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The primate APOBEC3 gene locus encodes a family of proteins (APOBEC3A-H) with various antiviral and anti-retroelement activities. Here, we trace the evolution of APOBEC3H activity in hominoids to identify a human-specific loss of APOBEC3H antiviral activity. Reconstruction of the predicted ancestral human APOBEC3H protein shows that human ancestors encoded a stable form of this protein with potent antiviral activity. Subsequently, the antiviral activity of APOBEC3H was lost via two polymorphisms that are each independently sufficient to destabilize the protein. Nonetheless, an APOBEC3H allele that encodes a stably expressed protein is still maintained at high frequency, primarily in African populations. This stable APOBEC3H protein has potent activity against retroviruses and retrotransposons, including HIV and LINE-1 elements. The surprising finding that APOBEC3H antiviral activity has been lost in the majority of humans may have important consequences for our susceptibility to retroviral infections as well as ongoing retroelement proliferation in the human genome.
A substantial fraction of the human genome is comprised of active and extinct retroelements, a result of numerous germline insertions by both endogenous retroviruses and non-LTR retrotransposons (such as LINE and SINE elements) (Lander et al., 2001). Due to the potentially detrimental effects of the integration of mobile elements, such as insertional mutagenesis and an increase in the likelihood of ectopic recombination events (Boissinot et al., 2006), restricting the replication of mobile retroviral elements by host genes has likely been an ongoing source of genetic conflict during primate evolution. In particular, primates have evolved an expanded locus of genes, the APOBEC3 locus, that encodes seven genes in humans (APOBEC3A to APOBEC3H) that can inhibit retroviruses and retroelements through cytidine deamination and other mechanisms (Malim and Emerman, 2008). The role of APOBEC3 proteins in limiting the success of retroviral invaders in vivo has been demonstrated in mice where APOBEC3−/− mice have increased susceptibility to the replication of the mouse retroviral pathogen, mouse mammary tumor virus (MMTV) (Okeoma et al., 2007).
The human immunodeficiency virus (HIV) can successfully infect APOBEC3-expressing cells because the virally encoded Vif protein is able to cause destruction of APOBEC3 proteins by targeting APOBEC3 proteins to a proteaseome-mediated degradation pathway (reviewed in (Harris and Liddament, 2004; Malim and Emerman, 2008)). Despite efficient inactivation by HIV Vif, APOBEC3 proteins remain important in vivo because viruses that no longer encode a functional Vif protein are sensitive to APOBEC3-mediated restriction and bear hallmarks of hypermutation due to cytidine deamination (Simon et al., 2005).
Genetic conflict between host and pathogen drives rapid change in interacting host and pathogen proteins (rapid evolution) as they attempt to increase or decrease interactions with one another in the battle for dominance. Therefore, a signature of positive selection is often observed for host proteins that are directly involved in pathogen defense. APOBEC3G and APOBEC3H genes, in particular, demonstrate strong signatures of positive selection in primates that are likely the result of successive sweeps of APOBEC3 alleles with antiviral activity against an ever-changing array of rapidly evolving retroviruses and retroelements (OhAinle et al., 2006; Sawyer et al., 2004). These signals of adaptive evolution are present throughout primate phylogeny, suggesting that the conflict of primate APOBEC3 genes with exogenous retroviruses and endogenous retroelements is ancient and has taken place over many millions of years. This ancient conflict has shaped the evolution of a landscape of APOBEC3 genes with diverse antiviral functions and has important implications for the susceptibility of humans to current retroviral pathogens, such as HIV.
Despite the fact that the APOBEC3H gene is conserved in mammals and shows positive selection throughout primate evolution, previous work from our lab and others showed that whereas rhesus macaque APOBEC3H is an efficient retroviral inhibitor, human APOBEC3H shows little activity against retroviruses and LINE-1 elements (Dang et al., 2008; Dang et al., 2006; Kinomoto et al., 2007; Muckenfuss et al., 2006; OhAinle et al., 2006; Virgen and Hatziioannou, 2007) . The lack of antiviral activity of human APOBEC3H correlates with its low steady-state expression at the protein level although mRNA levels between human APOBEC3H and macaque APOBEC3H are similar (OhAinle et al., 2006). Moreover, the protein is stable and enzymatically active in bacteria (OhAinle et al., 2006). Thus, it appears that humans have lost a functional APOBEC3 gene that has potent antiviral activity in other primates.
Here, we investigated the evolutionary history of human APOBEC3H. We found that the stability of the human APOBEC3H protein was independently lost twice during recent human evolution through the acquisition of two different amino acid mutations that decrease the half-life of the protein. Moreover, we reconstructed the sequence of the human ancestral APOBEC3H protein and showed that it is well expressed and is very active against retroviruses and the prolific primate non-LTR retroelement, LINE-1. Despite the presence of two inactivating mutations in APOBEC3H alleles in humans, a sequence survey of the APOBEC3H alleles from several different human populations reveals the existence of a stable APOBEC3H allele currently circulating in the human population. This allele lacks either destabilizing mutation, is active against LINE-1 elements and HIV, and is sensitive to the HIV Vif protein. This stable and active APOBEC3H allele is more prominent in certain world populations, particularly in African populations and may reflect local selective pressures on APOBEC3H alleles across world populations. Together, these data suggest that our recent human ancestors possessed an APOBEC3H allele that was active against modern retroviruses and retrotransposons, but whose function is now lost in the majority of the human population.
Rhesus macaque APOBEC3H is an efficient inhibitor of retroelements, but human APOBEC3H is not (OhAinle et al., 2006). We previously showed that the lack of antiviral activity of human APOBEC3H corresponds with poor steady-state protein expression (OhAinle et al., 2006). To track the origin of the instability of the APOBEC3H protein in primate evolution and, therefore, the likely cause of the loss of antiviral activity, we cloned and expressed a panel of hominoid APOBEC3H proteins by transient transfection and evaluated their steady-state expression levels by western blotting. All hominoid APOBEC3H proteins, except human, are expressed at levels similar to that of macaque APOBEC3H (Figure 1A). Importantly, chimpanzee APOBEC3H, the hominoid APOBEC3H protein with the highest amino acid identity to human APOBEC3H, is expressed at high levels suggesting that the stability of human APOBEC3H was lost sometime after human-chimpanzee speciation. We also cloned and tested the expression of a second human APOBEC3H allele that differs from the original human allele at four amino acid sites (R18L, G105R, K121D and delN15 - amino acid numbering is maintained based on the original clone). The expression of the second human APOBEC3H protein (human_B) is similar to that of the original human APOBEC3H protein (human_A) and is much less than that of chimpanzee APOBEC3H protein (Figure 1B).
To ask if stable expression correlates with antiviral activity, we tested all the APOBEC3H proteins for inhibition of the mouse retrotransposon, MusD. All primate APOBEC3H homologs, except for human, were found to inhibit the replication of MusD (shown in Figure 1C) as well as other retroviruses and retrotransposons (HIV vif-, SIVagm vif-, and the L1.3 LINE-1 elements; data not shown). Thus, the human APOBEC3H protein was uniquely incapable of inhibiting the retroviruses and retroelements tested.
We then performed a pulse-chase analysis to determine if the lack of steady-state expression is due to increased protein turnover. A 30-minute pulse with 35S-Cys/Met resulted in approximately equivalent levels of labeled chimpanzee and human APOBEC3H proteins, suggesting that equivalent amounts of both APOBEC3H proteins were synthesized (Figure 1D). However, both human APOBEC3H proteins showed a dramatic decrease during the 6-hour chase (Figure 1D). Quantification of the rate of degradation showed that both human APOBEC3H proteins have a half-life of less than 30 minutes, while the chimpanzee APOBEC3H protein has a half-life of over 6 hours (Figure 1D & 1E). Thus, we can attribute the lack of activity of human APOBEC3H protein to the evolution of a dramatically increased rate in protein turnover that occurred in the human lineage after speciation from a common ancestor with chimpanzees.
Macaque and human APOBEC3H proteins differ at 31 single amino acid sites. Further, macaque APOBEC3H encodes a 27 amino acid C-terminal tail that is missing in human and chimp APOBEC3H due to a premature termination codon (PTC) that arose in the APOBEC3H coding sequence in the common ancestor of human and chimpanzee (Figure 2A). Recently it was suggested that the PTC may have a role in determining relative levels of APOBEC3H protein (Dang et al., 2008). However, we found that while both human and chimpanzee APOBEC3H alleles encode the PTC, these alleles differ dramatically in the rate of protein turnover (see Figure 1D & 1E), leading us to deduce that the PTC is not a major determinant of turnover of the APOBEC3H protein. We specifically addressed the role of the C-terminal tail in protein stability and antiviral activity of APOBEC3H by first deleting the 27-residue C-terminal tail from the macaque APOBEC3H protein (Macaque−tail) and then by fusing the C-terminal tail to the human APOBEC3H protein (Human +tail). While the tail modifications did result in changes in expression levels of the proteins (Figure 2B; compare Macaque to Macaque −tail and Human to Human +tail), the relative increase or decrease was not nearly as dramatic as the tail-independent differences in expression levels of the human and chimpanzee proteins (see Figure 1A).
On the other hand, the C-terminal tail did dramatically alter the subcellular localization of APOBECH proteins. Truncation of the C-terminal tail from macaque APOBEC3H altered its localization from cytoplasmic to nuclear while addition of the C-terminal tail to human APOBEC3H altered its localization from mostly nuclear to mostly cytoplasmic (Figure 2C). It appears that cytoplasmic localization may be necessary for the antiviral activity of macaque APOBEC3H, since truncation of the C-terminal tail impairs its ability to efficiently restrict HIV-1 (Figure 2D; compare Macaque to Macaque−tail). In contrast, the native chimpanzee APOBEC3H protein lacks the C-terminal tail, yet retains robust antiviral activity (see Figure 1C). More importantly, the human APOBEC3H chimera with the macaque C-terminal tail does not restrict HIV-1 (Figure 2D), likely due to a lack of sufficient expression (see Figure 2B). Although not explored further, we note that there is a possible Nuclear Export Sequence (NES) in the C-terminal tail that is conserved in other APOBEC family members, such as APOBEC1 and AID (Ito et al., 2004) (Figure 2A). Thus, although the C-terminal tail of primate APOBEC3H appears to play a role in protein localization, we conclude that the major determinants of stability of primate APOBEC3H proteins do not lie within the C-terminal tail.
Since the instability and lack of antiviral activity of human APOBEC3H is not determined by the C-terminal tail, we hypothesized that changes in the APOBEC3H coding sequence must distinguish human and chimpanzee APOBEC3H orthologs. We used parsimony to infer those sites in the APOBEC3H coding sequence that have changed specifically in either the human or chimpanzee lineages, using the gorilla sequence as an outgroup. We identified four amino acid changes that have been fixed in the chimpanzee lineage (upside down black flags in Figure 3A) and three amino acid changes that have been fixed in humans (short black flags in Figure 3A). Based on polymorphisms currently segregating in the human population, the human_A protein differs from chimpanzee APOBEC3H at 2 additional sites (open flags in Figure 3A) and human_B differs at 3 additional sites (grey flags in Figure 3A). In sum, human and chimpanzee APOBEC3H proteins differ at 9 and 10 sites, respectively, with some changes shared in common between both human proteins and other changes specific to each allele. By creating chimeric proteins between the chimpanzee APOBEC3H and human APOBEC3H proteins, we mapped the determinants of the instability of both human proteins to the N-terminal two-thirds of APOBEC3H (Figure 3B). Next, we experimentally tested all remaining non-synonymous changes that distinguish the chimpanzee from the human APOBEC3H proteins. We found that only reversion of the R105G polymorphism in human_A could stabilize this protein (Figure 3C).
The fact that the R105G mutation was able to stabilize the human_A APOBEC3H protein was surprising because this change is not found in the human_B APOBEC3H protein, suggesting that an independent change is responsible for its high instability. Indeed, we found that by reverting the deletion of a single amino acid residue (delN15), we were able to restore stable expression of the human_B protein (Figure 3C). In addition, we found that either polymorphism (R105G or delN15) is independently sufficient to destabilize the otherwise stable chimpanzee APOBEC3H protein (Figure 3D). Thus, remarkably, the human species independently lost stable expression of APOBEC3H twice through different mutations (G105R and delN15) that are each independently capable of destabilizing the APOBEC3H protein.
The mutations that cause the instability of human APOBEC3H proteins are both polymorphic in the human population. To determine whether or not stable and active alleles of APOBEC3H might still circulate in the human population, we sequenced the APOBEC3H coding exons from a panel of individuals from diverse world populations (Figure S1). The major haplotypes (set of polymorphisms that are inherited together on a single chromosome) were then inferred using PHASE 2.1.1 software (Table S1) (Stephens and Donnelly, 2003; Stephens et al., 2001). Four predicted haplotypes were found at frequencies > 10% in our panel (haplotypes I – IV in Figure 4A; and in bold in Table S1. The total counts for all haplotypes are listed in Tables S2). Amino acid polymorphisms in haplotypes I–IV are diagramed in Figure 4A.
To ask if any of the major human APOBEC3H haplotypes identified in our panel might be stable and active, we reconstructed the four major haplotypes by site-directed mutagenesis of the human_A and human_B cDNAs. Three of the four haplotypes (hap I, hap III and hap IV) contain one of the destabilizing polymorphisms (either R105G or delN15) identified in Figure 3. As expected, all three haplotypes encoding either of the destabilizing mutations were found to encode unstable APOBEC3H proteins (Figure 4B). However, a single human APOBEC3H haplotype (hap II) encodes an APOBEC3H protein that is as stable as chimpanzee APOBEC3H (Figure 4B). Therefore, in spite of the two destabilizing polymorphisms that arose during recent human evolution, a stable APOBEC3H allele still persists in the human population.
To ask if the stable human APOBEC3H protein is capable of retroviral inhibition, APOBEC3H proteins were tested for activity against both wild type and vif-deficient HIV-1 (HIV vif-). Rhesus macaque APOBEC3H is a potent inhibitor of HIV-1 and is not neutralized by Vif from HIV-1, but is neutralized by Vif from SIV from african green monkeys (SIVagm) and rhesus macaques (SIVmac) (OhAinle et al., 2006; Virgen and Hatziioannou, 2007). Consistent with our observed correlation between stability and antiviral activity of APOBEC3H proteins, the human APOBEC3H haplotypes that encode unstable proteins have no effect on HIV replication (Figure 4C). In contrast, both chimpanzee APOBEC3H and the stable human APOBEC3H (hap II) proteins can inhibit HIV-1 (Figure 4C) but are both counteracted by HIV-1 Vif (although to a less extent than APOBEC3G) (Figure 4C; compare white to black bars). Thus, an active form of APOBEC3H does exist in the human population and, like APOBEC3F and APOBEC3G, it is sensitive to inactivation by HIV-1 Vif.
To demonstrate that the loss of stability and antiviral activity of APOBEC3H occurred specifically during recent human evolution, we reconstructed and tested the predicted ancestral states of human APOBEC3H since human-chimpanzee speciation. Using the gorilla and chimpanzee APOBEC3H sequences as outgroups to the human APOBEC3H alleles, we constructed both a hypothetical human ancestor sequence (referred to as human ancestor) as well as a hypothetical human/chimpanzee ancestor sequence (referred to as human/chimp ancestor) based on parsimony (Figure S2). Specifically, human APOBEC3H protein sequences were aligned with both chimpanzee and gorilla APOBEC3H. Sites that are polymorphic in humans were reverted to the ancestral state, as predicted based on the chimpanzee sequence, to create the human ancestor APOBEC3H protein. Similarly, amino acid sites that differ among humans and chimpanzees were identified and, using the gorilla APOBEC3H sequence as the outgroup, the ancestral state at each position was inferred by parsimony.
Next, we used this information to define the evolutionary path of changes that occurred in the APOBEC3H coding sequence during recent human evolution (Figure 5A). Four amino acid changes were fixed in the chimpanzee lineage (black flags in Figure 5A along the chimp lineage), three non-synonymous changes were fixed in the human lineage (black flags in Figure 5A above "human ancestor"), and six non-synonymous changes occurred in recent human evolution that have not been fixed in the human population and remain polymorphic in humans (open and grey flags in Figure 5A). The ancestral APOBEC3H cDNAs (circled nodes in Figure 5A) were then reconstructed in vitro by site-directed mutagenesis of human and human/chimpanzee APOBEC3H constructs. Both the human/chimp APOBEC3H ancestor and the human APOBEC3H ancestor encode stable APOBEC3H proteins that are expressed at levels similar to that of the native chimpanzee protein and much higher than the 'unstable' human APOBEC3H proteins (Figure 5B). Further, both the ancestral APOBEC3H proteins efficiently inhibit retroviral replication, as indicated by their potent activity against vif-deficient HIV (Figure 5C, open boxes).
We consistently find that the ancestral versions of APOBEC3H are more active than either the extant human (Figure 4C) or chimpanzee versions of this protein (Figure 5C, open boxes). We also tested the Vif-sensitivity of the reconstructed human/chimp ancestor of APOBEC3H and the reconstructed human ancestor of APOBEC3H. We find that both are at least partially counteracted by a modern version of HIV-1 Vif (Figure 5C, solid boxes). These results suggest that while there has been some degradation of function of APOBEC3H in both the chimpanzee and the human lineage, the APOBEC3H protein has still retained a surface that is important for interaction with a modern day HIV-1 Vif.
Since APOBEC3H is conserved in eutherian mammals and appears to have evolved under constant positive selection in primates (OhAinle et al., 2006), it is likely to have evolved to counteract a more ancient and pervasive pathogen than lentiviruses. Endogenous retroelements, such as the LINE and SINE non-LTR retrotransposons, represent such a constant, pervasive threat in human evolution. Therefore, we tested both modern and ancestral human APOBEC3H proteins for the ability to block the replication of the human LINE-1 element. We find that all stably expressed APOBEC3H proteins inhibit the replication of LINE-1 (Figure 5D). In contrast, the unstable proteins have no impact on LINE-1 replication. Similar results were obtained with an Alu element (data not shown), consistent with previous findings that APOBEC3G potently blocks Alu transposition (Bogerd et al., 2006; Hulme et al., 2007). Significantly, the ancestral human APOBEC3H protein is as good or better than any of the extant hominoid versions of this gene in terms of its anti-LINE1 activity (Figure 6D). Thus, recent human ancestors encoded an APOBEC3H protein with potent activity against retroviruses and LINE-1 elements, but this activity now persists in only a fraction of the human population.
To determine the approximate frequencies of stable and active alleles across human populations, we genotyped both the inactivating polymorphisms of APOBEC3H (R105G and delN15) in a larger panel of unrelated individuals from three American populations with diverse ancestry, African Americans (AA), European Americans (EA) and Han Chinese from Los Angeles (CA) (Hinds et al., 2005). Both destabilizing human polymorphisms are found at significant frequencies in the populations we sampled (Figure 6A). Specifically, the N15 deletion is found at relatively similar frequencies in all three populations (0.27 in African Americans, 0.21 in European Americans and 0.30 in Han Chinese) (gray bars in Figure 6A), while R015G differs in frequency among populations (0.22 in African Americans, 0.61 in European Americans and 0.65 in Han Chinese) (black bars in Figure 6A). Pairwise comparisons of FST values, a measure of population differentiation that reflects either demographic effects or regional selection (Lewontin and Krakauer, 1973), indicate that there is little to no difference in frequencies of the N15 deletion polymorphism between the three populations (Figure 6B). In contrast, the R105G polymorphism is found at significantly higher frequencies in European Americans and Han Chinese as compared to African Americans (black bars in Figure 6A). The pairwise FST values for the APOBEC3H R105G SNP are 0.26 and 0.31 for comparisons between African American and the other two populations, respectively (Figure 6B); these values are higher than ~90% of human SNPs compared in the same populations in a genome-wide study (Hinds et al., 2005)
We were concerned about the potential confounding effects of admixture in the 3 populations we had thoroughly analyzed. Therefore, we examined the incidence of both destabilizing polymorphisms in the HapMap dataset that examined a larger panel of humans, that is relatively devoid of admixture. . These included the Yoruban (YRI) panel from Nigeria, the Caucasian (CEU) panel of Utah residents with ancestry from Northern and Western Europe and a combined East Asian (JPT/CHN) population that included Japanese individuals from Tokyo and Han Chinese individuals from Beijing (more information about these populations is available from www.hapmap.org). We tabulated the incidences of both the R105G polymorphism (already typed by the HapMap consortium) and the N15 deletion. Again, we found no significant discordance in the incidence of the N15 deletion allele frequencies across these three populations (Figure 6C). The N15 deletion frequencies ranged from 0.28 to 0.4 with no significant discordance in allele frequencies (low Fst values) between any two populations. In contrast, we found that the R105G polymorphism existed at a frequency of 0.689 and 0.608 in the HCB and CEU populations respectively, but only at an incidence of 0.075 in the YRI population (Figure 6C). This is an even more dramatic discordance in allele frequencies than what we have typed in our smaller dataset (Figure 6A, 6B). The Fst values that highlight this discordance between African (YRI) and non-African populations (CEU, HCB) are presented in Figure 6D.
Next we asked if the stable APOBEC3H alleles are found at a significant frequency in any of the six subpopulations that we had sampled in Figure 6A and 6C. Based on our phasing analysis, the loci encoding the two destabilizing mutations (R105G and delN15) are tightly linked and, therefore, are rarely found on the same chromosome (only 2 of 86 chromosomes analyzed in our panel are predicted to encode both polymorphisms -- see Table S2). Therefore, summing the frequencies of both polymorphisms within a population gives a robust estimation of the frequency of active (stable) versus inactive (unstable) APOBEC3H alleles in each subpopulation. The relative frequencies of active (gray) and inactive (black in Figure 6C) APOBEC3H alleles show that the frequency of active APOBEC3H alleles varies significantly between populations, from 51–52% in African Americans (AA) and Yorubans (YRI) respectively, 10–18% in Europeans (CEU) and European Americans (EUR) respectively and only 3–4 in East Asian (JPT/CHN) and Chinese-American populations respectively (Figure 6E). Thus, a gradient of active APOBEC3H alleles exists with increasing distance from Africa, from nearly 53% for the African population (YRI), to only 3% for the East Asian population (JPT/CHN) (Figure 6). Therefore, the ancestral active APOBEC3H allele has been maintained at higher frequency in Africa and has been lost with increasing distance from Africa.
Remarkably, the trend of loss of APOBEC3H activity in human populations is similar to that observed for another APOBEC3 gene, APOBEC3B. APOBEC3B is found at a frequency of nearly 100% in African populations but is present in only 40% of East Asians due to a large structural deletion (Kidd et al., 2007). Since APOBEC3B and APOBEC3H are present in the same genetic location, we wished to ascertain whether there was any correlation between the unstable APOBEC3H alleles and the loss of APOBEC3B. Therefore, we typed the presence or absence of the APOBEC3B gene in the same individuals from all three subpopulations (Table S3). We did not find a significant correlation between non-functional alleles of these two genes within individuals (2 by 2 contingency χ2 test; p =0.08). This either means that the loss of APOBEC3B and APOBEC3H were independent events or that our test was not powerful enough to identify any linkage between the two loci. Nevertheless, whether independent events or not, populations that lack the APOBEC3B gene, in particular East Asian populations, are also more likely to encode inactive APOBEC3H proteins.
We present evidence of the evolutionary history of APOBEC3H in humans in which two independent mutations arose during recent human evolution and gave rise to an unstable APOBEC3H protein with no detectable antiviral activity. This is in contrast to our finding of stable expression and antiviral activity of other hominoid APOBEC3H proteins. Both of the inactivating mutations occur in distinct regions of APOBEC3H, but similarly act to decrease the half-life of the APOBEC3H protein. Reconstruction of the ancestral human APOBEC3H gene suggests that recent human ancestors encoded a very powerful anti-retroviral and anti-retroelement version of this gene. While most humans possess unstable APOBEC3H alleles, we found that a minor fraction of humans still encode a stable allele of APOBEC3H that still retains activity and can be targeted by the HIV Vif protein.
Our finding of an APOBEC3H allele with variable activity among individuals may have important consequences for the interaction of the human APOBEC3 locus with HIV. First, in those individuals homozygous or heterozygous for the active APOBEC3H haplotype, there may be an additional APOBEC-mediated block to infection of HIV target cells in addition to the activities of APOBEC3F and APOBEC3G. However, we must point out that although APOBEC3H mRNA is present in the appropriate cell types (T cells), (OhAinle et al., 2006)) it is still unclear if the APOBEC3H protein is present. Second, our finding that HIV Vif can inhibit the active APOBEC3H allele argues that HIV Vif may have specifically evolved to counteract the active APOBEC3H protein in addition to the other active APOBEC3 proteins. The mechanism by which HIV Vif recognizes and inhibits APOBEC3H may or may not overlap with the mechanisms of inhibition of other APOBEC3 proteins. Therefore, successful replication of HIV in individuals with active APOBEC3H alleles may further challenge Vif evolution even though HIV-1 Vif may be sufficient to counteract any potential benefit of active APOBEC3H in humans. Significant differences in HIV-1 progression have been shown to be correlated with APOBEC3G variation (An et al., 2004), however, we do not yet know if individuals heterozygous or homozygous for active APOBEC3H are more resistant to successful HIV replication. On the other hand, since the stable APOBEC3H proteins appear to be able to inhibit multiple retroelements (against both LINE-1, Alu elements and retroviruses), it is also possible that APOBEC3H served an ancient role and has no modern impact on HIV-1.
In light of the multiple protective functions served by human APOBEC3H (against both LINE-1, Alu elements and retroviruses), it is puzzling that a majority of humans have lost this activity and even more puzzling that loss of activity happened twice in recent human evolution. This suggests the interesting possibility that non-functional APOBEC3H alleles have been actively selected for in humans. The "less-is-more" hypothesis suggests that loss of function mutations can be maintained at high frequency during a shift of environmental pressures under which they may be selectively advantageous, particularly if the functional allele presents a cost to the host (Olson, 1999). For example, there may be a significant cost associated with maintaining active APOBEC3 proteins at high levels in the cell as APOBEC3 proteins present a risk to the host if they were to deaminate the wrong targets, such as cellular mRNA or genomic DNA. Consistent with this hypothesis, APOBEC3H was recently shown to hypermutate human papillomavirus, a double-stranded DNA virus thought to closely resemble nuclear DNA (Vartanian et al., 2008). We speculate that APOBEC3H may be particularly toxic relative to other APOBEC3 family members as no duplications of the APOBEC3H-like domain have been tolerated in any mammalian genome, in striking contrast to the multiple duplications of the non-APOBEC3H domain (Conticello et al., 2005; OhAinle et al., 2006). Further, the expression of APOBEC3H in germline tissues (OhAinle 2006) poses a mutagenic threat with serious evolutionary consequences and, therefore, could be particularly costly to the host. In this context, APOBEC3H may be especially 'costly' in humans and chimps, compared to other primates, because nuclear localization of the APOBEC3H protein occurred only recently during the evolution of the protein in hominoids, corresponding with loss of the C-terminal domain in the human/chimpanzee ancestor. Therefore, the nuclear localization and potential risk of increased genomic DNA editing are greater for humans and chimpanzees than for other primates. The benefits of maintaining stable and active APOBEC3H alleles (due to significant selective pressure from an active exogenous viral infection or from active endogenous retroviruses or retrotransposons) might outweigh the costs associated with the stable APOBEC3H protein in chimpanzees but not in humans. This is plausible given that retroviral infections and retroelement amplifications are known to be distinct among even these two closely-related species (Jern et al., 2006; Polavarapu et al., 2006; Yohn et al., 2005).
Taken together, our findings suggest that there may be significant evolutionary pressure to downregulate levels of APOBEC3H protein in the cell in times of decreased pathogenic pressure. Under this scenario, maintaining a stable and active APOBEC3H protein capable of retroelement inhibition would have to be balanced with the costs associated with maintaining high doses of this protein in the cell. Other APOBEC3 proteins that are localized to the nucleus (and therefore may also contribute to higher genomic mutation) might also display evidence of this increased cost. Consistent with this model, APOBEC3B has also been lost in a significant fraction of human populations (Kidd et al., 2007), while the start codon of the APOBEC3A gene is missing from chimpanzees (unpublished data). These debilitating events appear specific to the APOBEC3 genes that have a nuclear localization, supporting the 'costly defense' selective scenario for the acquisition of destabilizing mutations in human APOBEC3H.
Nonetheless, there are important differences between the loss of APOBEC3B and APOBEC3H activities in humans. While APOBEC3B has been lost due to a genomic deletion, APOBEC3H still appears to be expressed but encodes an unstable protein. We cannot rule out the possibility that instability, rather than loss of APOBEC3H function, is what has been selected. Even the unstable APOBEC3H alleles may still be sufficient to carry out some antiviral function that we have not assayed. A simple 'loss-of-function' hypothesis might be insufficient to explain the high prevalence of the unstable alleles (~50%) even in the African populations, where APOBEC3H activity still appears to be selectively retained. Instead, our findings suggest that the two unstable alleles appear to be independently under selection, based on the discordance in their allele frequencies in different human populations.
Independent of the true nature of selective pressures shaping the human APOBEC3 locus, the consequences of these pressures on the antiviral repertoire are significant. Combining the presence of two 'loss of stability' mutations in APOBEC3H and the loss of the APOBEC3B gene in humans, we can conclude that at least two APOBEC3 antiretroviral proteins are maintained at higher frequencies in African populations and have subsequently been lost to varying degrees in European and Asian populations. This is consistent with a model in which a lower pathogenic burden outside Africa may allow for the accumulation of inactivating mutations in antiviral genes outside of Africa (either due to genetic drift or due to selection). This implies both that a relatively recent (and perhaps ongoing) pathogenic pressure is maintaining APOBEC3H and APOBEC3B activity in African populations, and further that these populations might have an associated higher risk of genomic mutations. Regardless of the cause, it is clear that much of the redundancy of an expanded APOBEC3 locus that built up over primate evolution to defend the genome against the deleterious effects of retrotransposition or unknown retroviruses has been lost from a substantial fraction of the human species.
APOBEC3H cDNAs were cloned by RT-PCR or nested RT-PCR (QIAGEN One-Step RT PCR Kit) from RNA derived from chimpanzee (Pan troglodytes verus – Coriell GM03448), gorilla (gorilla gorilla – Coriell AG05251), orangutan (Pongo pygmaeus pygmaeus - Coriell AG05252) and gibbon (Nomascus leucogenys leucogenys - GM051496). Primers used were as follows: chimpanzee APOBEC3H was amplified using an initial round of RT-PCR (50 cycles) with primers JAK012/JAK015 followed by a second round of 30 cycles (Pfu Turbo – Stratagene) with primers JAK012/MO136. Gorilla APOBEC3H was amplified with an initial round of RT-PCR using primers JAK012/JAK015 followed by a second round of 30 cycles with JAK012/MO143. Orangutan APOBEC3H was amplified in a single round of RT-PCR (50 cycles) using primers JAK012/MO173. Gibbon APOBEC3H was amplified in a single round of 50 cycles using primers JAK012/JAK015. RT-PCR products were gel purified, TOPO cloned (Invitrogen) and sequenced. Cloning of human (= human_A) and macaque APOBEC3H cDNAs were described previously (OhAinle 2006). The "human_B" APOBEC3H cDNA was amplified by RT-PCR from a Druze individual (Coriell #11521) using primers JAK012/JAK015. GenBank accession numbers for the primate APOBEC3H sequences described here are EU861357 through EY861361.
All APOBEC3H cDNAs were cloned into the XhoI/EcoRI sites of pcDNA3.1 (Invitrogen) with a primer-encoded 5' HA tag (MYPYDVPDYA). Chimeric human/macaque APOBEC3H plasmids were constructed by PCR using primers that match both the macaque and human APOBEC3H sequences. The Human +tail plasmid was constructed by PCR using overlapping primers specific for both the human APOBEC3H C-terminus and the macaque APOBEC3H C-terminal tail domain. First, these primers were used with external primers to create human APOBEC3H and macaque tail fragments, followed by amplification with external primers to create the chimeric Human +tail plasmid. The truncated macaque APOBEC3H plasmid (Macaque −tail) was constructed by amplification of macaque APOBEC3H with the human APOBEC3H 3' primer, resulting in truncation of macaque APOBEC3H at the STOP found in the human sequence. Chimeric human/chimpanzee constructs were created by digesting chimpanzee and human APOBEC3H constructs with EcoRI/BamHI and BamHI/XhoI, purification of plasmid backbone and both fragments and triple-ligation. All constructs were confirmed by sequencing. Point mutants were constructed by site-directed mutatgenesis of plasmids using the Quickchange kit (Stratagene). Primers used in this study are described in Table S4.
HEK293T and HeLa cell lines (ATCC) were maintained in DMEM/5% Pen/Strep/10% FBS at 37°C in a CO2 incubator. Transfections were performed with TransIT-LT1 transfection reagent (Mirus Bio) at a reagent:plasmid DNA ratio of 3:1. For Western blot analysis, cells were lysed in ice-cold NP40doc buffer (1% NP40, 0.2% Sodiumdeoxycholate, 0.12M NaCl, 20mM Tris pH 8.0) with Protease Inhibitors (Roche Complete Mini, EDTA-free tablets), resolved by 12% SDS-PAGE, transferred to PVDF and probed with anti-HA (Santa Cruz Biotech) and actin (Sigma) antibodies.
Single-round HIV-1 infectivity assays were performed as previously described (OhAinle 2006)(Yamashita and Emerman, 2004). All assays were performed by transfection of 1.25 × 105 293T cells in 24-well plates with a 1:1 ratio of pcDNA/APOBEC plasmid (250ng) to BruLuc2 env- proviral plasmid (250ng). MusD and LINE-1 retroelement assays were performed essentially as previously described (Esnault et al., 2002; Ribet et al., 2004). For MusD assays, HeLa's were plated at 2.5 × 105 cells/mL in 2 mLs in 6-well plates and triplicate wells transfected with 0.75 µg MusD plasmid and 0.75 µg APOBEC expression plasmid. At Day 3 post-transfection, cells were replated @ 1.5 × 105 cells/mL in 2 mLs of a 6-well dish. At Day 4, cells were selected in G418+ media (0.4 mg/mL) and fresh G418+ media added every 2 days until single colonies remained. For LINE-1 assays, HeLa's were plated at 5 × 104 cells/mL in 2 mLs in a 6-well plate and transfected with 0.3 µg LINE-1 plasmid and 0.6 µg APOBEC expression plasmid.
HEK293T cells were plated at 2 × 105 cells/mL in 6 cm poly-L-lysine plates (Becton Dickinson) and were transfected one day later with 5 µg pcDNA/APOBEC plasmids. One day post-transfection, the pulse was initiated by incubating cells in 1mL Starvation media (Met/Cys-free DMEM (Invitrogen)/5% Dialyzed FCS + L-Glutamine). After 30 minutes, starvation media was removed and 750 µL Pulse media was added. Pulse media is Starvation media plus 440µCi EasyTag EXPRE35S35S Protein Label (PerkinElmer) per plate. Following a 30-minute pulse, pulse media was removed and 1mL warm Chase media (Met/Cys-free DMEM + 5%FCS + 2mM Met + 2mM Cys) was added to all plates. Cells were harvested at 0, 30 min, 1 hr, 2 hr and 6 hr timepoints by washing 1x in PBS and lysing cells directly in 500µL ice-cold NP40doc buffer (1% NP40, 0.2% Sodiumdeoxycholate, 0.12M NaCl, 20mM Tris pH 8.0) with Protease Inhibitors (Roche Complete Mini, EDTA-free tablets). Debris was removed by spinning 5 minutes at 16,000g @ 4°C and supernatants were frozen @ −80°C until use. HA-tagged APOBEC3H proteins were Immunoprecipitated using Protein G-Sepharose beads (Zymed) conjugated to RAW HA.11 antibody. Lysates were pre-cleared by incubating lysates with unconjugated beads for 1 hr @ 4°C with rocking and antibody was pre-conjugated with beads for 1 hr @ 4°C with rocking. Beads were washed twice in NP40doc and spun out for 2 min @ 4000g @ 4°C. 0.01% BSA was added to antibody-conjugated beads which were incubated with pre-cleared lysates for 2 hours @ 4°C with rocking. Beads were spun out (2 min @ 4000g) and washed in a series of wash buffers with protease inhibitors (Roche Mini, EDTA-free tablets) as follows: RIPA (10mM Tris pH7.5, 150mM NaCl, 1% NP40, 1% sodiumdeoxycholate, 0.1% SDS), High Salt Buffer (2M NaCl, 10mM Tris pH7.4, 1% NP40, 0.5% sodiumdeoxycholate), Low Salt Buffer (0.5% NP40, 0.1% SDS in PBS) and RIPA. Bead slurries were resuspended in 20µL 2X Protein Loading Buffer (125mM Tris pH6.8, 4% SDS, 20% Glycerol and 10% β-mercaptoethanol), boiled for 10 minutes, beads spun out @ 16,000g for 1 min and supernatants used for 12% SDS-PAGE. Radioactivity was detected using a Typhoon Trio imager (Amersham Biosciences) and quantitated using ImageQuantTL software (Amersham Biosciences).
HeLa cells plated onto coverslips in a 6-well dish at 2 × 105 cells/mL were transfected the next day with 1.5 µg pcDNA/HA-APOBEC expression vectors. 24 hours post-transfection slips were fixed in 4% Paraformaldehyde, permeabilized in 0.3% Triton-X/PBS and blocked in 10% FBS/PBS. HA-tagged proteins were detected using RAWHA.11 antibody (Santa Cruz Biotech) at a 1:100 dilution followed by anti-mouse-TexasRed conjugate (Invitrogen). Nuclei were stained with DAPI in mounting media (Vectashield; Vector Labs)and images were collected on an Olympus DeltaVision microscope.
The human/chimpanzee ancestor APOBEC3H protein sequence was inferred using the gorilla sequence as an outgroup. The human/chimpanzee ancestor APOBEC3H cDNA was then constructed in vitro by site-directed mutagenesis (QuikChange Site Directed Mutagenesis Kit – Stratagene) using the chimpanzee/human_A chimera as the template. The human ancestor APOBEC3H protein was defined by determining all changes that are fixed in the human population and reconstructed in vitro using the human hap I cDNA as a template by site-directed mutagenesis.
The human APOBEC3H coding sequence was amplified/sequenced from genomic DNA samples with the following primer pairs (see Table S4 for primer sequences): Exon 1- Ex1_For/Ex1_Rev, Exons 2/3 -Ex2&3_For/Ex2&3_Rev and Exon 4 - Ex4_For/Ex4_Rev. A second round of sequencing was performed to obtain missing data with the following primer pairs: Exon 2 - MO276/MO271, Exon 3 - MO272/MO273 and Exon 4 - MO274/MO275. Human genomic DNA samples (Coriell) sequenced were: African Pygmy (GM10469 – 10473/GM10492 – 10494), South American (GM10965 – 10969), Central American (GM10975/6/8 & 9), Caucasian (GM893, 946, 1310, 1805, 1806, 1814, 1953, 8428, 9948 and 14492), African South of the Sahara (NA17341 – 17349) and African North of the Sahara (NA17378 – 17384). Extended genotyping of the delN15 and R105G polymorphisms was performed from the following Coriell samples: Han Chinese from Los Angeles (NA17733–17747, 17749, 17752 – 17757, 17759, 17761), African American (NA17102 – 17116, 17125, 17133 – 17140) and European American (NA06990, 07019, 07348, 07349, 10830, 10831, 10842 – 10845, 10848, 10850 – 10854, 10857, 10858, 10860, 10861, 12547, 12548, 12560, 17201). For the R105G polymorphism, genomic DNA was amplified (Supermix; Invitrogen) with primers JAK08 and JAK10 for 30 cycles (94°C – 30 sec.; 58°C – 30 sec; 72°C – 1.5 min.) and PCR product diluted and directly sequenced. For the delN15 polymorphism, genomic DNA was amplified with primers MO285 and MO286 with Taq for an initial denaturing for 5 min at 94°C , followed by 30 cycles (94°C – 15 sec; 58°C – 1 min) and a final 5 min extension at 72°C. DNA Fragment Analysis was performed on an Applied Biosystems 3730xl DNA Analyzer with a 36 cm capillary array and analyzed by GeneMapper 4.0 software (Applied Biosystems) to detect the presence or absence of the 3 bp deletion. Calculation of Hardy-Weinburg equilibrium (HWE) was used to assess genotyping accuracy; the data did not result in a significant rejection of HWE for both the delN15 and R105G polymorphisms in each population.
SNP data was input to PHASE 2.1.1 software (http://stephenslab.uchicago.edu/software.html) and was used to infer phasing of haplotypes over five iterations. Human haplotypes found at > 10% frequency in the panel were reconstructed by site-directed mutagenesis (Quickchange; Stratagene) of human_A APOBEC3H and human_B APOBEC3H plasmids. Complete primer sequences used in this study are found in Table S4. FST values were calculated for each pairwise comparison using the following method of calculation: (MSP − MSG)/[MSP + {nc −1)MSG] (Akey et al., 2002).
We thank John Moran for the LINE-1 retrotransposition plasmid, Thierry Heidmann for the MusD and Alu retrotransposition plasmids, Rasmus Nielsen for advice on the FST analyses, Adam Geballe and Morgan Hakki for assistance with the pulse-chase analysis, Evan Eichler and Jason Kidd for access to the HapMap samples, the FHCRC Genomics and, Scientific Imaging Shared Resources, and Jaisri Lingappa, Evan Eichler, and Rasmus Nielsen for comments on the manuscript. This work was supported by NIH grant R37 AI30937 (M.E.), and Searle Scholar and Burroughs-Wellcome Investigator Awards (H.S.M). J.A.K. was supported by an NIH Postdoctoral Training Grant and M.O. was supported by a National Science Foundation Graduate Research Fellowship.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information 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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Originally written on Fox Sports Detroit | Last updated 5/1/13
DETROIT (AP) -- The Tigers have put left-handed reliever Phil Coke on the 15-day disabled list and have recalled right-hander Jose Ortega from Triple-A Toledo. Detroit announced the moves Wednesday. The Tigers also reinstated outfielder Avisail Garcia from the 15-day disabled list and optioned him to Toledo. Coke has been bothered by a left groin strain, and he goes on the DL retroactive to April 26. He is 0-3 with an 8.31 ERA for the Tigers this season with 10 strikeouts in 8 2-3 innings. Detroit still has left-handers Drew Smyly and Darin Downs in the bullpen. Garcia has missed time because of a bruised right heel.
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Dondi Star Photography Photographer
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The Furious Improvisation
How the WPA and a Cast of Thousands Made High Art out of Desperate Times
By Susan Quinn
(Walker & Company, Hardcover, 9780802716989, 336pp.)
Publication Date: July 8, 2008
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A vivid portrait of the turbulent 1930s and the Roosevelt administration as seen through the WPA's Federal Theater Project.
Under the direction of a five-foot redheaded firecracker, Hallie Flanagan, the Federal Theater Project managed to turn a WPA relief program into a platform for some of the most inventive and cutting-edge theater of its time. This daring experiment by the U.S. government in support of the arts electrified audiences with exciting, controversial productions. Plays like "Voodoo Macbeth" and "The Cradle Will Rock" stirred up politicians by defying segregation and putting the spotlight on social injustice, and the FT P starred some of the greatest figures in twentieth-century American arts--including Orson Welles, John Houseman, and Sinclair Lewis. Susan Quinn brings to life the politics of this desperate era when FDR, Eleanor Roosevelt, and the chain-smoking idealist Harry Hopkins furiously improvised programs to get millions of hungry, unemployed people back to work. Quinn's compelling story of politics and idealism reaches a dramatic climax with the rise of Martin Dies and the House Un-American Activities Committee, which turned the FTP into the first victim of a Red scare that would roil the nation for the next twenty years.
Susan Quinn is the author of two award-winning biographies, about Marie Curie and Karen Horney, as well as Human Trials, which recounts the emotion-laden process of developing a drug for a difficult disease. She lives in Brookline, Massachusetts.


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Meirion Matthias
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a winter scene with an avenue of trees leading along a rough track to houses in the background. Textured and toned for a vintage feel.
August 24th, 2011
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love the pathway dividing trees silhouetted against grey with golden tones above matching ground..
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This is so beautiful...v/f and watch
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so lovely.....vf...barb
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beautiful capture and finishing
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Congratulations on your sale Meirion!!
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Amazing photo! Really dig that vintage feel :)
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Super post processing!!! Makes me want to stroll up that road. I'm a big fan of textures and you've added just the right touch, Meirion. Very nice.
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super composition and love your use of textures.
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fantastic processing and presentation..
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- Simple game play that is hard to master!
- Google Play Games Leaderboards!
- Amazing achievements!
- Easy to share scores!
- Pure evil....
What's New
Trainz Driver
It's time to pack more horsepower in your pocket and take Trainz Driver with you everywhere you go. With a new user interface, controls, and a mix of realistic routes and arcade style sessions, there is something for everyone aged 4 to 104.
Here's what our customers are saying:
***** Shirley Hoffman
Trainz driver Awesome man!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
***** Eric Brubaker
Love it Best simulator i have played,
***** Jaden Campbell
Nice game This is a awesome game!!!
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Good This is the best train game I've ever played on a phone. Keep it up
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Awesome This is the game I've been waiting for. I'll keep it simple... This app works great and I've had no problem with it! I love it... LG Nitro ICS 4.02
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Great Well worth the money. A highly involving and thoroughly enjoyable game simulator. Runs brilliantly on my Galaxy tab 10.1 ICS.
***** noxo
Great game Very happy with game on Samsung Galaxy Note N7000! Super good graphics and nicely boxed controls for mobile.
Trainz Driver puts you in the cab of a variety of locomotives from around the world. Choose from the "Easy Mode" speed controls, or the more advanced "Realistic Mode" control system. With multiple camera modes you can fly alongside your favourite train, admire the view from the cab, or watch from trackside all while you interact with industries and passenger stations, picking up and dropping off freight wagons, people, and more!
Expand your experience: You can also create your own Routes and Sessions in Trainz Simulator for Tablet devices (available for separate purchase on Google Play) and then share them with Trainz Driver users through the Download Station. Hundreds of user-created routes are already available, free to download.
Note: Samsung S3 device owners report incompatibility with the (m0) chipset. S3 + Android 4.1.2 CM10 is reported to solve this issue for some users.
Unfortunately there are just so many Android devices in the market and suppliers update their specs all the time. We have to make the best judgment as to which devices will work with our product outside of our quality assurance. If you are experiencing issues on your device we would love to hear from you. Please contact support so we can attempt to resolve it.
Car Valet
The objective of the game is to get your brand new car out of the way by sliding the other cars, buses and taxis out of the way. You will have to strategically move your car to get out of the traffic jam ! It's comes with 2000 puzzles. As you accumulate coins, you will be able to unlock level packs and various hidden features in the game bonus store.
Each of the 2000 levels requires logic, skill, and patience. Do you have what it takes to help me your car out of these rush hour traffic jams ?
All the fans of Mouse Trap can rejoy, with this exciting new block-sliding puzzle game, we made sure to add stunning HD graphics that look amazing on Android Tablets !
Car Valet will provide you with hours of fun. Will you be able to find a way to unblock all the challenging puzzles ?
We are committed to updating the game on a regular basis with new and challenging levels.
Trainz Simulator
With Trainz custom built input system to get the most out of the touch functionality you'll find driving Trains, laying tracks, creating layouts and completing scheduled tasks a breeze. Trainz Simulator THD is packed full of content, routes and sessions to give you the best Train experience on any mobile platform. Trainz Simulator THD is a world first for Android!
Trainz Simulator includes everything you need to build and operate your own dream railroad. You can build a fun layout in minutes, faithfully recreate a railroad from days gone by or develop a fully fledged prototypical route; the choice is yours.
• Touch input makes driving Trains, laying tracks, creating layouts and completing scheduled tasks easy.
• More Accessible! Take Trainz with you anytime, anywhere.
• Drive Loads of Trains.
• Operate a complete working rail line.
• Build and customize your own living railroad using our advanced route creation tool called "Surveyor".
• Trainz Simulator THD offers advance visual effects such as lighting, weather effects, fog, higher detail Trains, beautiful water, increase performance and 2 new routes with all new sessions.
Previous Upgrades
- List scrolling functionality
- Accuracy for touch interface in Surveyor.
- Free Roaming View camera panning
- Save Session display the time of save.
- Scroll around Map View while in driver mode.
- Display hint windows in various parts of the game.
- Camera control toggled on/off.
- Thumbstick 'navigation' controls.
- Camera Options for Tutorial 2.
- Sander icon displays correctly.
- Merging routes.
- Rail Yard sleep state.
- Sound burst before a session resumed or reloaded.
Note: Unfortunately there are just so many Android devices in the market and suppliers update their specs all the time. We have to make the best judgment as to which devices will work with our product outside of our quality assurance. If you are experiencing issues on your device we would love to hear from you. Please contact support so we can attempt to resolve it.
My First TRAINZ Set
Introducing My First TRAINZ Set!
Get the creative juices flowing as you build winding tracks up, around and through real life rooms in a house! Sit back and take in the view as you watch trains steam around your masterpiece. You can even interact with different industries to pickup and drop off passengers and inventory.
Instead of kids fighting over a violent video game, imagine your thrill when they are feverishly working together to build train tracks all over the bedroom, living room, kitchen or garage!
My First TRAINZ Set brings the joy of model train building to touch devices in a format that is kid-friendly, creative and fun.
* You're the designer! Build winding, climbing tracks and build it over, under, around and through more objects than you can poke a stick at!
* Build Team-Work! Your kids will never get along better than this as they work together to build tracks that will WOW you.
* Nine realistic trains to drive! From old chugging steamers to shiny bullet trains.
* Let your children's (and your!) creativity run wild. Four huge rooms, Countless Items to build on / through / under, Nine Trains and a Track Builder mean that there is virtually limitless gameplay!
* Developed by the company that has been making the very best Train Simulation games for over 10 years!
* Enhanced graphics and effects for NVIDIA Tegra powered devices.
Note: Unfortunately there are just so many Android devices in the market and suppliers update their specs all the time. We have to make the best judgment as to which devices will work with our product outside of our quality assurance. If you are experiencing issues on your device we would love to hear from you. Please contact support so we can attempt to resolve it.
Epic Trains
Build your own train stations to connect the different cities and facilities of you Epic City Buildings! Make transportation easier for the citizens and the trade of goods! Show you dedication to your people! Are you prepared?
*Powered by Dolby® on supported devices*
Train Driver - Train Simulator
Want to learn how to drive a train? Give our train simulator a go!
Our apologies that we've not updated the game in so long. We have been busy with other projects to earn our bread. But we're back and we're bringing you a new update to Train Driver that we hope you guys like!
Issues with sounds stopping on some 4.3 systems are caused by a bug in the Android system:
It will be fixed in next system update.For now, here are things you can do if you are affected:
Use Android version 4.2.2 for now.
Upgrade to latest custom 4.3 (quite complicated).
Wait for google to release official update (should be soon, you can check for it in Settings->About device->System update menu)
Think driving a train is too easy and boring? We dare you to try it with Train Driver simulator!
Wait for the departure signal, close the doors and off you go. Just pay attention to the signs. And don't worry, if you know nothing about driving a train, our tutorial will guide you via an introductory ride. After this ride, the tracks are randomly generated, so you get a different ride every time you play.
In the future, we aim to improve and optimize graphics, and also introduce localized routes with country-specific signals, tracks and trains (with strong emphasis on realism), including High Speed Rail. For now, we have the Polish signals and default train is based on clunky old Polish EN57 EMU.
Train Driver is still a work in progress, but we figured it's already fun to play, so we might as well release it now and get some feedback from you. Feel free to visit us at http://mobiletraindriver.blogspot.co.nz and leave comments or suggestions, how we can improve it.
If you would like early access to new versions and become a beta tester, just join us here:
and follow the instructions. Warning! Beta versions may contain bugs!
Simulator was tested on Asus Nexus 7, Samsung Galaxy S2 (works nice and smooth) and HTC Wildfire (a bit choppy, but still perfectly playable). If you encounter any problems on your device, please let us know via our webpage, or email us, so we can resolve it as fast as possible.
If Train Driver crashes, please send a crash report, stating what it does, along with what actual device you are using. Often, we need to know what the device is, so we can fix it. Google is supposed to tell us this information, but they don't... It's always useful to hear what's going on at your end, and along with the info they do give us we can fix the problem in the next update.
Baby Hazel Kitchen Time
Baby Hazel is very much interested to try something at the kitchen. So today our little angel has decided to make her afternoon meal. Can you be of her help in preparing delicious meal for her. As a first thing, shop for required ingredients, tools and utensils to prepare her food. Then go to kitchen and help her in food preparation. Finally set up the food cart and then feed our princess with the delicious home made food. Happy Kitchen time with Baby Hazel.
Traffic Master
You need to manage the traffic lights to avoid collisions and let the drivers to reach their destinations on time.
Two play modes: time attack and unlimited time.
Version with 24 levels and no ads.
Full Version is free. Ads are only on the main menu! No ads will ever appear during game play!
If you enjoy this application considering purchasing NESDroid or GambatteDroid to support our next projects!
Does not require root!!
Cycle accurate and higher compatibility than Gensoid, required better hardware though.
Multi-touch support!
GENPlusDroid is an open source Sega Genesis emulator powered by GENPlus. Runs Sega Master System and Sega Mega Drive games. High compatibility, games like Virtual Racing and Phantasy Star work full speed!. Devices with a good GPU can use shaders to increase the graphic quality. Real time frame by frame rewinding of game play. Complete customization over multi touch input (size and position). Use a keyboard or gamepad with custom key bindings if you have one (WiiMote and SixAxis IME work!).
Cheats/Game Genie coming next!
Visit us at:
- Developers cannot respond to app comments, so if you need help please email me or visit our forums. I do respond to emails quickly and would love to field all requests (firstname.lastname@example.com). I cannot get extra feedback from a comment without talking to you.
- Supports Android 2.0+, 2.3.X, 3.X, 4.X
- Sega Mega Drive / Genesis, Sega Master System
- High compatibility: Virtual Racing, Phantasy Star, etc
- Sega 6 Button Support
- Multi Touch/Keyboard/Gamepad controller supported
- Custom key bindings
- 2Player support
- Custom multi touch input location and size
- Real Time rewind (see youtube for video)
- Fast Forward
- Auto Save, phone calls won't ruin your game
- Move and resize the touch input controls to your liking!
- Load/Browse compressed archives (*.zip, *.7z)
- Custom ROM directory, RomFetcher support
- PAL support
- Shaders! (hq2x, super eagle, 2xSaI, etc).
- Tablets, many tablets are known to be working perfectly
- The list of working devices grew to big to display here.
- All Android 2.X, 3.X devices should be working.
- After installation, start GENPlusDroid and follow the welcome screen instructions.
- Some tablets it is tricky to locate where GENPlusDroid creates its external directory. If you are having trouble note that in the settings of the app you can set the rom directory to any directory you want.
- Manually loading the auto state fails sometimes
Report all issues to me via email (firstname.lastname@example.com) or post them at:
- Compile yourself, instructions coming soon!
- Portrait Mode
- Native bluetooth support
- Stereoscopic 3D (only on supported devices)
- Shader performance increase for all devices
- Game Genie
- Haptic Feedback
- Email me for feature requests!
This product is not affiliated with, nor authorized, endorsed or licensed in any way by Sega Corporation, its affiliates or subsidiaries. Sega Genesis game software sold separately. Sega and Sega Genesis© are trademarks or registered trademarks of Sega Corporation. All rights reserved. Company and product names are trademarks or registered trademarks of their respective companies. All brands/names/images/etc are copyrighted by their respective owners. Images are shown for documentation purposes only. Halsafar is not affiliated with, nor authorized, endorsed or licensed in any way by software/hardware companies.
TrainOnMap JR Yamanote
In map view mode, trains move on the map. In train view mode, the map behind the train moves by translation of the train.
In higher zoom level, each train shows as three cars.
Each translation of trains is based on timetables of stations. Currently, only timetable for weekdays is supported. If you want to see train moving more clearly, please try high time count ratio and zoom in.
This app is toy app. please don't use it for business and mission critical use.
Map view mode keeps map position. Trains move on the map. Train view mode keeps train position. The map behind the train moves.
A train is touched, app changes train view mode for the train.
Center of operation menu shows train types. Touch it to change train.
Left bottom of operation menu shows station name. Touch it to change station view on map view mode.
+/- are used for zoom-in/zoom-out. Station of view point list is shown at the center of the map.
Selection list of clock with cross mark is used for offset hours from now.
Selection list of clock with x mark is used for time count ratio to 30 times from real time.
Roadmap/satellite photo are used for map type selection.
Right top corner shows language setting. Touch it to change language.
Train Puzzle:Akita Shinkansen
Train Puzzle:Wilderness
Steam locomotives were first developed in Britain and dominated railway transportation until the middle of the 20th century. From the early 1900s they were gradually superseded by electric and diesel locomotives.
Guaranteed without advertising!
Crazy Dentist Salon-Girl Game
Choose your patient first and do a crazy dentist salon carefully. Don't hurt them and you will help them and recover their healty teeth.
You can do Oral Spray and breath spray by yourself. Clean the dirty teeth, pull out teeth. That's really a salon game!
Play our new dentist salon game now and you will get fun!
Physics Sandbox FREE
The play ground awaits.
From simple falling balls to complex physics-driven contraptions, they can all be made in this 2D physics sandbox.
-Simple primitives: simply select the circle or square then drag on screen to create and scale it. Then just let go and physics will do the rest.
-Freeform drawing: just draw the shape you want on the screen using your finger and it will turn into a real physical object. It can be anything from a rock to a rocky mountain. If you can draw it we can create it.
-Joints: for those times when you want to connect objects together we have a simple fixed joint and a spring. You can make things like swinging arms, car suspensions, bridges or any other thing you may think of.
-Edit: you can edit parameters for the objects you create, like mass, friction* and bounciness*.
-Control: in edit mode, adjust the torque slider then use the arrows to rotate the selected object. Perfect for controlling wheels on cars but you can use it for anything you want.
-Camera follow: what it says on the tin. The camera will follow the object you want. Select the object with edit mode then click on the tiny camera in the toolbar. To unfollow just switch to Pan mode and pan away.
-Duplicate(PAID VERSION): tap on the duplicate button on the edit panel then tap anywhere on screen to create a copy of the currently selected object. Does not work for freeform objects yet.
-Time control(PAID VERSION): stay in control by slowing down time or doubling its speed. The time control slider is at the bottom of the edit panel when selecting an object.
-Play/pause: stop the physics simulation so you can build your scene or contraption to your heart's content without having all your stuff fall over. Then hit play and watch the mayhem.
-Pinch zoom: because it's nice.
So jump into the sandbox and knock yourself out.
Car Drift - Police Drift Car
★ Features:
✓ Car Drift is a game designed for players of all ages to exercise the brain response capability. Players can enjoy the game, there is no time limit.
✓ Provides online world rankings. You can also view friend's scores and rankings, to compete with them!
✓ High quality HD graphics. Tablet PC can also be used.
✓ Best suitable for family, friends, and the kids play with the Car Drift free puzzle game.
✓ Funny music and cool sounds accompany the amazing game.
✓ Are you fond of expensive cars? Craving for speed and acceleration? Do you like crazy races? Then Car Drift is made for you!
✓ Car Drift suitable for all, girls, kids, boys and adults alike.
✓ Improve children's memory capacity, and ability to concentrate. The development of children's cognitive skills, and improve the level of education.
✓ Pretty cute game pictures, interesting and rich game content.
✓ Test your reflexes and highway drive skills.
✓ Relax music and sound will make you feel very relaxed and happy, and enjoyable leisure time.
★ Communication:
If you enjoy it, please take a moment to rate it. It would help us a lot.
Thank you for your support!
More from developer
CPU Benchmark
CWMR Touch Installer
Note: This is not the touch edition being built directly by Koush.
Support Devices:
- HTC One
- Motorola Xoom (all variants)
- Verizon Incredible 4G
- AT&T Galaxy Note 2
- Nexus 10
- Nexus 7 & Nexus 7 GSM
- Nexus 4
- Samsung Galaxy S III (all US Variants)
- Galaxy Nexus (LTE/CDMA) (Sprint and Verizon)
- Galaxy Neuxs (GSM)
- View Sonic GTab w/ 1.2 bootloader
- AT&T Skyrocket HD (Galaxy S II)
- T-Mobile Hercules (Galaxy S II) (currently identifies as Skyrocket, but it works fine).
This application does require root.
If you have any questions or problems please first contact: firstname.lastname@example.com
PictureCast for Chromecast
Currently, PictureCast supports two view modes:
- Fullscreen Mode: As the name implies, fullscreen mode utilizes all available space to display your photos.
- Stacked Mode: In this mode, your photos appear in a stack, as if you're throwing your printed photos at your TV screen.
Transitions (only in fullscreen):
- Fade: A simple fade in/out animation to transition between photos.
- Slide: Have a little fun and let your photos slide in and out of view.
- Zoom: Want to mix it up more? Have your photos zoom in and out of view.
Locations you can share from:
- Local: Have a great photo already on your phone/tablet? No problem. Downloaded a fantastic animated GIF of a cat? Share it with ease!
- Google+/Picasa: Do you automatically back up your photos to Google+? Maybe you upload your point and shoot photos to Picasa. Choose an album and share away!
- Facebook: Choose any album from your Facebook profile for immediate view on your TV.
- Reddit: Do you love the pics on /r/funny or do you need a smile from /r/aww? Your favorite subreddit is now only a click away from your TV.
- Note: Support for Instagram, Flickr, Dropbox and more coming soon.
Additional features:
- Slideshow Mode: Did you take 500+ photos on your last vacation and don't want to click through them one by one? We understand. In Slideshow mode, photos can be viewed in order or randomly. You just sit back and enjoy.
- Multi-user Mode: Have a group of friends over who all want to share photos? No problem. PictureCast allows multiple users to connect at once and share photos (really fun in Stacked view!)
- Animated GIF's: Want to see your favorite GIF's from reddit and else where on your big screen? There is no easier way than PictureCast
In-app Purchases explanation:
PictureCast allows for free sharing of your local content in any view mode. Slideshow and Multi-user modes are also available at no cost. To unlock all of the Premium features, we use a one-time In-app purchase. This purchase is good for all devices you own!
Permissions explanation:
android.permission.INTERNET - required for access your online photos
android.permission.READ_EXTERNAL_STORAGE - required to read your local photos
com.android.vending.BILLING - required for in app purchase
android.permission.ACCESS_NETWORK_STATE - required for the Chromecast
android.permission.ACCESS_WIFI_STATE - required for the Chromecast
android.permission.GET_ACCOUNTS - required for loading photos from Google+/Picasa
android.permission.USE_CREDENTIALS - required for loading photos from Google+/Picasa
Please note, this application requires a Google Chromecast to function properly.
Planets Live Wallpaper
Koush Yacht Escape
Small World Notifier
With SWN you can pick a custom notification tone and decide if vibration is your thing or not.
That's it! Go play some Small World man.
Math Warp
Koush Head 2: Return of Koush
This is an alpha, so be gentle but report bugs.
DashClock Fortune Extension
* Open Feint w/ 1000 achievement points!
* Online Leaderboards
* Multiple power ups
* Support for Tablets and Phones!
Knights Of TheButton
Use this application to help you track the status of the button and if it is in danger of hitting zero.
Get push notifications of status updates and major events.
Koush Head
Rainbow Words Live Wallpaper
Works great on phones and tablets both!
To use:
HOME/MENU/Wallpaper/Live Wallpapers/Rainbow Words
Works for phones with live wallpaper feature.
Rule The Air Live Wallpaper
BGB Shout: Virtual Bacon
1-Press a button to hear Bacon
2-Long press a button to set Bacon as your ringtone!
CPU Benchmark (No Ads)
- Benchmark results leveled. Note, multiple runs can still lead to improved results due to cache improvements.


text:
Member Since: 12/04/08Send This Person a Message Give this person a Gift Add This Person as a Friend
Armed Forces Supporter Proud to Know Proud Grandpa American Patriot Proud Father Indianas Finest Hard Charger Blue to the Bone Dedicated Professional Brother in Christ Warrior and Friend Father Husband True Patriot Stay Safe
You can reach this milestone by accruing more than 50 reputation points in the last 7 days
You can reach this milestone by completing the quiz Would You Pass the Police Exam?
You can reach this milestone by completing the quiz Good Cop / Bad Cop
You can reach this milestone by completing Stew Smith's Fitness Challenge
You can reach this milestone by starting a group that is one of the top 100 most active groups.
You can reach this milestone by inviting at least 5 friends who join PoliceLink.
The first 1,000 PoliceLink members to earn one star are considered Founding Members
This user is a PoliceLink Forums Moderator
You can reach this milestone by being one of the top 100 users on the Reputation Leaderboard.
Only sworn LEOs are eligible to receive access to restricted areas.
96harley: Friends
- Chris_Cosgriff
- bump237
- Anonymous
- sdc81
- Robocop33
- oldcop6
96harley: Groups
- Christian Cops Supporters
- Gun Buffs
- Deer Hunting In INDIANA
- Indiana LEOs
- Christian Cops Association
- Struggling with Holidays
- Gun Collectors and Gun Lovers
- Followers Of Christ An Invitation
- Hunters Across America
- Cops for Christ
- Scripture of the day
- 911 Never Forget
- The Great Outdoors
- Remember When
- In Remembrance Friends of Officers lost in the line of duty and otherwise
- Daily Devotional
- GODs Cops
- Future Detectives
- International Police Collectors Group
- Stalkers House
- Christian Knights of the Rose KOR
- Those Who Didnt Vote For Obama
- Duty And Dedication
- Blue Knights MC Group
- Career Advice
- Current Issues
96harley: Gifts
COPS Support Ribbon
COPS Support Ribbon
- Location:
- Martinsville, IN
- Gender:
- Male
- Service:
- Police Officer / Deputy Sheriff / Other Sworn Position
- Status:
- Actively Serving (has access to L.E. restricted areas)
- Relationship Status:
- Married
- Hobbies:
- Patch Collecting, Shooting, Hunting, motorcyles
- Favorite Movies:
- The Searchers
- Favorite TV Shows:
- American Rifleman
- Favorite Music:
- Country Gospel
96harley: References
96harley: Activity
Satan divides and then conquers. He is doing an excellent job with those who claim clergy status. Those who, even among the same race, sacrifice re...
I had a citizen post a thank you on my FB account. I told them we appreciate their loyalty and support. I know the citizens I serve. I have had fol...
The ones I have encountered look anything but "special agents or investigators". One in particular has no law enforcement background.
Comment Wall
Add this person as a friend to write on their Comment Wall.
about 1 month ago
47024 Comments
Thanks for the friendship. Have a great week and stay safe.
over 5 years ago
70012 Comments
about 6 years ago
18328 Comments
I promised many of my PoliceLink friends that I would keep you updated on my new mystery/suspense trilogy. I'm pleased to announce my first novel, Child Finder, has debuted this month. It is available online at Barnes & Noble, and
Amazon. It has already received some great reviews, to include a 5-Star rating by Armchair Interviews!
I am hosting a formal launch and book signing event at Borders bookstore, Chapel Hills Mall, Colorado Springs, CO on Sunday, June 7th, from 12:00 noon to 4:00 pm. It would be great to see you there, but if you cannot make it, then I hope you still get your hands on a copy of my book.
Please check my website where I have a pretty cool book trailer movie posted, as well as much more detail about the trilogy and upcoming book signing events: www.childfinder.us
see more Lolcats and funny pictures
about 6 years ago
MyHotComments Walking thru the hood saying hello how are you.


text:
Photo Coverage: Meet the Contestants on Episodes 1 and 2 of THE X-FACTOR!
|Related Links
|Scoop: THE GOLDBERGS on ABC - Tonight, December 24, 2014
December 24, 2014
|Scoop: THE GOLDBERGS on ABC - Wednesday, January 7, 2015
December 23, 2014
|Scoop: THE GOLDBERGS on ABC - Tonight, December 17, 2014
December 17, 2014
|Related: FOX, THE X FACTOR
Here's a look at the hottest contestants from episode one and two of the X-FACTOR on Fox! Tune in on Wednesdays, 8:00-10:00 PM ET live/PT tape-delayed and Thursdays, 8:00-9:00 PM ET live/PT tape-delayed).
Expect the unexpected when the world's only $5 million audition returns with two new superstar judges and more surprises and twists. Season Two of THE X FACTOR features global pop icon Britney Spears and multi-talented artist Demi Lovato on the judging panel, alongside returning judges Simon Cowell and L.A. Reid.
THE X FACTOR, last season's No. 1 new unscripted series, gives viewers the opportunity to help discover the next global superstar or breakout music group. The superstar team of judges travels the nation searching for talent 12 years old or over – both solo artists and vocal groups – who are willing to brave the panel for a chance to make their dreams come true. Auditions for THE X FACTOR were held this spring in Kansas City, MO; Austin, TX; San Francisco, CA; Greensboro, NC; and Providence, RI.
In a departure from other singing competition series, contestants not only audition in front of THE X FACTOR judges. Perhaps more importantly, they sing for an audience of thousands, which is often referred to as the "fifth" judge, because their enthusiasm – or lack thereof – can help determine the fate of these hopefuls. This initial audition is the ultimate test to prove they have the vocal ability, charisma and stage presence it takes to become a global superstar or breakout music act and win a $5 million recording contract with Syco/Sony Music.
Those contestants who survive the first auditions graduate to "boot camp" and will be divided into four categories – Boys, Girls, Over 30s and Groups. Each category will be mentored by one of the show's judges. Not only is it a competition between the hopefuls to stake their claim for the coveted win, but it's also a showdown among the judges as to whose acts will dominate the competition and make it to the finals. The judges may have their say in how the competition progresses, but it will be up to America to decide who ultimately has THE X FACTOR.
Photos: FOX
Don Philip performs
Paige Thomas performs
Quatrele Da'an Smith
Reed Deming
Demi Lovato (L) and Jillian Jensen (R)
Jillian Jensen
Kaci Newton
Paige Thomas
Reed Deming
About Author
- CNBC to Air THE MAKING OF DIG, the USA Network Event Series, 3/1
- NBC to Air One-Hour Season Finale of CHRISTINA MILIAN TURNED UP, 3/1
- NBC Universo to Present ROMEO SANTOS DESDE VINA Concert Exclusive, 3/1
- Kelly Clarkson to Visit CBS SUNDAY MORNING, 3/1
- VIDEO: Clark Gregg Teases New AGENTS OF S.H.I.E.L.D. Season Using Emojis
- Dakota Johnson to Host SNL Tonight; Alabama Shakes to Perform
- NBC Sports to Air Coverage of U.S. GRAND PRIX FROM PARK CITY
- Discovery Family to Premiere New Episodes of TRANSFORMERS RESCUE BOTS, Today
- Telemundo to Present FUTBOL ESTELAR: LEON VS SANTOS, Today
- Telemundo to Give Viewers Sneak Peek at Upcoming Programming on LO NUEVO, Today


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The UK's fastest-growing regional news network
See the full forecast for your area.
Sponsored by Britelite.
Home Kent Sport Article
Olympic champion Lizzy Yarnold received a hero's welcome on her bus tour through Kent on Friday.
Yarnold, who won skeleton gold in Sochi, was greeted by crowds of cheering pupils at several schools in the west of the county.
And many people took to the streets to salute the former Maidstone Grammar School for Girls student, who posed for pictures and spoke to supporters.
The bus route took in Sevenoaks, Otford, Eynsford and the iconic Brands Hatch circuit before finishing at West Kingsdown C of E Primary School.
Yarnold said: "It's been a lot of fun. It's really surprised me how many people in the local community have come out on the streets. When they're driving along, they're honkng their horns. I think they're proud of what I've achieved and they're a part of it as well.
"It does mean a lot and it surprises me all the time, that it's not just me that cares about winning and representing my country, it's everyone."
Yarnold was presented with a miniature gold postbox in West Kingsdown and her open-top bus was followed by the Arriva bus which was sprayed gold to commemorate her Sochi success.
Her skeleton win meant Great Britain have now won medals in that event in the last four Winter Olympics. Alex Coomber claimed bronze in 2002, Shelley Rudman took silver in 2006 and Amy Williams won gold in 2010.
So what is it about Brits in the skeleton?
"It's down to so many things," Yarnold said. "We've got a great programme, we pick the best talent through the talent searches at UK Sport.
"I think we can go from strength to strength and hopefully I'll defend it in Pyeongchang."
Click here for more sport from Kent.
Click here for more news from around the county.


text:
Seattle, WA
Comment, Like, Favorite
Expert Printing and Assembly
Shipping Within 2 - 3 Business Days
Millions of Satisfied Customers
African Gerbera Standout
Mike Reid
Photograph - Photography
March 25th, 2013
Viewed 1,040 Times - Last Visitor from Beverly Hills, CA on 06/30/2015 at 2:03 AM
Sales Sheet
More from Mike Reid
Silverthorne, CO - United States
Beautiful.....Congrats on your sale!
Toms River, NJ - United States
Beautiful congrats on your sale!
Barataria, LA - United States
Beautiful composition congrats on your sale
Stevenson, MD - United States
Beautiful color and composition. F/L
Brookeville, MD - United States
Glorious work of art, Mike ... so beautifully composed ... and I just love the colors and mood!!! l/f
New York, NY - United States
Beautiful shot.
Pianoro, Bo - Italy
Fantastic capture, congratulations on your sale!
San Francisco, CA - United States
Beautiful image, congrats on your sale!
Nagpur, Ma - India
Great and Congratulation on sale announcement.
Wooster, OH - United States
Beautiful macro. Congratulations.
Spokane, WA - United States
WOW beautifully done....fantastic composition. Congrats on your sale. SE f
Boise, ID - United States
This is so beautiful. Congrats!
Keene , On - Canada
Just beautiful! Congrats and best wishes moving forward.
University Place, WA - United States
Beautiful work....congrats on your sale!
Honolulu, HI - United States
Beautiful! Congratulations!
Casa Grande, AZ - United States
Congrats on Your Sale..............Gorgeous
Seattle, WA - United States
Thanks everyone....found this amazing patch of color down in Carmel.
Miami, FL - United States
Lovely! Congrats on your sale!
Binghamton, NY - United States
Beautiful color and dof, congratulations!
San Luis Obispo, CA - United States
Wow, that is beautiful!
Markham, ON - Canada
Exciting piece, congratulations on selling.
Galt, Il - United States
Beautiful image!! Congratulations on your sale!!
San Francisco, CA - United States
Lovely soft colors, congrats on your sale!
Seattle, WA - United States
Thanks so much for all your kind words
Lyman, ME - United States
Love this pic. The slightly out of focused flowers in the back are a great backdrop.
Blue Springs, MO - United States
beautiful! Congrats!!
Palm Desert, CA - United States
Nice work! Congratulations on your sale!
Canton, Ga - United States
What beautiful colors! Congratulations on your sale!
Lewiston, ME - United States
Congratulations on your sale! Beautiful!
Chalmette, LA - United States
Absolutely beautiful! Congratulations!
London - United Kingdom
Beautiful work! Congrats on your sale! v
Seattle, WA - United States
Hey thanks everyone....love the dramatic hues in this shot
Calgary, AB - Canada
Hi Mike. Lovely. Congratulations on your sale.
Colorado Springs, CO - United States
This is really dramatic! Congatulations on your sale!
Asheville, NC - United States
Beautiful! Congratulations!
Lockport, IL - United States
Mike a beautiful image! Congrats on your sale!
Selah, WA - United States
Oslo, Os - Norway
Congratulations on your Art sale, Mike!
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