!
Not yet reviewed by any member.... You can be the FIRST one to write a review for AJ Help Desk Software - Customer Support and Ticketing Software


text:
Red Party Hat With HeartsItem #: 17281
4,99 €incl. VAT, plus shipping
Order fast! Only a few items left
Delivery until 21.11. possible**
Fitting for this:
Add the product directly to your basket by clicking the checkbox
Add the product directly to your basket by clicking the checkbox
Red Party Hat With Hearts
Red party hat made of felt with many colorful hearts. The red party hat in the form of a classic melon is the hammer among the carnival hats and is always gladly put on to spread a good mood.
The funny, red party hat has a standard size for adults. The red carnival melon is made of felt and has roundly colored colorful hearts and is additionally decorated with a red hat band. Whether you're a fun clown or a nice gentleman, the colorful party hat is always a party. Matching ours would be Clown shoes blue and yellow with flames And a red cheeky Foam clown nose ,
- Material: 100% polyester


text:
Extreme Couponing
by: Pregasaurus • 511
All Android applications categories
by: Pregasaurus • 511
Extreme couponing teaches you how to save $100s on your grocery bill. Download our free app and learn the secrets of the Extreme Coupons pros.
With our app, you get new grocery coupons right on your phone for free so you can save money on the go.
Our free app gives you a lesson plan that teaches you every trick in the book to save money and start becoming an Extreme Couponer.
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text:
hawk hill press website re-design
The Re-Design
- Retooled the existing logo for more presence
- Added beautiful top banner for interest
- Changed text/ background for better readability
- Hawk Hill books are full color, stunningly beautiful coffee table books, but their old website did nothing to showcase this to potential customers. We fixed this by creating a photo gallery for each one of their books
- Added reviews and awards for each book
- Added PayPal Shopping Cart
- Custom shopping cart buttons
- Custom Banner for PayPal page
SEO (Search Engine Optimization)
As for the SEO on this site, Hawk Hill was in pretty desperate shape in the beginning. Even typing "Hawk Hill Press" into Google (or one of their book titles) showed only links to Amazon.com. After working with Blue Oak, not only do they show for their chosen keywords, they get 1st page results!
As you can see, having a website professionally re-designed can make a huge difference to your potential customers. Interested in a website re-design? Contact us.


text:
User:Jacqueline Ohmura
From OpenWetWare
I am a new member of OpenWetWare!
Contact Info
- Jacqueline Ohmura
- Koch Institute
I am currently a third year Biological Engineering PhD student in the Belcher lab, this is my second semester of assisting with the 20.109 course.
- 2016 (In progress), Biological Engineering PhD, MIT
- 2011, Materials Science and Engineering BS, The Ohio State University
Research interests
- Biomolecular materials
- Enzyme catalysis


text:
Published on Amazon? If you have a book, e-book, or audiobook available on Amazon.com, we'll promote it on WritingForums.org for free. Simply add your book to our Member Publications section. Add your book here or read the full announcement.Dismiss Notice
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I have never been very careful with my words.
It sounds so much better in Greek.
In Greek a lot of sentences and phrases sound nicer in context then they do in English.
I phrased some stuff really bad in English, I didn't know they sounded rude cause in Greek they don't sound as rude and jerkish. Well now saying what I did everyone is mad at me now.
I really didn't know it was that rude.
I'm such a screw up sometimes. Now I think I have lost a some people as friends and I have lost some respect too.
I didn't mean to.
I really didn't it.
I don't want lose anyone, but I feel like this is going to effect in a very negative way. And the people that were my friends are going leave me. I know its the internet, but I found a place where I belonged or at least a place where I could talk to people, but I messed that up just as much as I messed the real world up for myself.
I'm such a screw up.
And they probably don't want to talk to me any more. I'm upset at myself and I don't want to lose anyone.
Because that means that I'm going to be lonely again and hated.
I don't want to be hated.
Why do I always screw up the good things? Why?
I hate myself sometimes and this is one of those times.
I needed someone to talk or write out how I feel so I put it here.
I want to apologize, but I don't feel I have done anything wrong.
And to apologize would mean that I have done something wrong.
But I didn't do anything wrong.
At least I don't, but I know those people think I have done something wrong.
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MOVIE SYNOPSIS [ General Audiences ] Directed by Michael Habernig, F. Holmes Skip Atwater heads the cast of this feature film originally of USA, with original script in English, whose premiere was scheduled for 2009. You can watch The Path: Afterlife online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2009.
Rating: 5 by 5 users./
Runtime: 71 minutes
F. Holmes Skip Atwater, Patty Ray Avalon, William Buhlman, Brian Campbell, Thomas Campbell, Robert Colby, Paul M. Corrigan, Jeannette Duffau, Bernard Gray, Nate Gray, Kelly Habernig, April Hannah, Marian Hannah, Brenda Jenks, Joseph Nagele and Beth Netter.
71-minute film (full movie) directed by Michael Habernig.
The original audio of The Path: Afterlife has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2009.
Michael Habernig.
April Hannah.
Path 11 Productions.
Without commercial breaks, the full movie The Path: Afterlife has a duration of 71 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, The Path: Afterlife in FULLTV Movies.


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Country girl Jan bends over the tree to be fucked from behind tough
Description:Pack of Porn sex clip provides you with too horny amateur slut. This pale brunette with big tits lives in a village and has no other hobby than sucking hot tasty lollicocks for sperm. Too voracious nympho seduces a stranger to get her wet cunt drilled from behind right on the country road.
Category:+18 only (Adult)
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Winter Solstice - Performance/Accompaniment CD
To play the media you will need to update yourFlash plugin.
Composed by Ruth Elaine Schram. Concert. Performance/accompaniment CD. Heritage Music Press #99/2576H. Published by Heritage Music Press (LO.99-2576H).
Item Number: LO.99-2576H
From the collection "Every Day's a Holiday!" (30/2477H) emerges this haunting, almost-mysterious musical description of the longest night of the year. Information explaining this celebration (and the text of the song) is included, making this a wonderful cross-curricular teaching piece for young and developing choirs and a unique selection for your winter or holiday concert.


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Discussion in 'Introduce Yourself Here' started by CANDY HIPPIE, Jan 30, 2013.
hit us up if ur in the atl ga and north of atl ga
Hello Lovely Angel, I want to know besides the size do you prefer your guy to be exlusively very DARK, Ebony toned, muscular and of course hung for the "TRUE" BLACK experience and the hot, stark, erotic contrast of your Lily white body pressed up to a deep Ebony guy. If you prefer this post me
how long is ur cock and where u from
I am a tall, 6'1, lean muscular build, deep DARK, Ebony guy at about 8.5in and 4in thick. So how about it, do you only prefer your guy to be very Dark skin and masculine/muscular for the stark, erotic contrast? If you prefer this post me Doll
and ur from where
I'm from Chicago. but will be in ATL on March 5 and 6 for a job conference and hang out there with family on regular basis. Still interested? And do you prefer your guy to be solid DARK, Ebony toned, muscular and of course Hung Let me know
Separate names with a comma.


text: Preliminary figures show that total revenues for the Canaveral Port Authority have topped $50m for the first time, reaching the $51 million mark in Fiscal Year 2006. Records were achieved in overall revenue, operating revenue, cruise revenue, cargo revenue, multi-day cruise passengers, and cargo tonnage. A decade ago, total revenues were $18.1 million. Total revenues last year were $44.6 million. In the latest economic impact report, Port Canaveral contributed 34,000 jobs and more than $1.5 billion to Brevard County. Regionally, the Port's impact contributed 50,000 jobs and $2.3 billion to the central Florida economy.


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You Want to Try Again?
"You want to try again?"
(Thanks for your comments and emails yesterday. The support found in these kinds of patient communities help so many of us to keep trying … again and again and again. Thanks for what you've done for me, and what you do for so many. Now go outside and roll down a hill, because it's pretty fun. Just be sure to wear shorts under your dress.)
She does a somersault way better than I can! (and sidenote: could stores PLEASE offer up more superhero options for girls underwear? Or at least some options in NON-PASTEL colors? My daughter wanted the bright and primary-colored batman/green lantern/superman/ thor set, and all they had it in was boy's briefs, so that is what we got. We do what we've got to do to encourage potty training!)07/2/13; 4:17 pm
I hope this doesn't get sent to spam because of the link but your post reminds me of this video – http://www.youtube.com/watch?v=xcIvIladNnQ.
Maybe it should be your new theme song?07/21/13; 1:53 pm


text: got to fondle one of these at the mountain of ganders for $899. I have to say that I was very impressed. Then I looked at buds gun shop and they have them for $737. why oh why. I haven't even paid off my first 1911 and I feel the disease taking a hold. What are your guys thoughts on this one. If I hadn't already bought 2 guns this month I think it would have come home with me.


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The Voice of Corporate Finance
Welcome to Acquisition International's Deal Diary, the place where we showcase exciting deals from across the globe. With each diary entry, we'll be taking a comprehensive look at the inner workings of the deal in question and will be venturing behind the scenes to take a look at the dedicated professionals involved in ensuring its success.
Here you will discover the most active deal makers and a comprehensive catalogue of the transactions they have been involved with.
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Within 24 hours of booking, we'll upload your deal and company profile - including contact details and links to your homepage - right here on our site, putting you and your firm in front of40,000 visitors every month!
You will, of course, get a full company bio, including your contact details and fully clickable links direct to your website, and to make sure the people who matter will be able to find every deal you and your firm have been involved in at the click of a button, all of the deals posted on the Deal Diary site are incredibly easy to search by firm name, individual name, sector or region. We even have an automatically updated deal leaderboard (right).
Tier 2: The Newsletter - Direct to 108,000 subscribers!
Your involvement in the deal will be featured on our weekly Deal Round-Up newsletter, published via email every Thursday to a global audience of 108,000. Your deal write-up will also include hyperlinked images and logos, taking readers straight to your homepage. That's a potential 108,000 clicks every month!
Tier 3: The Publication - Sent to 108,000 people in 170 countries!
The deal and your involvement will appear in the Deal Diary section of our monthly magazine, distributed to 108,000 people in 170 countries around the world. This is where you can tell us all about the deal and how you made it happen, providing the perfect opportunity to shout about your firm's experience, expertise and the great work you've been doing.
Deloitte (7)
Merrill Datasite (6)
Allen & Overy (5)
J.P.Morgan (3)
Merlin Piscitelli (3)
Irmantas Norkus (1)
Christian Atwood (1)
David Zhang (1)
Dave Rogers (1)
Vanity Fair Brands Europe, owned by Fruit of the Loom, has a high-end positioning in its industry and Merrill DataSite was very proud to be selected as the virtual data room provider on this project. Merrill DataSite is the high-end, marketing leading VDR solution and was pleased to help "showcase" this prime asset sale.
Registered in England and Wales.
No. 07400916.
1st Floor, Suite F,
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Sometimes sex can be hard work and sometimes you're just too tired to put forth the effort. If you're more interested in having a short love-making session so you can get to dreaming, one option is to fake it. Some women have no qualms about pulling a Meg Ryan, especially if they're in a new relationship, but other women wouldn't dare fake a good time, namely because they wouldn't be able to make it seem believable.
So how do you feel about faking an orgasm? Is it something you do often or every once in a while? Would you do it to build your partner's confidence or do you think it's wrong to pretend like you're having mind-blowing sex when you aren't?


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San Francisco Sales Ops Happy Hour – March 2016
The first SF Sales Ops Happy Hour, sponsored by Salesloft, Datafox and Nova, and hosted by AlwaysHired and Datahug was a big success. We had 130 people in attendance.
The panel was moderated by Arik Pelkey (Sr. Director of Product Marketing, Datafox). Sales Ops leaders such as Courtney Hays (Sr. Sales Ops Manager, Marketo), Tobias Muellner (Head of Sales Operations, Anaplan), Stephanie Max (Co-founder, AlwaysHired), and Taft Love (Manager of Sales Development and Sales Operations, PandaDoc) discussed the role of Sales Ops and the skills you need for optimizing sales teams' effectiveness.
- Sales Ops is involved in a little bit of everything. Data management, strategy execution, and sales process measurement are just a few.
- Sales Ops isn't always about putting out fires. It can be about preventing fires from happening in the first place.
- The biggest challenges in Sales Ops are a) finding the right people and b) companies investing in Sales Ops too late.
- The most important thing in Sales Ops is helping to put measurments in the sales process.
Here is a summary of some of the best questions and answers from the session.
Q: What is Sales Ops? What do you do on a daily basis?
Courtney: For me, it's a lot of meetings – a lot of decision making. My team is responsible for data management, systems, platform processes etc. We're a little bit of everything. Sales, at the end of the day, are our priority. We're there to help you execute and win business for the company.
Tobias: What is Sales Ops not? We're the people at the front lines. We ensure salespeople can sell, stuff is booked, and strategy is outlined and executed. I think when you think about the whole sales cycle, Sales Ops supports everything along the way.
Stephanie: I think Sales Ops is very different depending on the size of organization you have. For me, Sales Ops is about making salespeople's lives more efficient and effective. It's more about supporting the sales team than anything else.
Taft: Day-to-day, my job is split between running an SDR team, putting out fires, and helping plan out sales processes. The most important thing for Sales Ops, in my opinion, is helping put measurements in the sales process because ultimately, you can't manage what you can't measure. We help sales teams make improvements that lead to more revenue.
Q: What would you say are some of the biggest challenges you faced in your role?
Courtney: A big challenge for us is working cross-functionally. It's really key that you build relationships with each department. We're really investing in our partnerships with the other departments.
Tobias: The biggest challenge I see is that a lot of companies invest too late in Sales Ops. Also, it's hard to find the right people to build the team around. You need to have a team that sticks together.
Stephanie: When you come into a company, you're often cleaning up a huge mess that's not your making. Additionally, there are always fires to put out. I think at the end of the day, the most difficult part is making sure you're not becoming the enemy of the sales teams. You're trying to make their lives better and you're measuring all these things to show them how they can get better.
Taft: Once you find your feet, I think the single biggest challenge is actually identifying the problems worth investing time in and solving. Take a step back, look at the system that you put in place, and ask lots of questions about what's broken.
Q: What would be your advice for companies that do not currently have Sales Ops?
Courtney: For me it was making sure we have those key people in place. It's making sure you have that somebody in the starting position that is willing to build a foundation and work hard to roll up those sleeves. Figuring out what the problems are, creating processes even if the company doesn't want them – that's part of our job.
Tobias: When it comes to really building an infrastructure, I think a lot of people are trying to patch problems with tools – which is never going to work. Build your infrastructure as early as possible; build it while looking forward. What problem do you want to solve tomorrow and not just the problem that you want to solve today?
Stephanie: The biggest struggle with infrastructure and what companies should do is to make sure the person who's building out their Salesforce knows what they're doing. I think the biggest mess you walk into as a Sales Ops person is a completely disastrous Salesforce implementation that you have to try to debug. Making sure you have the right people trumps the infrastructure.
Checkout Datahug's New eBook
B2B Sales Operation PlaybookLearn more
Taft: Step one for me is to lock the stakeholders in a room and have a very specific goal. If you don't have that, you're never going to have a process that works because people will end up bringing in tools to patch problems.
Q: How do you take Sales Ops up a level and add strategic value to the company?
Courtney: For me, it's really important that you help a company scale and grow. A good example of this is when we implemented a CPQ (configure price quote) tool. It saved our sales team hours of manual data entry.
Tobias: I think Sales Ops should be in any strategic discussion that happens in a company in regards to sales strategy. Why? Because you're at the heart of the business; you own the data. We know what's really going on in the company.
Stephanie: I think getting to the strategy is the most important part and any good Sales Ops leader will tell you it's the hardest part to get to. You're not a firefighter – instead of putting out fires, you're supposed to be building things that prevent fires.
Taft: The way I created value was to first map out what is and isn't right. The second is to measure where we are right now. You define success in a quantitative way and once you do that, it's pretty easy to prove your value.
Audience Q: How do you justify expansion of hiring and spend?
Courtney: We implemented case management inside Salesforce so all of our work goes through that. I can track my team to make sure what they're doing is right, what they are working on, etc.
Audience Q: How do I as an SDR suggest implementing new apps? What should I do to show the tool is an investment?
Taft: It depends on the tool. First, you need to identify if you are fixing the symptom or the disease. If you can show that you identified the disease, then it's usually very easy to demonstrate the opportunity cost of extra time spent that this tool can save. If it's something that's not quantifiable, it's probably a hard case to make.
Audience Q: How do you work your way up and what are the resources you found in order to get to where you are? There's not a lot of knowledge or resources for "getting there" so how do you climb your way up in order to bring a lot of value?
Courtney: I've gotten to where I am thanks to an amazing team that has helped me get there. But on your own, you can't stop asking questions. Just being passionate about it was huge. I also had a great mentor throughout my beginning stages, which was very helpful. If you don't have Sales Ops in your company, I suggest finding a mentor outside of your company of someone that is in Sales Ops.
Audience Q: The whole idea of Sales Ops is to be proactive and give your organization a competitive advantage. How do you get people to change their mindset from today's problem to what we're going to be doing in the future?
Stephanie: I think the turnaround campaign you have to lead is about communicating and showing the value of the other things you want to do. I call it ruthless prioritization of things I would say yes to. I know that I'm going to add more value long-term if I say no to a lot of things now.
Next Event: Don't miss the next San Francisco Sales Ops Happy Hour on 5/12/2016!
See below some related articles and resources that might help too:
Sales Ops Blackbelt Checklist – eBook
Why You Should Think of Sales Operations as Sales Optimization – By Don Otvos
Four Sales Activities That Kill Sales Performance – By Don Otvos


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by Kara-Leah Grant I watched Madonna on the Grammy's a few years ago and I almost wept.Who was this shell of a woman, her papery-thin and paper-coloured skin stretched tight across her face? We all know she's nearly 60, where's the deviant wrinkles, the glorious salt & pepper hair, the relaxed winks as she settles into enjoying her undisputed reign as Queen of Pop?Instead, she's morphed into a caricature of her once saucy and irreverent self. I yearn for the Maddy of old, who was … [Read more...]
Exploration of the no-self: A total waste of time?
by Kara-Leah Grant I started this article over two years ago... and never finished it.But thanks to suggestions from a YLB commenter, I found and read a book by Jed McKenna.That was enough to give me the missing piece of the puzzle... I finally got what No-Self is.What follows is that article, written two years ago, with a fresh conclusion.*******There's been a great discussion going on in the comment section of Suggestions on what to do if you've had a Kundalini Awakening. … [Read more...]
Exploring the Niyamas: How does Svadhyaya affect our lives?
by Kara-Leah Grant I've been pondering the yamas and niyamas for a few years now. Reading about them, thinking about them, writing about them... and of course, living them out. The niyama on my mind right now is Svadhyaya, often translated as study. Seems to sum up my entire approach to yoga really - the study of the practice and the study of the Self. Without that, there is no yoga, just a bunch of fancy moves thrown together on a mat.Svadhyaya is the difference between rotely moving … [Read more...]


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Over 1.8 million nonprofits and charities for donors, volunteers and funders
Causes: Arts & Culture, Arts Education, Arts Services, Performing Arts Schools
Mission: For over sixteen years, Multi-Arts, an innovative arts program for children ages 4-18, has fulfilled its mission of inspiring, guiding, and educating youth in the performance and visual arts. What started as a summer camp for twelve children in the director's garage has grown to be a year-round program that inspires hundreds of students each year through an outreach network that includes public spaces and schools in a number of communities, a beautiful website, and, most recently, through the Children's theater company. There is no other organization in this area doing what we do so effectively to engage young people in becoming published and presenting artists.
Direct beneficiaries per year: over 300 students. 10 children art exhibitions.
Geographic areas served: Western Massachusetts
Programs: Innovative arts program for children 4-13 years old.


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Earlier today, the official Call of Duty channel accidentally set their 2 new DLC gameplay videos to Public. Five minutes later, however, they put it back on Private.
Don't be saddened, though, a quick YouTuber was able to download the videos for screenshots.
By the way, Liberation has a minigun.
And here's Piazza.
Source charlie INTEL


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Mobilizing for War . Again…. Building an Army…. Selective Training and Service Act (1940)- First peacetime draft in U.S. history! At first, men 21-35 had to register. Later men 18-45 were included (up to 65) 16 million Americans served in the military (10 million were draftees)
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Selective Training and Service Act (1940)- First peacetime draft in U.S. history!
At first, men 21-35 had to register. Later men 18-45 were included (up to 65)
16 million Americans served in the military (10 million were draftees)
At one point, 200,000 men were being drafted per month!
1 million African Americans served
300,000 Mexican Americans served
Production boomed factories made guns, jeeps, tanks, ammunition
War Production Board (WPB)- Supervised the conversion of factories to wartime production
Banned production of cars (1942-1946)
Later becomes the Office of War Mobilization (OWM)
Organized scrap drives
Rationing (controlling) amounts of goods available for use
Used ration coupons for coffee, gas, meat, shoes
Large families got more coupons
Office of War Information (OWI)- kept public aware of government policies
Warned of German and Japanese spies!
Government increased taxes- Everyone paid for the first time! (paid 40% of our expenses)
Sold war bonds (borrowed the rest)
Went to work!!- 19 million women went to work in factories, agriculture, transportation, office work, government jobs
300,000 women in the Armed forces
Women's Auxiliary Army Corps (WAAC)
Women's Airforce Service Pilots (WASPs)
Helped run communications, flew planes, nurses
African Americans still faced discrimination
A. Philip Randolph- African American labor leader, organized a march on D.C. to protest unfair treatment of African American workers
Fair Employment Practices Committee (FEPC)- created to prevent discrimination in war industries and government jobs
Mexican Americans also faced discrimination
Braceros- Mexican workers allowed to enter the U.S. to help fill job vacancies
200,000 came to work
Zoot-Suit Riots- June 1943
Sailors and Marines in LA attacked Mexican American men wearing Zoot Suits
Mobs roamed the city attacking men randomly
Benefitted from economic opportunities and higher wages
Continued to face discrimination and unequal treatment
People were very ANGRY after Pearl Harbor!!
Fearing spies and sabotage the U.S. government began the process of internment in 1942
Government forcibly relocated and imprisoned Japanese Americans
Moved to camps and kept under guard


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To let me and Randy Paynter know that Care 2 Members are interested and waiting for a reply to the petition and email sent to him.
The body of the email I sent to Randy today (23 September 2012) reads as follows:
Dear Randy
I understand Randy that the concerns of your Care2 Members are very important to you. As such I sent a petition to you by email, some weeks ago, signed by Care2 Members "Do our 'clicks to donate' to breast cancer fund research that experiments and tests on animals?". Many of those who signed commented, with quite some passion and concern, about this question.
If you have misplaced this petition or my email (I am sure you receive many emails), I can send this to you again.
I understand Randy that you are very busy, which is why I have allowed a few weeks before sending you this follow up email. However, I posted on the Care2 site, a message informing members that I had sent the petition on to you. As such Care 2 Members are waiting for me to post an answer from you. In fact I have had a number of inquiries asking if I have heard from you as yet.
Please reply email if you need me to resend the petition to you or if you need a little more time to answer the question that this petition raises.
Yours in good faith
Susan Griffiths
Care2 Member
Care2 animal lovers do not want to 'click to donate' to research that experiments and tests on animals!
Dear Care 2 members and visitors
My passion is the welfare and rights of animals.
I like to 'click to donate' when I visit this site but given my passion for animals I am very concerned about' clicking to donate' to breast cancer research. When we click to cure breast cancer 100% of the donations go to the Susan G. Komen organisation to manage and support breast cancer research.
By signing this petition you will be asking the Care2 Management team to investigate and advise us if Susan G. Komen uses the money we donate to support research that experiments and tests on animals.
I have asked Care2 this question before but have received no response from them. So please join me in asking again.
When this petition closes, I will send it to the Care2 Management team and advise you all of their answer to this vital question. If they can not assure us that our donations do not go to research that experiments and tests on animals - then I hope you will join me again in taking further action.
By signing, you accept Care2's terms of service.
Having problems signing this? Let us know.


text: Find latest reviews and retail prices of Knives in India today. Reviewable found 511 Knives in online stores. Find user reviews, advantages & disadvantages of Knives along with specifications, key features, photos. Reviewable finds selling prices from all major online stores like Flipkart, Naaptol, Indiatimes, Snapdeal, Homeshop18 in India for an all round comparison.