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Despite recent evidence suggesting that histone lysine acetylation contributes to base excision repair (BER) in cells, their exact mechanistic role remains unclear. In order to examine the influence of histone acetylation on the initial steps of BER, we assembled nucleosome arrays consisting of homogeneously acetylated histone H3 (H3K18 and H3K27) and measured the repair of a site-specifically positioned 2′-deoxyuridine (dU) residue by uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1). We find that H3K18ac and H3K27ac differentially influence the combined activities of UDG/APE1 on compact chromatin, suggesting that acetylated lysine residues on the H3 tail domain play distinct roles in regulating the initial steps of BER. In addition, we show that the effects of H3 tail domain acetylation on UDG/APE1 activity are at the nucleosome level and do not influence higher-order chromatin folding. Overall, these results establish a novel regulatory role for histone H3 acetylation during the initiation of BER on chromatin.
Cellular DNA is constantly exposed to genotoxic agents arising from both exogenous and endogenous sources. If left unrepaired, the resulting DNA lesions can lead to genetic instability and alterations that are at the heart of various diseases, including cancer, cardiovascular diseases, and neurodegenerative diseases, as well as aging1,2,3,4,5. The front-line defense against damaged nucleobases is the base excision repair (BER) pathway6. BER is initiated by DNA glycosylases that are responsible for recognizing and excising specific nucleobase lesions via cleavage of the glycosidic bond7. For example, 2′-deoxyuridine (dU), which can result from the deamination of cytosine8, is recognized and excised from DNA by uracil DNA glycosylase (UDG). Removal of the damaged base results in formation of an abasic site, which is subsequently cleaved by apurinic/apyrimidinic endonuclease 1 (APE1) in order to generate a nicked substrate for further downstream processing6.
Like all genomic processes, BER must take place within the structural constraints of chromatin. In eukaryotic cells, the basic unit of chromatin is the nucleosome, which consists of ∼147 base pairs (bp) of DNA wrapped nearly two times around a protein core comprising two copies of each histone H2A, H2B, H3, and H4. In general, nucleosomes are repressive structures that restrict DNA-binding factors from accessing the underlying DNA9,10. Indeed, nearly every step in the BER process is inhibited to varying degrees by the packaging of DNA into nucleosomes, and the extent of inhibition depends largely on the rotational setting of the lesion in relation to the histone octamer surface11,12,13,14,15. In addition, individual nucleosomes are connected by stretches of linker DNA to form chromosome-sized nucleosome arrays that fold and condense into more compact higher-order structures that further impact DNA accessibility and BER16,17,18. For example, the combined catalytic activities of UDG and APE1 are inhibited by as much as 20-fold upon folding of nucleosome arrays into compact 30 nm chromatin fibers19.
Given the impact of chromatin structure on BER, it is not surprising that histone posttranslational modifications (PTMs) have been implicated in the repair process20,21,22,23. In particular, histone acetylation has been shown to directly impact nucleosome stability, mobility, and unwrapping24,25,26, as well as higher-order chromatin folding27,28, supporting a model in which histone acetylation directly regulates BER by controlling DNA accessibility. Indeed, emerging evidence supports a role for site-specific histone acetylation in cellular BER29,30. However, due to challenges associated with manipulating the chemical structure of chromatin in cells, functional relationships between histone acetylation and BER remain mostly unexplored. A few pioneering studies have investigated the influence of histone acetylation on BER using homogeneously acetylated mononucleosomes as model substrates30,31. Uncoupled from the more complex cellular environment, reconstituted mononucleosomes enable quantitative interrogation of the BER pathway in the context of specific PTMs. Although these studies have provided novel insight into how acetylation potentially regulates BER in vivo, mononucleosomes fall short of being an ideal chromatin model. In particular, they cannot be used to assess the impact of histone acetylation (or other PTMs) on inter-nucleosomal interactions and higher-order chromatin folding, which are likely key factors that regulate BER at the chromatin level19,32.
In the present study, we assembled novel nucleosome arrays consisting of both site-specifically damaged DNA and homogeneously acetylated histones in order to investigate, for the first time, the impact of histone acetylation on BER within higher-order chromatin structures. Specifically, we reconstituted 12-mer nucleosome arrays consisting of one of two homogeneously acetylated H3 histones (H3K18ac and H3K27ac) and measured the repair of a site-specifically positioned dU residue by the combined activities of UDG and APE1. By comparing acetylated chromatin substrates, including mononucleosomes, to their unmodified counterparts, we find that H3K18ac and H3K27ac modulate the activities of UDG and APE1 in a context-dependent manner, indicating that acetylation of histone H3 has a site-specific effect on BER. Given that H3K18 and H3K27 are targets of the acetyltransferase CBP/p300, this work potentially establishes a novel regulatory role for CBP/p300 in the BER pathway.
Results and Discussion
Assembly of chemically defined nucleosome arrays
The nucleosome arrays described in this work utilize a DNA template composed of 12 copies of the “ Widom 601” nucleosome position sequence33, each of which is separated by 30 bp of linker DNA (Figs 1a and S1a). This array architecture has been shown to reconstruct the native chromatin environment and the spatial relationship between neighboring nucleosomes34,35, and has been used extensively to investigate the regulation of chromatin structure by histone PTMs28,36,37,38. Furthermore, while the 601 DNA sequence has a high affinity for the histone octamer, mononucleosomes and arrays assembled using 601 DNA remain highly dynamic and undergo spontaneous conformational fluctuations that increase DNA accessibility (i.e. site exposure) on a biologically relevant timescale39,40,41. We designed the 12 × 601 template (12-601-Nb) to contain two proximal nicking endonuclease recognition sites (Nb.BbvCI) within the nucleosome-bound region of the fifth 601 repeat (N5), which enabled site-specific incorporation of single dU residue using our previously reported “plug-and-play” approach (see “Methods” section and Fig. S1)19. The minor sequence variations resulting from the inclusion of these restriction sites do not significantly alter the rotational positioning of the DNA relative to the native 601 sequence19. Treatment of 12-601-Nb with Nb.BbvCI resulted in formation of a short gap upon heat denaturation, which was subsequently filled in with a DNA oligonucleotide carrying the desired dU residue (Table S1). The remaining nicks were then resealed by T4 DNA ligase, resulting in generation of the fully intact DNA template (12-601-dU49). Complete incorporation (>95%) of the dU-modified oligonucleotide was confirmed using an electrophoretic mobility retardation assay (Fig. S1b). The final position of the dU residue (49 nucleotides from the N5 dyad; dU49) was such that upon reconstitution of DNA template into chromatin, the lesion would be facing outward relative to the underlying nucleosome surface (Fig. 2b). UDG/APE1-mediated digestion of dU at this position is highly sensitive to magnesium-induced chromatin compaction, suggesting that this position is ideal for examining the impact of histone PTMs on the initial steps of BER19.
The dU containing DNA template (12-601-dU49) was reconstituted into 12-mer nucleosome arrays using recombinant human histone octamers containing either wild-type (WT) H3 or homogeneously acetylated H3 (H3K18ac or H3K27ac)42,43,44. The acetylated H3 proteins were produced via unnatural amino acid mutagenesis using an amber suppression-based approach (Fig. S3)45. Optimal reconstitution conditions were determined by histone octamer titration, and fully saturated nucleosome arrays were purified via selective magnesium-induced precipitation (Fig. S4a)43,46,47. Native gel electrophoresis of the reconstituted products revealed, in all cases, a single band consistent with a homogeneous population of nucleosome arrays (Fig. 2a). Partial digestion with micrococcal nuclease (MNase) confirmed the presence of 12 positioned nucleosomes (Fig. 2b), and restriction enzyme digests of the reconstituted arrays demonstrated full nucleosome occupancy (Fig. S4b). Together, these data suggest that formation of nucleosome arrays is not affected by the presence of a single dU residue and acetylated histone H3, which is in agreement with several previous reports11,19,30,32.
Uracil removal by UDG and APE1 is dependent on site-specific histone acetylation
We investigated the impact of H3 acetylation (H3K18ac and H3K27ac) on the combined activities of UDG/APE1. We chose these lysine residues because they are preferred targets for the acetyltransferase p300 and closely related CBP (CBP/p300)48,49. However, additional sites on the histone H3 tail (e.g. K9, K14, and K23) can also be acetylated by CBP/p300. CBP/p300 has been shown to interact with and acetylate a number of key enzymes in the BER pathway, including several DNA glycosylases, APE1, and polymerase β (pol β)50,51,52,53. Thus, it is possible that these BER proteins could facilitate the recruitment of CBP/p300 to sites of DNA damage to acetylate histones and potential stimulate BER54. However, this has yet to be experimentally demonstrated.
We examined UDG/APE1-mediate repair of dU under both low (0.2 mM) and high (2.0 mM) Mg2+ concentration. At 0.2 mM Mg2+, 12-mer arrays take on an extended “beads-on-a-string” structure representing an accessible chromatin state. However, at 2 mM Mg2+, these same arrays fold into compact “30 nm” fibers55,56. Magnesium has been shown to impact the activities of UDG enzymes and is required for APE1 catalysis6,57,58,59. Magnesium also influences nucleosome dynamics and stability60,61. Therefore, the rate of BER on each substrate type (naked DNA, mononucleosomes, and arrays) was measured at both Mg2+ concentration and only datasets obtained at the same Mg2+ concentration (i.e. chromatin structural state) were compared to each other.
We first examined the ability of UDG/APE1 to digest the various chromatin substrates (Fig. 1b) under low magnesium conditions (0.2 mM Mg2+), where nucleosome arrays are mostly in an extended state55,56. Both naked DNA (12-601-Nb) and nucleosome arrays (10 nM, Fig. 1b) were treated with UDG and APE1 (1 nM each), and cleavage of the dU-containing strand was measured over time (Figs 3a,b and S6a). A similar set of digestion reactions was also carried out on either unmodified or acetylated (H3K18ac and H3K27ac) mononucleosome substrates containing an identically positioned dU lesion (Figs 1c and S2, S6b). We note that the reaction conditions used here have been used extensively to study the impact of nucleosomes on UDG/APE-mediated BER11,12,62,63. In agreement with our previous study19, UDG/APE1-mediated digestion of dU49 was significantly inhibited on both mononucleosomes and nucleosome arrays relative to free DNA (Fig. 3a and Table 1). Importantly, the activity of UDG/APE1 on the chromatin substrates was dependent on histone H3 acetylation. While H3K18ac had no effect on digestion of dU49 by UDG/APE1, H3K27ac resulted in significant inhibition of UDG/APE1 activity relative to WT histone H3 (Fig. 3b and Table 1). This trend was similar for both mononucleosomes and nucleosome arrays, suggesting that the influence of H3K27ac on UDG/APE1 activity is likely the result of intra-nucleosomal interactions rather than interactions between neighboring nucleosomes. This is consistent with previous observations that acetylation of the H3 tail domain primarily regulates DNA accessibility at the nucleosome level27. This is also in agreement with our previous observation that accessibility of dU49 in extended arrays is almost exclusively dictated by the underlying histone octamer19. These different outcomes were somewhat surprising given the close proximity of these two lysine residues to each other on the histone H3 tail (Fig. 1c). Furthermore, previous work has shown that acetylation of lysine residues near the N-terminus of the H3 tail domain (e.g. H3K14 and H3K18) does not induce significant changes in nucleosome dynamics (i.e. DNA unwrapping)30,64. Nevertheless, acetylation has been shown to alter H3 tail dynamics and tail-DNA contacts65,66. Therefore, it is possible that H3K27ac, but not H3K18, results in the repositioning of the H3 tail domain in such a way that accessibility of dU49 to UDG/APE1 is reduced. Further experiments are needed to verify this model.
Next, we repeated the UDG/APE1 digestion experiment described above in the presence of 2 mM Mg2+, which induces compaction of the extended array into a “30 nm” chromatin fiber55,56. Consistent with our previous observations19, increasing the Mg2+ concentration from 0.2 to 2.0 mM stimulated UDG/APE1 activity on both free DNA and mononucleosomes (Table 1). Under these conditions, we found that acetylation of either H3K18 or H3K27 did not significantly affect the rate of UDG/APE1-mediated cleavage of dU49 on mononucleosomes (Fig. 3c,d and Table 1). The extent of digestion (∼80%) was also independent of acetylation (Fig. 4). Digestion of compact arrays by UDG/APE1 resulted in the formation of substantially less incised product overall compared to corresponding mononucleosome substrates (regardless of the acetylation state), which is consistent with our previous observation that folding of chromatin into a 30 nm fibers is a major obstacle for BER (Fig. 4)19. Importantly, the amount of dU49 digested by UDG/APE1 after 60 minutes on the compact arrays was highly dependent on the acetylation state of histone H3. Acetylation of H3K18 resulted in increased product formation relative to the unmodified array, whereas acetylation of H3K27 had the opposite effect (Figs 3c,d and 4). Notably, product formation was inhibited by only ∼30% on H3K18ac containing arrays (57.1 ± 7.6% cleavage) relative to mononucleosomes (83.0 ± 0.7% cleavage) after 60 minutes, whereas ∼70% inhibition of product formation was observed on H3K27ac arrays (24.5 ± 2.1% cleavage) (Fig. 4). Interestingly, despite differences in the amount of product formed, we found that the initial rate of dU49 incision was similar for each of the array substrates (Table 1). A possible explanation for this observation is the existence of at least two distinct substrate populations within each compact array: one in which dU49 is directly accessible to UDG/APE1 and one where it is not, which is consistent with the dynamic nature of nucleosome arrays39,67. Accordingly, our data suggests that acetylation of histone H3 influences the distribution of these states in a site-specific manner, with acetylation of H3K18 shifting the population towards a more accessible state. However, based on our steady-state analysis, we cannot rule out that product inhibition plays a role.
It was previously reported that acetylation of the histone H3 tail domain (H3K14) does not affect the combined activities of UDG/APE1 on damaged mononucleosome substrates30. These experiments were carried out under “high Mg2+” conditions (5 mM), and are in general agreement with our mononucleosome data presented in Fig. 3. However, by expanding these studies to include oligonucleosome arrays, which more accurately represent chromatin in vivo, the above results demonstrate that histone acetylation can have a dramatic affect on the initial steps of BER on compact chromatin. To the best of our knowledge, this is the first direct experimental evidence demonstrating that histone acetylation is capable of modulating UDG/APE1-mediated excision of dU from chromatin.
Acetylation of the histone H3 tail domain regulates BER at the nucleosome level within compact chromatin
In our previous study, we showed that digestion of dU49 on nucleosome arrays by UDG and APE1 was highly dependent on the structural state of the array19. In order to determine if structural differences exist between unmodified and acetylated arrays that may explain the observed variation in UDG/APE1 activity, we utilized a previously reported fluorescence resonance energy transfer (FRET)-based assay that measures the distance between nucleosome 5 (N5) and nucleosome 7 (N7) within the arrays (Figs 5a and S7)39,68. Mg2+-induced compaction (2.0 mM) of both WT and H3 acetylated arrays resulted in a dramatic increase in the FRET signal (>10-fold) compared to naked DNA (Fig. 5b), consistent with the formation of compact 30 nm chromatin fibers. Importantly, no significant difference in the extent of compaction (i.e. distance between nucleosomes) was observed for acetylated arrays relative to unmodified arrays, indicating that the higher-order structure was not perturbed by histone H3 acetylation.
Next, we measured restriction enzyme (RE) accessibility within compact arrays (2 mM Mg2+) containing either WT or acetylated histone H3 (Fig. 6). Previous studies have shown that RE digestion serves as a sensitive assay to interrogate the influence of histone PTMs on chromatin structure and DNA accessibility27,69. We first assessed the rate of cleavage of the BstXI restriction site, which is positioned within the linker DNA between the fifth (N5) and sixth (N6) nucleosomes in the array (Fig. 1a). We found no significant difference in the rate of cleavage by BstXI between these arrays, indicating that H3K18ac and H3K27ac do not cause significant changes in the overall structure of compact arrays (Figs 6a and S8a). This is in agreement with previous work showing that installation of up to six acetylation mimics (i.e. K → Q substitutions) in the H3 tail domain does not increase accessibility within nucleosome arrays27,69. We then investigated the cleavage of two BbvCI restriction sites, which are positioned on either side of dU49 within the nucleosome (N5) core (BbvCI and Nb.BbvCI share the same recognition sequence) (Fig. 1a). In contrast to BstXI, cleavage by BbvCI within the nucleosome was significantly greater on both acetylated arrays compared to the WT array, indicating that acetylation of the H3 tail domain increases DNA accessibility near dU49 (Figs 6b and S8b).
Together, these data suggest that the observed effects of acetylation on UDG/APE1-mediate BER in compact chromatin is not due to changes in the higher-order structure, but rather acetylation of the H3 tail domain likely influences nucleosome-level accessibility of the DNA near dU49 in a lysine-dependent manner. Interestingly, these results show that RE accessibility and UDG/APE1 activity are not always correlated, further demonstrating the complex nature of BER on chromatin.
Despite the need for BER to occur throughout the genome, it is now clear that nucleosome organization and chromatin structure appear to restrict this pathway70,71,72,73. Indeed, several studies have observed an inverse correlation between BER and chromatin compaction in living cells72,73. For example, the mammalian 8-oxoguanine DNA glycosylase OGG1 is excluded from heterochromatin regions upon induction of oxidative DNA damage72. Furthermore, a recent genome-wide mapping study revealed that dU is enriched within the highly compact environment of human centromeres73. Our results obtained using nucleosome arrays are consistent with these observations in cells (Fig. 3), and show that inter-nucleosomal interactions and histone tail domains play a significant role in regulating UDG/APE1-mediated repair of dU within compact chromatin.
In order to investigate how histone acetylation impacts the repair of dU within chromatin, we assembled nucleosome arrays containing both site-specifically damaged DNA and homogeneously acetylated histones. Our results demonstrate that acetylation at a particular site does not have a general effect on UDG/APE1 activity, but rather each site must be considered individually. However, because we only examined a single positioned dU residue, we cannot rule out that H3K18ac and H3K27ac may have a different effect on the removal of dU at other positions, for example within the linker DNA. Similarly, acetylation of other lysine residues, including those on histone H4, may result in alternative outcomes. Nevertheless, the opposing roles of histone H3 acetylation during BER observed here highlights the complex nature of this relationship and suggests that regulation of BER in chromatin likely requires precise-tuning of the local acetylation state. Overall, these results further support the growing body of evidence showing the importance of epigenetic modifications in the BER pathway70,74.
The acetyltransferase CBP/p300 acetylates both H3K18 and H3K27 in vitro and in cells. Thus, our results suggest that acetylation of histone H3 may represent a novel regulatory role for CBP/p300 in the BER pathway. However, given the opposing influence of H3K18ac and H3K27ac on the removal of dU from compact chromatin, the precise role of CBP/p300 in BER (i.e. suppression vs. promotion) may ultimately depend on the presence of other factors that can target its acetyltransferase activity to a specific site. For example, pre-existing histone modifications (e.g. trimethylation of H3K27) may influence the substrate specificity of CBP/p300, possibly by blocking acetylation of a specific site or altering its binding affinity to the nucleosome75. In addition, interactions between CBP/p300 and its numerous binding partners, including BER proteins, may promote acetylation at a particular lysine on histones. For example, thymine DNA glycosylase (TDG) was shown to significantly alter the specificity of p300, increasing its preference for H3K1876. It is important to note that CBP/p300 itself (which was not included in our experiments) could potentially alter DNA accessibility independent of acetylation through binding of the H3/H4 tail domains65. Considering the observation reported herein, as well as the intimate association of CBP/p300 with several BER factors and chromatin, there remains a compelling need for future investigations on the role of CBP/p300 in the BER pathway.
Overall, this work bring us one step closer to understanding how histone acetylation influences BER and highlights the advantages of using chemically defined nucleosome arrays to investigate the BER pathway. In the future, it will be interesting to use this model system to examine how acetylation at other histone sites (and combinations thereof), as well as other histone PTMs, influence the BER pathway. Given that BER must occur within a variety of chromatin states (both structural and chemical) throughout the genome, these studies will provide a critical first step in unraveling the relationship between histone PTMs, chromatin structure, and BER in vivo.
Preparation of DNA substrates containing dU49
Assembly of a plasmid DNA (pUC-12-601-Nt) containing the unmodified 12 × 601 DNA template (12-601-Nb) was described previously19. A short oligonucleotide containing a single dU residue was incorporated into the plasmid using our previously reported strand-exchange protocol19. Briefly, plasmid pUC-12-601-Nt (500 μg, 160 pmol) was digested for 1 hour with 500 units of Nb.BbvCI (all RE were purchased from New England Biolabs) in a 700 μL reaction mixture according to the manufacturer’s recommended protocol using NEB buffer 3.1 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg/mL BSA, pH 7.9). To the nicked plasmid was added 3.2 nmol of the 5′-[32P]-labeled synthetic oligonucleotide insert (N5_dU49; Table S1) containing dU49 and 800 pmol of a locked nucleic acid (LNA) capture probe (N5_Cap.Nb; Table S1), which was required to sequester the unmodified strand. The reaction mixture was then heated at 80 °C for 20 minutes before being slowly cooled to room temperature. Following this annealing step (~1 hour), 400 units of T4 DNA ligase (New England Biolabs) and ATP (2 mM final concentration) were added to the mixture. The ligation reaction was allowed to proceed for 2 hours at room temperature, and inactivated at 70 °C for 20 minutes. Finally, the reaction mixture was desalted by ethanol precipitation and complete incorporation of the dU49-containing oligonucleotide was confirmed by 10% native PAGE (19:1 acrylamide:bisacrylamide) (Fig. S1b).
The unmodified DNA template (1-601-Nb) used to assemble mononucleosomes containing dU49 was prepared by PCR from the native 601 DNA sequence using primers N5_NCP_FWD and N5_NCP_REV (Fig. S2a and Table S1). The dU residue was then installed into this DNA using the same oligonucleotide exchange process described above, employing oligonucleotide N5_dU49 (Table S1). Complete incorporation of the modified oligonucleotide was confirmed by native PAGE (Fig. S2b).
Histone preparation and octamer refolding
Recombinant human histones (H2A.1, H2B.1, H3, and H4) were expressed and purified using established protocols77. Homogeniously acetylated histones (H3K18ac and H3K27ac) were expressed and purified from ΔCobB E.coli cells (BL21[DE3] strain) as descibed previously45, and acetylation was confired by mass spectrometry (Fig. S3). Histone octomers were refolded using standard protocol42,43,44, and purified by size exclusion chromatography using a SuperDex 200 10/300 GL column (GE Lifesciences). Purified histone octamers were stored at 4 °C in Octamer Buffer (2 M NaCl, 5 mM BME, 0.2 mM PMSF, 1 mM EDTA, 10 mM HEPES, pH 7.8).
Reconstitution of mononucleosomes and nucleosome arrays
Reconstitution of both mononucleosomes and nucleosome arrays was carried out according to procedures in our previous report19. Prior to reconstitution, the dU containing 12 × 601 DNA template (12-601-dU49) was removed from the corresponding plasmid backbone via digestion with EcoRV. The reaction mixture was also digested with DraI and HaeII (600 units each) in order to degrade the plasmid DNA to fragments ≤700 bp. Following digestion, the reaction mixture was passed through a QIAquick spin column (Qiagen) and the eluted DNA was used directly to reconstitute nucleosome arrays. Following the reconstitution step, oligonucleosomes were purified away from the plasmid DNA using selective Mg2+ precipitation (6 mM MgCl2). After addition of MgCl2, the samples were incubated on ice for 30 minutes and spun at 13,000 × g for 20 minutes at 4 °C. The pellet containing the pure arrays was then dissolved in 25 µL of low salt (LS) buffer (25 mM NaCl, 0.1 mM PMSF, 10 mM HEPES, pH 7.8) and stored at 4 °C until use. Reconstituted arrays were analyzed by 0.6% agarose gel electrophoresis (Fig. S4a) and reconstituted mononucleosomes were analyzed by 5% native PAGE (59:1 acrylamide:bisacrylamide) (Fig. S5).
Nucleosome saturation assay
Nucleosome saturation was confirmed by digestion of 0.2 pmol oligonucleosome array with 10 units of PflMI/BstXI in LS buffer supplemented with 2 mM MgCl2. The respective naked DNAs were also digested under the same conditions, and both sets of samples (naked DNA and arrays) were analyzed side-by-side by 5% native PAGE (59:1 acrylamide:bisacrylamide) (Fig. S4b). The presence of a nucleosome band as well as the absence of significant free DNA (<3%) demonstrates full nucleosome occupancies in these reconstituted arrays.
Partial micrococcal nuclease digestion of reconstituted arrays
The presence of 12 nucleosomes per array was confirmed through partial micrococcal nuclease (MNase) digestion. Arrays (0.4 pmol) were digested with 1000 units of MNase for 1 min on ice in presence of 5 mM CaCl2. Reactions were stopped with the addition of 0.2% (v/v) SDS and 20 mM EDTA and filtered through a QIAquick spin column. The eluted DNA was analyzed on a 0.5% agarose gel and visualized with ethidium bromide (Fig. 2b).
UDG and APE1 digestion reactions
Reaction mixtures contained 10 nM of the appropriate substrate, 25 mM NaCl, 0.1 mM PMSF, 10 mM HEPES, pH 7.8, and either 0.2 or 2 mM MgCl2. Reactions were initiated by adding UDG and APE1 (1 nM each, New England Biolabs) and incubated at 37 °C for the indicated times. For mononucleosomes, reactions were supplemented with 110 nM (11 equivalents) of undamaged mononucleosomes such that the ratio of damaged to undamaged mononucleosomes, as well as total number of 601 units, was identical to the 12 × 601 arrays. Aliquots were removed at different times and the reaction stopped with the addition of 1% SDS (v/v), NaBH4 (10 mM), and 5 units proteinase K. The mixture was then extracted with phenol:chloroform:isoamyl alcohol (25:24:1 v/v, ThermoFisher Scientific) and the DNA desalted by ethanol precipitation. The resulting DNA was digested with 5 units of Nb.BbvCI in order to excise the dU containing 5′-[32P]-labeled oligonucleotide insert from the large 12 × 601 DNA array. The digested DNA was then resolved by 10% denaturing PAGE (19:1 acrylamide:bisacrylamide) and visualized using a Typhoon FLA 9500 gel imager (GE Healthcare) (Fig. S6). The fraction cleaved was quantified using ImageQuant TL software (GE Healthcare, v8.1). At least three replicates were carried out for each substrate, and initial rates were calculated by fitting the digestion data in to a single phase exponential and multiplying each rate constant by the Ymax as previously described12,63.
Analysis of chromatin compaction by FRET
We prepared a fluorescently labeled 12 × 601 DNA template by installing Cy3 (Donor) and Cy5 (Acceptor) dyes within N7 and N5, respectively, as described in our previous report19. This DNA (12-601-FRET) was reconstituted using either WT histone octamers or octamers containing acetylated histone H3 (Fig. S7). FRET measurements on the resulting chromatin were carried out as previously described68. Briefly, nucleosome arrays (10 nM) were equilibrated in LS buffer in the presence (2 mM) or absence of MgCl2 at 37 °C for 5 minutes. A portion of each solution (20 μL) was then transferred to a Nunc 384-Well Optical Bottom Plate (Thermofisher) for imaging. Plates were siliconized with Sigmacote (Sigma Aldrich) according to the manufacturer’s instructions prior to use. Samples were imaged with a Typhoon FLA 9500 multimode imager (GE Healthcare Lifesciences) and all FRET intensities were corrected for spectral overlap as described previously68,78.
Restriction enzyme accessibility assay
The indicated nucleosome arrays (5 nM) were digested with either BstXI (0.1 U/μL) or BbvCI (0.2 U/μL) in LS buffer supplemented with 2 mM MgCl2 at 37 °C. Aliquots were taken at the indicated times and quenched with three volume equivalence of LS buffer supplemented with 0.1% SDS, 2 u/μL Proteinase K, and 8% glycerol and then were heated at 60 °C for 10 minutes. Digested DNA was resolved by 1% agarose gel electrophoresis and visualized/quantified as described above (Fig. S8).
All statistical analysis was conducted using GraphPad Prism (v7.03). Corrected FRET intensities were compared by an unpaired one-way analysis of variance (ANOVA). Rates and yields of UDG/APE1, BstXI, and BbvCI cleavage reactions at each Mg2+ concentration (0.2 mM and 2 mM) were compared using an unpaired one-way ANOVA. Prior to the analysis, each data set was separately screened for outliers using a Grubbs test (α = 0.01). Within individual Mg2+ concentrations, rates and product yields on mononucleosomes and arrays were then compared to free DNA using a Dunnett’s multiple comparisons test. Acetylated and non-acetylated substrates were compared similarly, as were arrays and corresponding mononucleosomes. P-values less than 0.05 were considered significant.
The data generated or analyzed during the current study are available from the corresponding author on reasonable request.
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The authors are grateful to Dr. Wenshe Liu for providing the pEVOL-mmAcKRS-tRNAUAG plasmid required for incorporation of acetyllysine into histone H3. J.T.S. is a CPRIT Scholar of Cancer Research supported by the Cancer Prevention and Research Institute of Texas (RR150038).
The authors declare no competing interests.
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Banerjee, D.R., Deckard, C.E., Zeng, Y. et al. Acetylation of the histone H3 tail domain regulates base excision repair on higher-order chromatin structures. Sci Rep 9, 15972 (2019). https://doi.org/10.1038/s41598-019-52340-0
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Thanks for visiting Alliance Electrical Services
As of April 1st 2021, Alliance merged with our sister company, McGill.
With our expertise in renewable technologies and excellent local reputation, and McGill’s vast experience and Scotland-wide coverage, together we have formed a strong and ambitious contractor based in the North-East of Scotland.
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text:
If you have diabetes, it’s important to understand your increased risk of stroke. Multiple studies have shown that people with diabetes are at greater risk for stroke compared to people without diabetes -- regardless of the number of health risk factors they have. Overall, the health risk of cardiovascular disease (including stroke) is two-and-a-half times higher in men and women with diabetes compared to people without diabetes.
What Is a Stroke?
Brain tissue needs a constant supply of oxygen and nutrients to keep nerve cells and other parts of the tissue alive and functioning. The brain relies on a network of blood vessels to provide it with blood that is rich in oxygen.
A stroke occurs when one of these blood vessels becomes damaged or blocked, preventing blood from reaching an area of the brain. When that part of the brain is cut off from its supply of oxygen for more than three to four minutes, it begins to die.
There are two types of strokes:
- Those that are caused by a rupture in an artery, also known as
hemorrhagic stroke, and
- Strokes that are caused by blockage of an artery, also known as
Risk Factors of Stroke
High blood pressure is the number one risk factor for stroke. Other risks include smoking cigarettes and high levels of LDL (“bad”) cholesterol.
When people with diabetes have a stroke, they often fare worse than individuals without diabetes. When the oxygen supply is cut off, other arteries can usually deliver oxygen by bypassing the blockage. In people with diabetes, however, many of the bypass arteries are also affected by atherosclerosis, impairing blood flow to the brain. Other risk factors may be responsible for poorer outcomes in people with diabetes following stroke, as well.
While overall death rates from strokes have decreased, it is unclear whether death rates from strokes have decreased in people with diabetes.
What Are the Symptoms of Stroke?
A stroke is an emergency whether you have diabetes or not. If you experience any of these symptoms, call for ambulance immediately. You must get to the hospital as soon as possible:
Sudden numbness or weakness in the face, arm, or leg
(especially on one side of the body)
Difficulty speaking or understanding words or simple sentences
Sudden blurred vision or decreased vision in one or both eyes
Sudden difficulty swallowing
Dizziness, loss of balance or becoming uncoordinated
Brief loss of consciousness
Sudden inability to move part of the body (paralysis)
Sudden, unexplainable, and intense headache
What Is the Treatment for Stroke?
One FDA-approved treatment for ischemic stroke is a clot buster drug called tPA. This drug must be given within the first three hours after stroke symptoms begin for it to work. This drug dissolves the clot that has clogged an artery and can reestablish blood flow to brain tissue. This drug is not appropriate for all ischemic stroke patients, especially for those with a history of major surgery in the last two weeks or recent head trauma.
Also, there are several new and experimental drugs that may stop -- and even reverse -- brain damage if administered immediately after a stroke.
Options for in-patient stroke treatment include carotid endarterectomy, or surgical removal of the plaque from inside the carotid artery (the artery that supplies much of the blood to the brain).
A less invasive treatment is a carotid angioplasty and stenting procedure, which may be appropriate for some patients who have blockages within the carotid arteries. This involves inserting a deflated balloon into the artery in order to expand the artery walls and then inserting a mesh structure (stent) to hold the artery open.
Angioplasty of the cerebral arteries can also be performed.
There are other ways to mechanically remove a blood clot in the brain. The FDA has approved the Merci Retrieval System and the Penumbra System for selected stroke victims. These devices can remove the blood clot after the stroke; however, improvement in stroke outcomes are uncertain.
How Can Stroke Be Prevented in Diabetes?
If you have diabetes and your doctor suspects that you have atherosclerosis (hardening of the arteries), he or she may suggest changes in diet and lifestyle, as well as certain medicines that may help to prevent the blockages that cause stroke. Other ways to reduce your risk of stroke include:
Keep your blood sugar levels controlled.
Maintain a healthy weight.
Have your cholesterol checked (especially your “bad” LDL-cholesterol) and if necessary, lower your levels by limiting the amount of fat and cholesterol you eat.
Limit the amount of alcohol you drink. Guidelines are one drink per day for women and two drinks a day for men.
Have your blood pressure checked and control your blood pressure, if necessary.
Follow your health care provider’s instructions for changing your diet.
Follow your health care provider’s instructions for taking preventive medicines.
Take daily aspirin therapy as prescribed by your doctor.
Low doses of aspirin (81- 325 mg of aspirin a day) are recommended for men and women with diabetes who are over age 30 and are at high risk for heart disease, peripheral vascular disease, and stroke. Talk to your doctor to determine if aspirin therapy is right for you. If you have certain medical conditions, aspirin therapy may not be recommended.

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Good Weather, Affordable Living & Studying Abroad
The City of Light: Lisbon is the capital of Portugal, known for its vibrant nightlife, formative history and amazing food. More importantly than this is that Lisbon is known for its inexpensive lifestyle, which comes in handy if you want to study here on a budget.
Lisbon offers a range of different options within this topic. Before start of the semester and depending on the availability of (your) accommodation, there is the possibility to either stay in a Youth Hostel, where prices for shared rooms range between 15 – 18 EUR/night (Camacho, Rocky , Flakus, Lep, Cavadas & Silva, n.d.) or AirBnB also offers a wide variety of rooms, or even private apartments, which can be offered for more or less than 500 EUR/month (for 4 people).
In regard to wanting to find something more permanent, there are other options available. When wanting to live near campus, the University often offers student residency halls or dormitories to stay in during your exchange. Next to this being the most sociable option since it is easy to find (international) friends extremely quickly, costs for such rooms often lay within the 400 EUR/month range. Such residency halls do set aside spots for international students, but they are on a first come, first served basis.
Surely, it is also possible to rent a private room or apartment. Again, there is no clear indication of price, since it depends on the budget and standards you set, but it should be doable to find a place within 450 EUR/month. An extra tip would be to find friends and students, wanting to share an apartment and split the rent. The positive aspects is that you are fully free in deciding where to live within the city (Educations.com, n.d.)
Above: Historical sites in Lisbon
Above: Picturesque streets in Lisbon
Food & Entertainment in Lisbon
As mentioned above, Lisbon is known for its outgoing student life and good-quality food. Surely, everybody knows that saving money in regard to food might be the easiest through buying weekly groceries in low-cost stores such as LIDL, and meal-prep for university days or trips. BUT, in such a city as Lisbon, eating out is very tempting. Pratos de dia, meaning “Special of the day” are daily and fair offers, which mostly all restaurants offer. So, when going out for lunch or dinner, always make sure to check the daily offers first, since they are way more affordable than the regular menu (Zara, 2017)
Moreover, it depends on preferences, but it is up to you to explore the city and find the best restaurants. One tip that can be given is: Stay away from main streets, since the restaurants there are the most popular and can, therefore, increase their prices just because of the optimal locations. But as an adventurous student, you will quickly find out that this is only popular amongst tourists.
Next to gastronomy, Lisbon has a very popular party street, called “Pink Street”. Within this street, a wide variety of clubs and bars open their doors each night and welcome a dozen of visitors. Even though it should be on your bucket list, the quarter Bairro Alto is way more attractive to students. Many bars (not clubs necessarily) offer free entry, there are long happy hours, and drinks only cost a few EUR.
Transportation around Lisbon
Luckily, Lisbon is a very walkable city. Most important quarters and places are reachable by foot. Especially, for students who travel on a low budget, this option is the most affordable AND adventurous, since you will be exploring parts of the city which you otherwise might not see. Biking would also be an option, but this is more or less hard, since Lisbon has many city hills, which might be hard to tackle by bike.
If walking is an absolute no-go for you, public transport is very affordable as well. There is this certain pass, called the “ Navegante Metropolitana”, which costs 40 EUR a month and lets you reach the metropolitan areas within the city (Buy, 2020) with public transport.
These tips and tricks should help you with your low-budget travelling plans and make the Erasmus one to never forget. Obrigada y até à vista!
Zara. (2017, May 07). Best cheap eats in Lisbon. Retrieved February 28, 2021, from https://bkpk.me/best-cheap-eats-in-lisbon/
Educations.com. (n.d.). Student accommodation & cost of living in Portugal. Retrieved February 28, 2021, from https://www.educations.com/study-guides/europe/study-in-portugal/living-costs-7821
Camacho, V., Rocky, A., Flakus, I., Lep, I., Cavadas, A., & Silva, A. (n.d.). Erasmus Portugal. Retrieved February 28, 2021, from https://erasmusu.com/en/erasmus-lisbon/erasmus-experiences/lisbon-how-to-survive-and-have-the-time-of-your-life-79787

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I-5 exit to West Seattle Bridge closed Friday through weekend | Regional news
The northbound I-5 exit to the West Seattle Bridge will close at 7 p.m. Friday, Aug. 23. The ramp will reopen by 5 a.m. Monday, Aug. 26. This round-the-clock closure is the first of four weekend-long closures on the South Spokane Street/Interstate 5 interchange.
During the four weekend closures in August and September, contractor crews working for WSDOT will replace six 50-year-old expansion joints on the northbound I-5 ramps to upper and lower South Spokane Street. They also will use jets of pressurized water to blast off the rutted roadway surface of the ramps before repaving them with new concrete.
This work is part of the I-5 Seattle Bridge Repair Project, which is scheduled to finish up in fall 2013.
Closure and detour information
- Thursday, Aug. 22 through morning of Friday, Aug. 23 - The northbound I-5 ramp to upper South Spokane Street/West Seattle Bridge will close from 10 p.m. Thursday through 5 a.m. Friday to prepare the roadway, and mobilize equipment for the full weekend closure.
- Friday, Aug. 23 through morning of Monday, Aug. 26 – The northbound I-5 ramp to upper South Spokane Street/West Seattle Bridge will close from 7 p.m. Friday through 5 a.m. Monday.
- Drivers will be detoured to the lower South Spokane Street/Sixth Avenue South off-ramp to First Avenue South to the West Seattle Bridge on-ramp. View the detour map.
There are two sold out Mariners games and a Sounders game this weekend. Drivers can stay on time by adding an extra ten or fifteen minutes to their trip and checking the Seattle traffic page for backups. The What’s Happening Now page,Twitter feed and mobile app also are reliable traffic-busting tools.

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That tagline up on the header right now pretty much says it all. If it’s not walkable, it’s MUNI for me and sometimes this proves an aggravating experience. Fully caffeinated on a Saturday morning I wander around taking pictures and then settle in at Mariposa and Bryant to get my $45 worth.
Elapsed wait time: 50 minutes
No. of busses that pass by going the opposite direction: 4
No. of other potential passengers who give up: 1, a MUNI driver clearly about to start her shift, who had even less confidence in the service than I did.
Possibility that San Francisco public transportation might prove so enticing that citizens will give up their cars: 0%
When the bus arrives it’s packed to the gills. Armed with the knowledge that the elusive 27 likes to hunt in packs, I wait five more minutes and climb aboard a car with plenty of breathing room.
Waves of shoppers take up every available inch of sidewalk, mistaking an afternoon inferno for “nice weather.” It’s always a relief to give the Market St. crowds the slip by turning the corner onto a deserted street where 49 Geary waits in the relative quiet. It feels like you’re privy to some undisclosed location ignored by maps and directories. I give the secret hand signal to the security guard at the desk and head up to Toomey Tourell. This is what makes it all worth it.
God damn it.
Too stubborn to give in, I grab some carpet and pout. Now I have to pee, which means begging a gallery attendant or owner for a key. Screw it. I go down the hall to see if there’s anything new at Steven Wolf.
One end of a huge painted landscape of a mountain vista is peeking out of the office, looking like a ship that’s just pulled into port. On the walls are a series of collages by brothers Kent and Kevin Young. The news article clippings give the feeling of free association on the subject of twins. Extracted bit by bit from the surrounding context of whatever was deemed timely and notable that day, they come across as the plunder of an obsessive’s scrapbook. “Is it just us,” ask the conspiracy theorists, “Or do twins hold a peculiar fascination for people?”
In the side gallery a Milton Bradley game is set up at a table. According to the website, this was to provide Bay Area twins the opportunity to try out their ESP aptitude by quizzing one another during the opening reception.
Kreskin: the original Dungeon Master.
Nearby four monitors tick off the results of a million die rolls, perhaps highlighting the craps game of genetics that every so often comes up “twins.”
Next time: Toomey better be open. Still need to use restroom.


text:
Tyranny starts of as a game of impossible choices. You are the Fatebinder, a man serving the Overlord’s court and handing out sentences to maintain his peace. This grim position constantly asks you to consider; do you want to be the oppressor who enslaves or the oppressor who assimilates?
A unique idea to say the least, Tyranny sets out to offer a different experience from that of most RPGs. Obsidian happily ignores the usual good vs evil spectrum and instead replaces it with more nuanced meters such as Wrath vs Favor and Fear vs Loyalty. And though the game pulls no punches when it comes to placing you in difficult situations; it fails to deliver a gameplay experience that compliments the narrative.
While the combat feels superior and smoother to that of Pillars of Eternity, another Obsidian isometric RPG title, it fails to enhance the immersion of the story. Consider this, one of the recurring actions that you land up doing, is seizing heavily guarded forts. Either you do this for personal gain or to further the Overlord’s goals. However, as the game is an isometric RPG, it basically asks that you and your three other companions, take a fort by yourself. Realistically, this feels…unrealistic. Not because you’re not powerful enough, but because in the interest of being balanced, the game pits you against 3-4 guards every encounter as you take this so called ‘heavily guarded’ fort. This does not seem believable and at some point the immersion just breaks.
This experience is worsened by the lack of enemy variety. 13-15 hours in, all my enemies felt as generic as the ones in a Call of Duty game. They were so tame that I eventually found myself looking away as the combat played out. Perhaps a higher difficulty would have engaged me further but unless it would have brought a wider range of enemies, perhaps ones that justified why an army couldn’t take a fort that I and my three companions could, I might have cared more.
Tyranny is not a bad game. Obsidian builds an excellent and unique world, the characters it populates it with are just as interesting and complex. But one can’t help but feel that developers fall short of the bar set by the writers.

domain: game


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Paris is the capital and most populous city of France, with an estimated population of 2,175,601 residents as of 2018, in an area of more than 105 square kilometres (41 square miles).
Paris is a major railway, highway, and air-transport hub served by two international airports: Paris–Charles de Gaulle (the second-busiest airport in Europe) and Paris–Orly. Opened in 1900, the city’s subway system, the Paris Métro, serves 5.23 million passengers daily; it is the second-busiest metro system in Europe after the Moscow Metro. Gare du Nord is the 24th-busiest railway station in the world, but the busiest located outside Japan, with 262 million passengers in 2015.
The Musée d’Orsay, Musée Marmottan Monet and Musée de l’Orangerie are noted for their collections of French Impressionist art. The Pompidou Centre Musée National d’Art Moderne has the largest collection of modern and contemporary art in Europe.


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There's something magical about watching a tree grow tall and strong, standing the test of time and weathering the elements. Among the majestic trees that grace our forests, the acacia tree stands out for its unparalleled beauty, durability, and versatility. But it's not just its aesthetic appeal that makes acacia wood so special; it's the journey from being a tree to becoming stunning tableware that truly captures our hearts.
**A Tale of Origins: The Acacia Tree**
Let's start at the very beginning of this enchanting journey - the acacia tree's origins. With over 1,300 different species spread across the globe, acacia trees boast an impressive diversity. From the African savannahs to the Australian outback, these trees have adapted to thrive in various environments, leaving us with a range of exquisite acacia wood varieties to choose from.
One of the most remarkable aspects of acacia trees is their rapid growth. They can reach towering heights in just a few years, making them a sustainable and eco-friendly choice for wood production. Since we can't help but care for the planet we call home, opting for products made from acacia wood is a small yet meaningful step towards preserving the environment.
**The Art of Craftsmanship: Transforming Acacia into Tableware**
Once an acacia tree reaches maturity and it's time for it to be harvested, skilled artisans embark on the transformative process of turning the wood into elegant tableware. Each piece is crafted with meticulous attention to detail, honoring the natural beauty of the wood and bringing out its unique patterns and colors.
The craftsmanship involved in creating acacia tableware is a true art form. Whether it's a sleek and modern dining set or a rustic farmhouse-style table, the distinctive grain patterns and rich hues of acacia wood add a touch of sophistication to any table setting. But it's not just about appearances; the durability and sturdiness of acacia wood mean that these pieces will stand the test of time, becoming cherished family heirlooms that can be passed down through generations.
**The Beauty of Imperfection: Embracing Nature's Flaws**
One of the most endearing qualities of acacia wood is its imperfections. Nature has a way of leaving its mark on this magnificent material, with knots, cracks, and unique variations in color that tell the story of the tree's journey through the years. Far from being flaws, these characteristics add character and depth to each piece, making it one of a kind.
Acacia wood's ability to embrace imperfections teaches us an essential lesson: true beauty lies in embracing our individuality and celebrating our unique qualities. Just as every piece of acacia tableware boasts its own distinct charm, each of us has something special that sets us apart.
In an era where sustainability and responsible consumer choices are paramount, acacia wood shines as an environmentally friendly option. Harvesting acacia trees for wood not only allows new trees to grow in their place but also supports the ecosystems they belong to.
Additionally, acacia wood is known for its remarkable resistance to decay and pests, reducing the need for chemical treatments and extending the lifespan of the products made from it. Choosing acacia tableware means investing in a sustainable and durable option that helps preserve our planet for future generations.
**Enhancing Your Dining Experience: The Magic of Acacia Tableware**
Bringing acacia tableware into your home is more than just a purchase; it's an experience. As you set the table with these stunning pieces, you'll feel a connection to nature and an appreciation for the craftsmanship that went into creating them.
Imagine the joy of gathering around a beautifully set table with your loved ones, sharing meals and creating memories together. Acacia wood's natural warmth and elegance create an inviting atmosphere, making every dining experience an unforgettable one.
Whether you're hosting a dinner party or enjoying a casual meal, acacia tableware effortlessly elevates the occasion. Its versatility allows it to blend seamlessly with various styles and settings, from casual and laid-back to formal and sophisticated.
**In Conclusion: Embrace the Beauty and Durability of Acacia Wood**
The journey from tree to table is a captivating one, where the acacia tree's beauty, durability, and sustainable qualities converge to create exquisite tableware. Choosing acacia wood for your dining experience is not only an aesthetically pleasing decision, but it's also an environmentally responsible one.
As you dine with loved ones around your acacia tableware, you'll feel a sense of harmony with nature and an appreciation for the craftsmanship that went into its creation. So, let's celebrate the imperfections that make each piece unique and cherish the journey of acacia wood, from its origins as a tree to its transformation into stunning tableware that enriches our lives and preserves our planet.

domain: artistic


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Construction projects in Austin, Texas that are larger than 5,000 sf are now required to recycle or salvage at least half of their refuse.
The new ordinance is expected to divert at least 76,000 tons of material from landfills. Another ordinance will mandate that demolition sites recycle beginning in 2019. About 1,500 projects that would now fall under the mandate had generated about 152,000 tons of concrete, metal, lumber, and other waste in 2014-2015.
The city estimates the ordinance will increase construction costs by 0.1% to 0.2%. Companies working on large projects will have to submit reports on the quantity of materials they recycle or salvage. Violators could be charged with a misdemeanor for noncompliance and pay a $500 fine.
City lawmakers developed the ordinance to forward Austin’s goal of diverting 95% of all waste from landfills by 2040. About 20% of the city’s waste is from construction and demolition materials.


GROSSE ILE, MI (October 11, 2016) -- Dearborn’s Evan Bowser hit a pitching wedge shot to four feet and made a birdie-3 on the second hole of a sudden-death playoff with Flat Rock’s David Vaclav to win the Michigan Tournament of Champions presented by Global Golf Post Monday at Grosse Ile Golf & Country Club.
Bowser, 21 and a recent graduate of Oakland University, and Vaclav, a Grosse Ile G&CC member, each shot 72 in regulation play.
“I hit a knock down shot, got a little hop left and it was in there close,” Bowser said. “It feels good to win, especially after I three-putted 18 for bogey after hitting a great shot in there.”
On No. 18 Bowser hit a 3-iron on the 230-yard par 3 to 12 feet, but rushed the first putt past five feet and missed again.
“I thought that might have been it right there, but I got another chance in the playoff,” he said. “I played pretty solid golf all day. It wasn’t playing easy. It was windy, cold and the greens were fast.”
Peter vanWezel of Lasalle and Nathan Clark of Charlotte each shot 73s to finish one shot out of the playoff.
Tom Gieselman of Commerce Township shot 74, and Ken Hudson of Bloomfield Hills and Joe Hooks of Commerce Township each shot 75.
Bowser plans to travel to Arizona to work on his golf game and attempt Web.com Tour qualifying as an amateur.
“I don’t want to turn professional until I have status,” said Bowser who earned his degree in finance at Oakland U. “I want to make it, but it’s not a life or death thing for me.”
Bowser won the GAM’s final tournament of the golf season to cap a year in which he won the Horton Smith Invitational, the Michigan Publinx Golf Association title and was part of the national championship GAM team representing Michigan in the USGA State Team Championship just over a week ago.
“Started the season great and ended it great,” he said.
View results for Golf Association of Michigan Tournament of Champions Golf Tournament


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Thrust into the limelight after winning the LVMH Prize, Dojaka has been picked by N°21’s creative director to design a capsule collection alongside Alfredo Cortese of AC9, a chance to showcase her style and personality.
Mr Porter’s on the search for new global design talent. The online menswear retailer has launched Mr Porter Futures, a “first-of-its-kind global and comprehensive talent search” for three aspiring menswear designers.
Alek Wek has developed a colorful new capsule collection with Weekend Max Mara. The Sudanese-born model worked with Max Mara’s design team to create a blend of her African roots, cosmopolitan lifestyle and artistic eye.
With the pandemic continuing to put the brakes on the fashion industry, a number of retailers are taking the time to re-evaluate their business models in response to consumer demand for more responsible products.


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Taylor gives a new look to basic black and white with a hint of sparkle to dress up for either day or night.
Taylor is a mix of silver, black, white, and silver glitter. Created July of 2020, Taylor went through a few changes over the months- first embellished with some extra sequins, I opted to go for a more minimal look.
I left the signature gold for a darker, nighttime feel with the Taylor- it felt like we were slowly moving downtown from the Upper West Side as I painted her.
Jonathan and Madison came after Taylor, introducing a new collection and brand new feeling- one I embraced after going back to New York. Letting go of old insecurity and fear was a brand-new feeling as I walked through Central Park, visited my old building, and caught up with old friends.
The Ugly Ducking statue felt symbolic, as it made me think about the feelings of self doubt I had that couldn't be covered up with gold paint and a red lipstick smile. Sometimes, you have to dig deep and face the thinks that scare you to come back out into the light.


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'Meredith dies tonight': Chilling text message predicted student's sex murder
Last updated at 23:27 10 November 2007
In a bizarre twist to the investigation into Miss Kercher's death, a chilling text message predicting her murder has surfaced.
According to reports in Italian newspapers, a man contacted police after receiving a message on his mobile from an unknown number.
It read: "For me, tomorrow or tonight Meredith dies."
The text flashed up on the man's mobile on October 30, two days before the student's semi-naked body was found in her bedroom in Perugia.
At the time, the man assumed the message was sent by mistake and he deleted it.
But earlier this week he heard reports of the murder, which has left Italy stunned, and contacted police in his hometown of Rome.
They are now trawling through vast quantities of phone data to retrieve the message and check if it was sent by one of the three suspects.
Phone records have already proved vital in the police investigation and it is known that in the hours after the killing there was heavy text and call traffic between Amanda Knox and boyfriend Raffaele Sollecito.
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Police say phone calls made by the pair expose their alibi as a lie.
Despite the couple's claims to have been elsewhere at the time of the killing, detectives say they used their phones in the vicinity of the murder scene around the time that Miss Kercher died.
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Best Beer On A Keto Diet
What Is The Keto Diet Regimen?
The keto diet is an extremely low-carb, higher-fat diet plan. It’s comparable in lots of means to other low-carb diet plans.Best Beer On A Keto Diet
While you eat far less carbohydrates on a keto diet, you preserve moderate healthy protein intake as well as might increase your consumption of fat. The reduction in carbohydrate intake places your body in a metabolic state called ketosis, where fat, from your diet regimen as well as from your body, is melted for power.
What Is Ketosis– The Scientific Research Behind Operating?
Ketosis is a metabolic state that includes the production of ketone bodies with the body fat as well as their in-turn consumption to generate energy.
The body typically uses carbohydrates (sugar or blood glucose) as the main resource of energy. Yet, when the carb consumption in the body is low (less than 50 grams a day), then the body becomes part of a phenomenon known as ketosis.
Burning fat is the body’s natural alternative for generating power when it does not get a sufficient quantity of glucose.
While the body is low on carbohydrates, the insulin degree in the body drops. A a great deal of fats kept right into the fats are launched right into the body, ultimately eating the fats. The fats are transferred to the liver, where the oxidation reactions transform the acids right into ketones (ketone bodies). These ketone bodies produce energy for the body.
Though the liver produces ketones consistently, ketosis customizes the price and quantity relying on the carbohydrate consumption. In this fashion, ketosis assists you to melt the stored fat to produce energy in the body– putting the unused fat books to valuable usage.
It deserves keeping in mind that the ketosis sensation has two advantages. Besides aiding you lose fat, it additionally offers you an increase of energy due to the fact that shedding the exact same amount of fat produces even more energy than shedding the very same quantity of carbohydrates.
When it comes to the best keto foods, you want to eat real low-carb foods and stick to foods with fewer than 5% carbs. Here are some examples.Best Beer On A Keto Diet
These are the foods that boost ketosis in the body by raising the fat focus against carbs.
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1.Baked products as well as bread.
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The Customized Keto Diet regimen is a program by Rachel Roberts that assists consumers to find the most effective consuming prepare for their objectives and their food choices. The program facilities around scrumptious food yet does not cause the participant to feel as though they are depriving themselves or needing to surrender a number of their preferred foods. Whether users are complying with a VLCKD [extremely low carbohydrate ketogenic diet] or a LFD [low-fat diet regimen], the designer of the Custom Keto Diet regimen details the seven reasons the Ketogenic Diet is the holy grail for weight loss.Best Beer On A Keto Diet
One of the hardest parts of sticking with any type of diet is the reality that there is way too much that individuals need to give up when they follow it. Deprival of these kinds of food can be helpful momentarily as the body begins to work from stored fat. Nevertheless, some people find self-discipline a challenge to uphold, leaving them vulnerable to giving up completely as well as returning to their days of poor nutrition.
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What Are The Pro Points Of The Custom Keto Diet Regimen Plan?
- High fat, reduced carbs:
The keto diet’s objective is to cut down upon the variety of carbs and also make your body enter the state of ketosis, where it begins to shed ketone bodies rather than carbohydrates.
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While you are on a keto diet regimen, on a route of slimming down, your body maintains muscle mass.Best Beer On A Keto Diet
The body burns fat to decrease weight and the overall metabolic weight stays the exact same due to the fact that you obtain muscles. That is an additional perk of being on a keto diet regimen. It aids you acquire the ideal form that you desire.
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Unlike fats, the ketone bodies produced with ketosis can quickly go across the brain-blood barrier. These ketone bodies give a good deal of energy to your brain. Thus, you might say that keto diet regimens assist you stand against anxiety.
Because of the good deal of energy that it supplies to your body, you experience a far better state of mind throughout the day.
The keto diet additionally assists you stand versus uneasyness and also exhaustion because of severe job patterns in daily life. It works with your sleep patterns and also thus, obtains you in total great form.
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domain: health


Publication of the CRES Discovery
April 20, 2015
The paper announcing the first detection of single-electron cyclotron radiation and the first application of the CRES technique has now been published in Physical Review Letters: Phys. Rev. Lett. 114, 1162501 (2015). The article was highlighted as an Editors' Suggestion. You can also read the Viewpoint commentary on the article written by Patrick Huber.
First Observation of Single Electron Cyclotron Radiation
August 25, 2014
The Project 8 collaboration is proud to announce the first observation of relativistic cyclotron radiation from single electrons! We detect individual electrons from a gasseous 83mKr source. Since the frequency of the relativistic cyclotron radiation is inversely proportional to the kinetic energy of the electrons, we use this technique to make a spectroscopic measurement of the 83mKr internal conversion electron lines. This new spectroscopic technique is called Cyclotron Radiation Emission Spectroscopy (CRES). Please see the preprint of our paper announcing these results for more details.
August 24, 2014
In anticipation of making public some very interesting results, we present this new website! Enjoy!


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Utility Fabric Cargo Netting
Creative possibilities for decorative projects are endless, when you use the Utility Fabric Cargo Netting 61''. Made of 100% polyester, this 61-inch cargo netting is sturdy, durable and easy to care for. Featuring fine mesh, it's ideal to make attractive and breathable totes for your picnics and beach outings. Adorn it with flowers, lace and stickers to enhance its visual appearance. Create decorative hats, holiday decor accents and baskets as well with this versatile cargo netting. You can also this utility fabric for various automotive uses like car seat coverings and back seat pockets. This utility fabric is sold by the yard.
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Overview of the Chilean summer fruit exports to the US, complemented by charts from Agronometrics. Original published on November 18, 2020.
“Retailers were eager to call out vitamin C and other health messages during our summer and fall citrus promotions,” said Karen Brux, managing director for North America of the Chilean Fresh Fruit Association. “As COVID-19 continues and we enter flu season, I expect they’ll continue to focus on health benefits.”
Brux said February is the key month for promotions as volumes will be at their peak. “In December we have small volumes of cherries, blueberries and grapes, while January brings larger volumes of blueberries. February is the key promotion month for blueberries, peaches and nectarines, as the cherry season comes to a close. We run some grape promotions – our biggest category – in February, but March and April are definitely bigger months for grapes.”
Josh Leichter, general manager at Los Angeles-based Pacific Trellis Fruit/Dulcinea Farms, said the season looks promising so far. “The good news is that Chile had a good amount of winter rain and snowpack in the mountains, so most growers aren’t facing the water issues they experienced this time last year. It’s been a good spring for fruit production and everyone’s optimistic about the size and quality of the crop.”
He said new varieties of grapes, and more recently stone fruit, combined with advances in post-harvesting handling and logistics, have added to the overall quality and consistency of the fruit.
With people cooking at home more and planning more meals in advance, Brux said now is the time to inspire with convenient, healthy and tasty ideas. “Our Fruits from Chile website offers recipes and usage ideas for everything from Blueberry Power Balls to Roasted Cauliflower with Chilean Grapes and Peach Pie Smoothie Bowls.”
Winter holidays and occasions such as Heart Health month in February offer even more promotion opportunities, Brux said. Fruits from Chile sends holiday or occasion-themed promotions to its more than 500,000 Facebook fans and 35,000 Instagram followers, and it distributes a consumer newsletter to more than 25,000 subscribers.
The association is also reviewing a number of e-commerce options to drive online sales of Chilean fruit.
Leichter said pairing Chilean fruit with wine and cheese remains his favorite cross-merchandising opportunity.
John Pandol, special projects director for Pandol Brothers Inc., Delano, Calif., notes that Christmas and New Year each fall on a Friday, which will encourage shoppers to stock up for long weekends at home. He predicts blueberry ads will feature larger clamshells than in the past.
“The second week of February we will be pushing both grapes and blueberries,” Pandol said, noting consumers buy fresh fruit for Valentine’s Day menus. Mardi Gras, even college bowl games, may also be celebrated in-house this winter.
An early Easter, which falls in the first week of April, should help also. “The earlier Easter falls, the stronger grape sales are,” Pandol said.
Pandol said one of the industry’s biggest challenges is how to adopt merchandising to the virtual world. “Grapes are an impulse item. People buy more when the grapes look good, and many taste a grape or two before buying. What happens when a customer does a search for grapes or blueberries and the same image comes up week after week, month after month? How will we introduce new varieties, especially those with unique flavor characteristics, without in-store sampling?
“Every produce manager needs to check the online version of his department the same way he walks the store and checks the displays,” Pandol said. “The online shopper who only sees a mediocre online catalog won’t buy, losing customers and sales.”
He said the training of online order fulfilment personnel, whether employees or third-party gig workers, is crucial.
While Leichter expected to see more demand for packaged fruit this season, he said customers who trialed additional packaging on certain items in the offseason found their shoppers still wanted to be able to see and touch the product. In addition, as misconceptions about COVID being spread by fruits and vegetables have been dispelled, he said the desire or need for additional packaging seems to have faded.
Here’s a closer look at some of the most popular winter fruits from Chile.
Brux expects peak cherry shipments to the US in weeks 51/52 a
nd said cherries provide great holiday promotion opportunities from November through January.
Bengard Marketing, Rancho Dominguez, Calif., starts its Chilean season in early November with air cherries. Sales manager Stephen Pearson anticipates the pandemic may cause delays and increase air costs due to lack of flights. Promotions for cherries arriving by vessel should peak in early to mid-January, Pearson said.
Chilean blueberries are available November through March, said Brux, with peak shipments to the US expected in weeks 2/3.
“The blueberry season is about a week behind last year’s season, but we anticipate the same volume, if not a little more,” said Brian Bocock, vice president of product management for Naturipe Farms, Salinas, Calif.
Naturipe has shifted its Chilean production toward organic. “Our acres of proprietary varieties are a bit firmer with more appealing flavor profiles than conventional,” Bocock said. “As they mature and produce more berries, these varieties will make up a higher percentage of our pack.”
The pint remains Naturipe’s most popular pack followed closely by the 18-ounce pack, he said. Blueberries also feature in the grower-shipper’s newest snack line, Bliss Bentos, which combine fresh blueberries or grapes with unique ingredients including chocolate-covered chickpeas for an indulgent hit of protein.
“Peaches and nectarines will have similar volumes as last season, and there will be an increase in plums (back to normal volumes), which were down last season,” Pearson said. He noted the best promotion time for peaches, plums and nectarines will be January through early March.
Brux expects nectarine and peach shipments to the U.S. to peak in weeks 4/5, with some early fruit flown in. Plums peak in weeks 7/8, the height of the Chilean stone fruit season.
“Some specialty varieties, such as lemon plums, present unique promotion opportunities as they’re only available for a few weeks,” she said.
Leichter said Chile’s newer stone fruit varieties, particularly nectarines from European breeding companies, focus on a better eating experience.
Chile’s grape season by vessel starts in mid-November, with overall volume to the U.S. expected to be about the same as last year. “Newer grape varieties will be coming in heavier volumes this season, as production plantings have increased over the past three or four years,” Pearson said.
Brux expects peak grape shipment to the US in weeks 12/13.
Leichter’s current favorites include green Sweet Globe, red Allison and black Iniagrape-One (a.k.a. Inia One), developed in Chile. “These new varieties bring excitement to the market not only for their quality and condition but also for their eating experience, which will ultimately lead to more sales and business for everyone.”
The News in Charts is a collection of stories from the industry complemented by charts from Agronometrics to help better tell their story.
Access the original article with this (Link)

domain: agronomy