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It has been established that regulation of chromatin structure through post-translational modification of histone proteins, primarily histone H3 phosphorylation and acetylation, is an important early step in the induction of synaptic plasticity and formation of long-term memory. In this study, we investigated the contribution of another histone modification, histone methylation, to memory formation in the adult hippocampus. We found that tri-methylation of histone H3 at lysine 4 (H3K4), an active mark for transcription, is upregulated in hippocampus one hour following contextual fear conditioning. In addition, we found that di-methylation of histone H3 at lysine 9 (H3K9), a molecular mark associated with transcriptional silencing, is increased one hour after fear conditioning and decreased twenty-four hours after context exposure alone and contextual fear conditioning. Tri-methylated H3K4 levels returned to baseline levels at twenty-four hours. We also found that mice deficient in the H3K4-specific histone methyltransferase, Mll, displayed deficits in contextual fear conditioning relative to wildtype animals. This suggests that histone methylation is required for proper long-term consolidation of contextual fear memories. Interestingly, inhibition of histone deacetylases (HDACs) with sodium butyrate (NaB) resulted in increased H3K4 tri-methylation and decreased H3K9 di-methylation in hippocampus following contextual fear conditioning. Correspondingly, we found that fear learning triggered increases in H3K4 tri-methylation at specific gene promoter regions (Zif268 and bdnf) with altered DNA methylation and MeCP2 DNA binding. Zif268 DNA methylation levels returned to baseline at twenty-four hours. Together, these data demonstrate that histone methylation is actively regulated in the hippocampus and facilitates long-term memory formation.
Experience-dependent transcriptional regulation is critical for the stabilization of long-term memory formation. While the role of transcription factors in the regulation of gene expression in memory have been the focus of intense research, an increasing amount of recent data have identified a role for chromatin remodeling in this process. Specifically, post-translational modifications of histones has been shown to be a mechanism for transcriptional regulation during long-term memory formation (Swank and Sweatt, 2001; Guan et al., 2002; Alarcon et al., 2004; Levenson et al., 2004a; 2005; Kumar et al., 2005; Levenson and Sweatt, 2005; Chwang et al., 2006; Wood et al., 2006; Bredy et al., 2007; Fischer et al., 2007; Lubin et al., 2008; Guan et al., 2009).
The regulation of chromatin structure is complex. This is partly due to the N-terminal tails of histones which are highly accessible for enzymatic transformation and are sites for multiple covalent post-translational modifications, including acetylation, phosphorylation, methylation, ubiquitination, and sumoylation (Peterson and Laniel, 2004; Berger, 2007). Histone modifications such as histone acetylation and phosphorylation facilitate the unraveling of DNA around the histone core resulting in the recruitment of the transcriptional machinery to mediate cell- and promoter-specific gene expression. However, other histone modifications such as histone methylation have different positive or negative effects on gene transcription depending on the amino acid residue modified and the number of methyl groups added (Berger, 2007).
The methylation of lysines can exist in three states: mono-, di-, and tri-methylated. Generally, transcriptionally silent regions contain di- and tri-methylation of histone H3 at lysine9 (H3K9) whereas active genes correlate with di- and tri-methylation of histone H3 at lysine4 (H3K4) (Sims et al., 2003; Margueron et al., 2005; Martin and Zhang, 2005; Vermeulen et al., 2007). Specifically, di-methylation of H3K9 correlates with transcriptional silencing, and tri-methylation of H3K4 is linked to active transcription (Santos-Rosa et al., 2002; Ng et al., 2003; Schneider et al., 2004). Recently histone methylation has been implicated in the regulation of chromatin structure in the nervous system (Tsankova et al., 2006; Huang and Akbarian, 2007; Huang et al., 2007; Kim et al., 2007). However, to date there have been no reports of histone methylation regulation in the nervous system functioning in the process of long-term memory storage.
Thus in the present study, we sought to investigate histone H3 methylation as an experience-driven mechanism of transcriptional regulation in hippocampus during consolidation of fear conditioned memories. We found that contextual fear conditioning induced both tri-methylation of H3K4 and di-methylation of H3K9 in hippocampus, suggesting active increases in gene expression and gene repression during memory formation. Interestingly, treatment of animals with the histone deacetylase (HDAC) inhibitor sodium butyrate (NaB) elevated tri-methylation of H3K4 and decreased di-methylation of H3K9. This suggests that histone acetylation is intricately linked to histone methylation in hippocampus during consolidation of fear conditioned memories. We also found that heterozygous deletion of a known regulator of histone methylation, Mll, led to significant deficits in memory consolidation. Finally, we found that fear conditioning triggered increases in tri-methylation of H3K4 at the Zif268 gene which were associated with altered DNA methylation at this gene promoter. Taken together these observations implicate experience-driven alteration of histone methylation in hippocampus as a mechanism contributing to long-term memory formation.
Young adult, male Sprague-Dawley rats (250–300 g) were used for all experiments with the exception of the Mll;eed mutant mice which were created as previously described by Kim et al. (2007). Briefly, the eed null allele employed in this study contained a N-ethyl-N-nitrosourea-induced L290P substitution in the second WD motif (Schumacher et al., 1996) and was maintained on a mixed genetic background largely derived from 101/R1 and C3Hf/R1. The mutant Mll allele employed in this study harbors a lacZ insertion in the third exon, resulting in a truncated protein (Yu et al., 1995). The mutant Mll allele was maintained on a mixed background with contributions from C57BL/6J, C3H/HeJ, and FVB/NJ. Experiments encompassed single and double heterozygous animals derived from intercrosses of eed and Mll single mutant mice. Homozygosity for the rd mutation was excluded by PCR. Animals were housed under light/dark 12 h/12 h and allowed access to rodent chow and water ad libitum. Five days prior to experiments, animals were handled for 3–5 minutes each and allowed to acclimate to laboratory condition. All procedures were performed with the approval of the University of Alabama at Birmingham Institutional Animal Care and Use Committee and according to national guidelines and policies.
Rats were transported to the laboratory at least 30 min prior to fear conditioning. Fear conditioned animals were allowed to explore the chamber for 2 min, after which they received a series of three electric shocks (1 s, 0.5 mA) at 2 min intervals. As controls, context exposed or Latent Inhibition plus fear conditioned animals were also placed in the novel training chambers. Context exposed animals were placed in the novel training chamber for 7 min without receiving the footshock. Latent Inhibition plus fear conditioned animals were pre-exposed to the context for 2 h before the same 7 min training protocol was administered as described for the fear conditioned animals. In all shocked groups the animals were allowed to explore the novel context (training chamber) for an additional 1 min after the receiving the final footshock prior to being returned to their home cage. Freezing behavior was recoded using Video Freeze software (Med Associates, St. Albans, VT). Another group of age-matched animals that were handled by the experimenter but did not receive any experimental manipulations were used as naïve controls in all experiments.
Exploratory activity, motor function, and sensorimotor processing were performed as described (Paylor et al., 2006). Behavioral experiments of eed x Mll investigation employed 91 female mice at 8–12 weeks of age (n=29 eed+/+;Mll+/+, 26 eed+/−;Mll+/+, 18 eed+/+;Mll+/−, and 18 eed+/−;Mll+/−). In the contextual fear paradigm (McIlwain et al., 2001), mice were placed in a chamber and after 2 min the mice received two conditioned stimulus (CS; 80 dB auditory sound, 30 sec) – unconditioned stimulus (US; 0.7 mA mild foot shock, 2sec) pairings separated by 2 min. Twenty-four hours later, the mice were assayed for freezing behavior to determine associative learning of the footshock with the contextual cues, which constitutes the training environment, and the discretely paired auditory sound. On Test day, the amount of freezing during the 3 min pre-CS period was subtracted from the freezing during the 3 min CS presentation.
Animals were sacrificed using a rodent guillotine. The brain was immediately immersed in oxygenated (95%/5% O2/CO2) ice-cold cutting saline (in mM: 110 sucrose, 60 NaCl, 3 KCl, 1.25 NaH2PO4, 28 NaHCO3, 0.5 CaCl2, 7 MgCl2, 5 glucose, 0.6 ascorbate). The whole hippocampus was then removed and area CA1 was dissected away from other hippocampal subfields under a dissecting microscope. Once isolated, area CA1 was frozen on dry ice then stored at −80C until processed.
Histone extractions were performed as in Lubin et al. (2007). Briefly, all procedures were performed on ice, and all solutions were chilled to 4 °C prior to use unless otherwise indicated. All centrifugation steps were performed at 4 °C. Hippocampal tissue was dounce homogenized in ice-cold homoginization buffer (in mM: 250 sucrose, 50 Tris, pH 7.5, 25 KCl, 0.5 phenylmethylsulfonyl fluoride, 1% protease inhibitor mixture (Sigma), 0.9 Na+-butyrate) using no more than 6 strokes of a glass pestle (Kontes Glass Co, NJ). Hippocampal tissue homogenates were centrifuged at 7,700 × g for 1 min and the supernatant (cytoplasmic fraction) was aspirated. The pellet (nuclear fraction) was then resuspended in 0.5 ml of 0.4 N H2SO4, incubated for 30 min and centrifuged at 14,000 × g for 10 min. The supernatant was transferred to a fresh tube, and proteins were precipitated with the addition of 250 µl of 100% trichloroacetic acid containing 4 mg/ml deoxycholic acid (Na+ salt, Sigma) for 30 min. Acid extracted histone proteins were then collected by centrifugation at 14,000 × g for 30 min. The supernatant was discarded, and the protein pellet was washed with 1 ml of acidified acetone (0.1% HCl) followed by 1 ml of acetone for 5 min each. Protein precipitates were collected between washes by centrifugation (14,000 × g, 5 min). The resulting purified histone proteins were resuspended in 10 mM Tris (pH 8) and stored at −80 °C until processed for western blotting.
Protein concentrations for each sample were determined using a DC protein assay (BioRad). Aliquots of sample were then normalized to ~2 µg/µl. Laemmli sample buffer [final concentration: 6.25 mM Tris, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 1.25% 2-mercaptoethanol, 0.1% bromophenol blue] was added to histone samples prior to performing SDS- polyacrylamide gel electrophoresis (PAGE). SDS-PAGE gels consisted of a 12% acrylamide resolving gel overlaid with a 4% acrylamide stacking gel. Histone proteins were transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting which consisted of incubating PVDF membranes in primary antibodies for 1 h at room temperature or overnight at 4 °C followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Detection of immunolabeled histone proteins was facilitated via electrogenerated chemiluminescence (ECL; Amersham Biosciences; SuperSignal, Pierce) and exposure to BioMax X-Ray film (Kodak Scientific Imaging Systems, Rochester, NY) and digitized (Epson Perfection 1240U). Antibodies—The primary antibodies used, and their dilutions were as follows: anti-H3K4me3 (1:1000, Upstate Biotechnology Inc.), anti-H3K9me2 (1:1000, Upstate Biotechnology Inc.), and anti-H3 (1:1000, Upstate Biotechnology Inc.). The host for all primary antibodies was rabbit. The secondary antibodies were horseradish peroxidase-conjugated goat Anti-IgG heavy and light chain (Jackson ImmunoResearch, West Grove, PA).
ChIP analysis was performed as previously described in Lubin et al. (2007). Briefly, the CA1 regions of hippocampus were microdissected and held in ice-cold PBS solution containing protease inhibitors [1 mM phenylmethysulfonyl fluoride, 1 µg/ml of protease inhibitor cocktail and phosphatase inhibitors (1 mM Na3VO4 and 20 mM NaF)]. Hippocampal tissue was immediately incubated in 1% formaldehyde in PBS at 37 °C for 10 min. Hippocampal tissue was homogenized in SDS lysis buffer (50mM Tris, pH 8.1, 10mM EDTA, 1% SDS). Chromatin was sheared using a Branson Sonifier 250 at 1.5 power and constant duty cycle. Lysates were centrifuged to pellet debris and then diluted 1:10 in ChIP dilution buffer (16.7 mM Tris, pH 8.1, 0.01% SDS, 1.1% Triton X-100, 167 mM NaCl, 1.2 mM EDTA). Immunoprecipitations were carried out at 4 °C overnight with primary antibodies (anti-H3K4me3 or MeCP2) or No antibody (control). Immune complexes were collected with an protein A-agarose bead/salmon sperm slurry and sequentially washed with low salt buffer (20 mM Tris, pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), high salt buffer (20 mM Tris, pH 8.1, 0.1% SDS, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA), LiCl immune complex buffer (0.25 M LiCl, 10 mM Tris, pH 8.1, 1% deoxycholic acid, 1% IGEPAL-CA630, 500mM NaCl, 2 mM EDTA), and TE buffer. Immune complexes were extracted in 1× TE containing 1% SDS, and protein-DNA cross-links were reverted by heating at 65 °C overnight. After proteinase K digestion (100 µg; 2 h at 37°C), DNA was extracted by phenol/chloroform/isoamyl alcohol and then ethanol- precipitated. Immunoprecipitated DNA was subjected to quantitative real-time PCR using primers using primers specific for 150–200 bp segments corresponding to promoters upstream of the rat Bdnf or Zif268: Bdnf promoter 1 sense, 5'-TGAGAAGTTGTGGGACCGCATA-3'; antisense, 5'- CGTCCTCTTCCCTTGGCTTTTT-3'; Bdnf promoter 4 sense, 5'- GTGGAGGAGAGGTGCCTTTTGA-3'; antisense, 5'- GTCCTCTGGGGACCATTTACCC-3'; Zif268 (ChIP 1) sense, 5'-ATGGGCTGTTAGGGACAGTG −3'; antisense, 5'-TTGGGGATTTAGCTCAGTGG-3'; Zif268 (ChIP 2) sense, 5'-CTCCCTCACTGCGTCTAAGG-3'; antisense, 5'- CCTAAAGAGGGGGACTTTGC-3'. The cumulative fluorescence for each amplicon was normalized to input amplification.
DNA was isolated from hippocampal tissue, purified and processed for bisulfite modification. For all our MSP data, we performed bisulfite treatment of genomic DNA an average of 5–6 times. Bisulfite treatment of genomic DNA converts cytosine to uracil, but leaves methylated 5'Cytosines unchanged. Quantitative real-time PCR was used to determine the DNA methylation status of the bdnf gene. Methylation-specific PCR primers were designed using Methprimer software (http://www.urogene.org/methprimer/)(Li and Dahiya, 2002). Methylation and unmethylated-specific PCR primers were designed to target putative CpG islands detected in silico in promoter or non-promoter regions of the Zif268 gene. Detection of CpG islands (≥ 200 bp) within Zif268 DNA was performed with the following primer sets. For CpG island region 1; Methylated Zif268 sense, 5'-ATTGCGGGGAAGGTTTAGGTC-3'; antisense, 5'- TATATTCCGAAAACCCCAAACG-3'; Unmethylated Zif268 sense, 5'- ATTGTGGGGAAGGTTTAGGTTG-3'; antisense, 5'-ATATTCCAAAAACCCCAAACAC-3'. For CpG island 2; Methylated Zif268 sense, 5'- TTTAGGATGACGGTTGTAGAATTTC-3'; antisense, 5'- GCGTAAAACTAAACGCTCCG-3'; Unmethylated Zif268 sense, 5'- GGATGATGGTTGTAGAATTTTGG-3'; antisense, 5'- CAAACACATAAAACTAAACACTCCA-3'. We also evaluated β-tubulin-4 DNA after context exposure or contextual fear conditioning and found no significant alterations in unmethylated β-tubulin-4 DNA. Thus, we used unmethylated β-tubulin-4 levels for normalization of Zif268 DNA methylation in the studies presented here. B-tubulin-4 sense, 5'-GGAGAGTAATATGAATGATTTGGTG-3'; antisense, 5'-CATCTCAACTTTCCCTAACCTACTTAA-3'. PCR reactions were in a iQ5 real-time PCR system (Bio-Rad, Hercules, CA) using the following cycling conditions: 95 °C for 3 min, 40 cycles of 95 °C for 15 sec and 58–60 °C for 1 min. Detection of the fluorescent products occurred at the end of the 15 sec temperature step. Samples were normalized to β-tubulin-4 and the comparative Ct method was used to calculate differences in gene expression between samples (Livak and Schmittgen, 2001).
Bisulfite-treated samples (as described above) were amplified by primers that amplify the same region of bdnf exon IV DNA, but independent of methylation status. The thermocycler protocol involved an initial denaturation cycle (5 min, 95 °C), 50 cycles of denaturation (1 min, 95 °C), annealing (1 min, 60 °C) and extension (1 min, 72°C), followed by a final extension cycle (5 min, 72°C) terminating at 4 °C resulting in a 258 bp PCR product for exon IV. BSP primer pairs used for the assessment of Zif268 cytosine residues within CpG island region 1 were as follows; Zif2568 sense, 5'-TTGTGAAGGAAGTGTTATTTTG-3'; antisense, 5'- CCAATCTAATAACCCCAAACTT-3'. The BSP products were then purified using a gel extraction kit (Qiagen), and sequenced using the reverse primer at the University of Alabama at Birmingham Genomics Core Facility of the Heflin Center for Human Genetics (http://www.heflingenetics.uab.edu). The percent methylation of each CpG site within the region amplified was determined by the ratio between peaks values of G and A (G/[G+A]), and these levels on the electropherogram were determined using Chromas software.
Behavioral data were analyzed with two-tailed unpaired t tests. Post-hoc comparisons after One-way ANOVA were made using Tukey post-hoc test where appropriate. Data for Mll;eed mutant mice were analyzed using individual one-way ANOVA with LSD post-hoc and simple-effects tests where appropriate for the context test and the CS test. Follow-up comparisons were made using least significance difference test. SPSS 10.0 software was used for statistical analysis of the data. All Western blots were quantified by densitometry (Scion) and GraphPad Prism software was used for statistical analysis of the data. For studies involving the assessment of histone or DNA methylation and mRNA levels were analyzed by two-tailed one-sample t tests when appropriate. Differences in BSP data were analyzed by Two-way ANOVA with Bonferroni post hoc test. Data are shown as the mean ± standard error of mean (SEM). Significance for all tests was set at p ≤ 0.05.
To begin exploring the potential role for histone methylation in memory formation we investigated whether contextual fear conditioning as an associative learning event triggers altered histone methylation within the hippocampus. For behavioral studies animals were fear conditioned in a novel context (training chamber) with a training protocol consisting of three 1-second, 0.5 mA shocks at 2-min shock intervals (Fig. 1A). As a control for our biochemical studies, we first confirmed that the context-plus-shock pairing produced an associative memory. As expected, 24 h after fear conditioning, re-exposing the animals to the training chamber without the footshock triggered recall of the associative memory, as measured by freezing behavior (Fig. 1B). As an additional control, novel context exposure alone produced no significant freezing when the animals were replaced in the training chamber (Fig. 1B).
To evaluate whether histone methylation was altered in response to contextual learning, histone extracts were prepared from area CA1 of hippocampus 1 h after training (Fig. 1A). Thus, we evaluated histone H3 methylation at lysine 4 (K4) as an active marker of transcription and observed a significant increase in tri-methylated H3K4 levels in the hippocampus 1 h after contextual fear conditioning compared to naïve controls (Fig. 1C). No changes in tri-methylated H3K4 levels were observed with context exposure alone (Fig. 1C). These results suggest that H3K4 tri-methylation is regulated in hippocampus after associative fear conditioning training and are consistent with prior observations that active transcription occurs during consolidation of associative contextual memories. These observations also implicate histone methylation as a potential positive regulator of gene transcription in memory consolidation.
We also examined whether or not contextual fear conditioning alters methylation of histone H3 at lysine9 (K9), a repressive marker of transcription, in area CA1 of the hippocampus. Western blotting analysis revealed that 1 h after contextual fear conditioning H3K9 di-methylation significantly increased in area CA1 of the hippocampus (Fig. 1D). Interestingly, context exposure alone produced similar increases in di-methylated H3K9 levels in area CA1 of hippocampus relative to naïve control animals (Fig. 1D). These findings suggest that H3K9 di-methylation is triggered in hippocampus with both novel context learning and associative contextual learning of fear. Together these results are consistent with the idea that histone-methylation-dependent active repression of transcription plays a role in the consolidation of contextual memories. It is interesting that context exposure alone triggered only the transcription-suppressing H3K9 di-methylation mark and not the transcription-activating H3K4 mark (see above); implying that the H3K4 mark may be an associative-learning specific signal.
Next, we determined whether regulation of hippocampal histone H3 methylation triggered with learning about the context or with associative contextual fear conditioning is a dynamic process. To address this, area CA1 of hippocampus was removed from animals 24 h after training and western blotting performed for histone H3 methylation that serve as activation (H3K4) or repressor (H3K9) markers of gene transcription (Fig. 2A). All groups were compared to naïve-handled animals that were not exposed to the training chamber. Twenty-four hours after training (without replacing animals into the training context) western blotting analysis revealed no changes in H3K4 tri-methylation in hippocampus after fear conditioning (Fig. 2B). In addition, levels of tri-methylated H3K4 were unchanged 24 h after context exposure alone when compared to naïve animal controls (Fig. 2B). These results suggest that tri-methylation of H3K4 is reversible, allowing the H3K4 tri-methylation state necessary for memory consolidation to return to basal levels after transient activation. Furthermore, these findings support the idea that a histone demethylase does exist for active demethylation of tri-methylated H3K4 induced in hippocampus after associative fear conditioning training.
Having established that the increase in H3K4 tri-methylation with fear conditioning was reversible, we then sought to determine whether or not the effect of contextual fear conditioning on H3K9 di-methylation was also plastic. Surprisingly, we found that H3K9 di-methylation significantly decreased 24 h after fear conditioning compared to naïve controls (Fig. 2C). We also observed a decrease in H3K9 di-methylation 24 h after exposure to context alone compared to naïve animal controls (Fig. 2C). These results indicate that learning about the context or fear conditioning to the context has effects on H3K9 di-methylation in hippocampus that persist at least 24 h. These findings are the first evidence to indicate a persistent change in a hippocampal histone modification following learning, lasting well beyond the 2–4 h memory consolidation phase.
The results obtained thus far suggest that H3K4 tri-methylation is induced by formation of an associative long-term memory wherein an animal associates a novel context with a footshock. However, it is also possible that the regulation of hippocampal H3K4 tri-methylation was due to a stress response to the footshock alone in the absence of associative memory formation. Therefore, a latent inhibition training paradigm was used to determine whether this event contributed to the regulation of H3K4 tri-methylation in hippocampus after fear conditioning.
The latent inhibition training protocol consists of pre-exposing the animal to a novel context (training chamber) for a long period of time prior to administering the unconditioned stimulus (footshock). This form of contextual learning is unique in that the animal does not form a strong association between the unconditioned stimulus and the novel context, making the actual latent inhibition training paradigm context-specific (Lubow and Josman, 1993; Atkins et al., 1998). Therefore, during a latent inhibition contextual learning the animal forms a spatial memory that attenuates the formation of an associative contextual fear memory. Nevertheless, in this protocol the animal still receives the identical footshock utilized for associative fear conditioning training.
Animals were placed into the novel training context (training chamber) 2 h prior to receiving the three-shock context fear conditioning training (Fig. 3A). The animals were then returned to their home cages. Twenty-four hours later, the animals were re-exposed to the training chamber with no footshock (Test). Latent inhibition fear conditioning inhibited the associative contextual fear memory as assessed by freezing behavior (Fig. 3B). We also found that the increase in hippocampal H3K4 tri-methylation normally observed 1 h after fear conditioning was blocked with the latent inhibition paradigm compared to naïve controls (Fig. 3C). These results suggest that the regulation of H3K4 tri-methylation after training was selective for the formation of robust associative contextual fear memories.
A number of enzymes for methylating or demethylating histones have recently been discovered (summarized in Allis et al., 2007). While few pharmacological agents are currently known to specifically affect these enzymes, genetic knock-out of these chromatin-modifying enzymes or their complexing partners allows us to further elucidate their specific role in learning and memory. Recently, Kim and colleagues reported altered patterns of histone methylation in eed+/−;Mll+/− mice (Kim et al., 2007). Eed interacts directly with the histone-lysine methyltransferase Ezh2 to target H3K27 or H1K26 (Kuzmichev, et al., 2004) and interacts with HDAC 1 and 2 (van der Vlag & Otte, 1999). Mll also interacts with HDAC 1 and 2 (Xia, et al. 2003) and acts as an H3K4-specific methyltransferase (Milne, et al. 2002).
To further assess the role of regulation of histone methylation in the process of memory consolidation, we assessed fear memory in eed and Mll and single and double mutant mice (Paylor et al., 1994; McIlwain et al., 2001; Kim et al., 2007) comparing their behavior to wild-type littermate control animals using an associative contextual fear paradigm. We found significant differences between genotypes during the context behavior test (Fig. 3D). In these comparisons we found that eed+/+;Mll+/− and eed+/−;Mll+/− animals displayed significantly reduced freezing compared to wild-type littermates (all comparisons p < 0.015). The effect on freezing behavior is stronger in heterozygote Mll mice compared to heterozygote eed mice. This is consistent with the idea that Mll is a H3K4-specific methyltransferase whereas Eed is a general component of the methyltransferase complex. Together, these results suggest in-part that the H3K4-specific methyltransferase, Mll, is essential for hippocampal-dependent long-term memory formation. Additionally, we assessed cued fear conditioning in eed+/−;Mll+/+, eed+/+;Mll+/−, and in eed+/−;Mll+/− mice and found no statistically significant differences in freezing behavior among genotypes [F(3,63)=0.664, P=0.57] (Supplemental Fig. 1H). Thus, eed and Mll are important for hippocampus-mediated learning and memory. Together, these findings indicate regulation of hippocampal histone methylation as a molecular mechanism underlying proper memory formation.
As additional behavioral controls, exploratory activity, motor function, and sensorimotor processing were assayed in eed and Mll mutant mice. While eed single heterozygous mice exhibited largely normal behavioral responses, Mll single heterozygotes as well as double heterozygotes exhibited defects in some tests of motor function (Supplementary Fig. 1F). In contrast, exploratory activity, anxiety-related responses, acoustic startle, and prepulse inhibition were normal (Supplementary Fig. 1).
Recent studies have revealed that the methylation state of histone H3 is directly linked to the hyperacetylated state of the histone H3 N-terminal tail in vitro (Zhang et al., 2004). In these studies, Zhang and colleagues report a synchronous relationship between hyperacetylated histone H3 at lysine-9 and hypermethylated histone H3 at lysine-4. Thus, we hypothesized that enhancing histone acetylation via HDAC inhibition might result in an enhancement in histone methylation as well.
To this end, we investigated whether treatment with the HDAC inhibitor sodium butyrate (NaB) might enhance the methylated state of histones in hippocampus during memory formation (Fig. 4A). We first confirmed the reported effect of HDAC inhibition on long-term memory formation. We found that 24 h after the training period, NaB-treated animals displayed significantly more freezing behavior than vehicle-treated controls (Fig. 4B). These results are in agreement with previous reports of the effect of HDAC inhibition on long-term memory formation (Levenson et al., 2004a; Fischer et al., 2007; Lubin and Sweatt, 2007; Guan et al., 2009). We then assayed histone H3 methylation in area CA1 of hippocampus 1 h after training and found that both NaB- and vehicle-treated animals exposed to the fear conditioning paradigm displayed increased H3K4 tri-methylation relative to naïve animal controls (Fig. 4C). However, there was no significant difference in H3K4 tri-methylation from NaB-treated animals relative to that of vehicle-treated controls.
We next examined the effect of enhancing histone acetylation on H3K9 di-methylation in hippocampus following fear conditioning. We found that NaB treatment significantly decreased H3K9 di-methylation in hippocampus relative to vehicle-treated animals (Fig. 4D). These results suggest that elevating histone acetylation via HDAC inhibition leads to suppression of the negative regulator of transcription H3K9 di-methylation. This surprising result suggests that one mechanism whereby HDAC inhibition might enhance memory formation is through secondary suppression of histone H3K9 di-methylation. Regardless of this specific possibility, the result suggests a dynamic cross-talk between histone acetylation and histone methylation in hippocampus.
To further support a role for histone methylation during memory formation, we determined whether fear conditioning triggers changes in chromatin structure directly at the level of individual genes. The Zif268 and bdnf genes are critical for the consolidation of several forms of memories including hippocampus-dependent memory formation (Bramham, 2007; Poirier et al., 2007). Thus, we investigated hippocampal tri-methylated H3K4 levels at the Zif268 and bdnf gene promoters 30 min after training, a time point known to be associated with transcriptional activation of these gene loci. Using chromatin immunoprecipitation (ChIP) assay combined with quantitative real-time PCR, we found that H3K4 tri-methylation increased at the Zif268 promoter after fear conditioning (Fig. 5 A , B). We also found that H3K4 tri-methylation significantly increased at bdnf promoter 1, but not at promoter 4, in area CA1 of hippocampus after fear conditioning, relative to naïve controls (Fig. 5C). Context exposure alone produced no significant changes in H3K4 tri-methylation at the Zif268 or bdnf promoters relative to naïve animal controls (Fig. 5 A–C). The selective increase in histone tri-methylation at the Zif268 promoter and bdnf promoter 1 is in good agreement with fear-conditioning-associated increased transcription of the Zif268 and bdnf genes (Lubin et al., 2008; Alberini, 2009). Together, these results demonstrate active regulation of H3K4 tri-methylation within specific gene promoters, and further demonstrate that H3K4 tri-methylation is regulated in response to fear conditioning.
There is evidence that H3K9 di-methylation represses gene expression via recruitment of DNA methyltransferase enzymes, which mediate methylation of CpG dinucleotides that then recruit repressive chromatin remodeling complexes (Fuks, 2005). In contrast to H3K9 di-methylation, H3K4 tri-methylation mediates active transcription and shows a strong correlation with activated RNA polymerase II and histone acetylation levels at gene promoters (Rice and Futscher, 2000; Nakayama et al., 2001; Rice and Allis, 2001; Li and Zhang, 2004; Zhang et al., 2004; Ruthenburg et al., 2007). Thus, given our finding that H3K4 tri-methylation is increased after fear conditioning, we next determined whether or not DNA methylation was altered during memory consolidation in a fashion that was indicative of an active promoter. Consistent with this idea, we previously have reported that bdnf promoter 1 shows decreased cytosine (DNA) methylation with contextual fear conditioning (Lubin et al., 2008). These previous findings are in good agreement with the idea that H3K4 tri-methylation levels at bdnf promoter 1 (Fig. 5C) correlates with DNA demethylation during fear conditioning. However, we also previously observed that fear conditioning is associated with decreased DNA methylation at bdnf exon 4 at 2 h post-training (Lubin et al., 2008), but this site did not manifest increased histone methylation (at 30 min post-training) in the current studies (Fig. 5D). Thus, the available evidence does not allow us to ascertain whether H3K4 trimethylation is universally correlated with DNA demethylation in the adult CNS.
Therefore in the present studies, we performed additional experiments and examined the DNA methylation state of the Zif268 promoter because increases in H3K4 tri-methylation levels in hippocampus were robust at this promoter region in response to associative contextual fear conditioning (Figure 5B). Furthermore, unlike bdnf DNA methylation (Lubin et al., 2008), the methylation state of the Zif268 gene has not been previously characterized in hippocampus during memory formation.
We first screened the Zif268 promoter region for CpG islands, which are defined as stretches of DNA (≥200bp) where there are a large number of cytosine-guanine dinucleotide sequences linked by phosphodiester bonds (reviewed in Goldberg et al., 2007). Using Methprimer software (http://www.urogene.org/methprimer/)(Li and Dahiya, 2002) two CpG island sites were found within the promoter region of the Zif268 gene immediately preceding the transcriptional start site (Fig. 6A). We first used methyl specific PCR (MSP) to screen DNA methylation changes at each CpG island site 30 min following fear conditioning. Our MSP results demonstrated that fear conditioning triggered changes in DNA methylation at CpG island region 1, but not region 2 in hippocampus (data not shown).
Next, we used direct bisulfite DNA sequencing PCR (BSP) to examine site-specific methylation changes at 20 CpG dinucleotides within Zif268 CpG island region 1 (Fig 6A). The results show that fear conditioning elicited an increase in methylated DNA levels across the region relative to context exposure alone or naïve animal controls (Fig. 6B). However, Zif268 mRNA levels 30 min after training were significantly increased in hippocampus in response to fear conditioning (Fig. 6C). Thus, these results suggest that increased H3K4 tri-methylation levels at the Zif268 promoter is associated with increases in DNA methylation resulting in active transcription of the Zif268 gene after fear conditioning. Overall these data support the idea that H3K4 tri-methylation can be associated with either increased DNA methylation (Zif268), or decreased DNA methylation (bdnf) at genes that are transcriptionally activated in memory consolidation.
Next, we sought to determine the effect of contextual fear conditioning on Zif268 DNA methylation 24 h later. We found that Zif268 DNA methylation and mRNA levels returned to baseline 24 h after fear conditioning compared to naïve controls (Fig. 7A–C). This suggests that the effect of contextual fear conditioning on Zif268 DNA methylation was also transient. Interestingly, we also observed an increase in Zif268 DNA methylation 24 h after exposure to context alone compared to naïve animal controls (Fig. 7A), which corresponds with a decrease in Zif268 gene expression 24 h after context exposure alone (Fig. 7B). These data support the idea that altered DNA methylation can be associated with either increased Zif268 gene expression 30 min after fear memory consolidation or decreased Zif268 gene expression 24 h after context exposure.
Although the findings of increased DNA methylation associated with increase Zif268 gene transcription with contextual fear conditioning were surprising, they are consistent with previous results by Chahrour et al. (2008) suggesting that methyl-binding proteins such as methyl-CpG binding protein 2 (MeCP2) can activate as well as repress gene transcription, specifically suggesting that MeCP2 associates with the transcriptional activator CREB1 at methylated promoter regions of activated gene target. Thus, we sought to determine whether or not the effect of contextual fear conditioning on Zif268 DNA methylation was associated with altered MeCP2 levels at the gene promoter. We found that MeCP2 significantly decreased at the Zif268 promoter region associated with increased H3K4 tri-methylation 30 min after fear conditioning or context exposure compared to naïve controls (Fig. 8A; ChIP1). These results indicate that learning about the context or fear conditioning to the context has effects on the recruitment of MeCP2 levels at the Zif268 promoter in hippocampus. We also observed an increase in MeCP2 at the Zif268 promoter region associated with increased DNA methylation 30 min after fear conditioning, but not after exposure to context alone, compared to naïve animal controls (Fig. 8B; ChIP2). Thus, Zif268 mRNA levels 30 min after training were significantly increased in hippocampus in response to fear conditioning (Fig. 6C). Together, these findings are consistent with the idea that increases in DNA methylation can represent an active signal for gene transcription.
In the studies presented here, we investigated the potential role of histone lysine methylation in memory formation and established several findings. First, we found that fear conditioning triggers changes in H3K4 tri-methylation (a transcriptional active marker) and H3K9 di-methylation (a transcriptional repressive marker) in area CA1 of the hippocampus. Second, we observed that H3K4-specific methyltransferase (Mll) deficient mice have a deficit in long-term memory formation. Third, treatment of animals with the HDAC inhibitor NaB altered histone methylation levels in hippocampus during memory consolidation, suggesting that altered histone methylation is coupled to HDAC inhibition. Finally, H3K4 tri-methylation significantly increased at the promoter of two activated gene targets (Zif268 and bdnf) during memory consolidation. Together, these findings support the role of histone methylation changes in the consolidation of long-term memory formation.
Covalent post-translational modifications of histones serve as an important mechanism for transcriptional regulation during consolidation of long-term memories. For example, increases in histone H3 phosphorylation and acetylation in hippocampus have been shown to be regulated during long-term memory formation (Levenson et al., 2004a; Chwang et al., 2006; Fischer et al., 2007). Additionally, regulation of histone modifications including histone methylation have been implicated in mental disorders including schizophrenia (Deutsch et al., 2008; Akbarian and Huang, 2009). However, investigation of the role of histone methylation in the process of long-term memory formation had not been explored prior to the current studies. Encouragingly, studies on abnormal gene expression in postmortem brain have revealed that histone methylation may be a viable avenue for early detection for some cases of schizophrenia (Akbarian and Huang, 2009). Therefore, the study of histone methylation in the regulation of memory formation is intriguing and will provide further insights into the epigenetic mechanisms that may be dysregulated in cognitive impairments.
We investigated the contribution of hippocampal histone methylation in the consolidation of long-term memory in a rodent model of fear conditioning. We were particularly interested in studying this form of histone modification because it is unique in that various specific sites of methylation of histones can have opposite roles in gene regulation; that is, methylation of H3K4 is associated with active transcription whereas methylation of H3K9 is associated with transcriptional silencing (Strahl et al., 1999; Lachner and Jenuwein, 2002; Schotta et al., 2002). Indeed, the process of storing stable long-term memories is likely to involve coordinated changes in both transcriptional activation and silencing of genes (Ressler et al., 2002; Levenson et al., 2004b). Now with the results presented here, we provide the first evidence for histone methylation changes in hippocampus in response to contextual learning, in both episodic learning and associative contextual fear conditioning. Furthermore, these results support the hypothesis that histone methylation plays a role in the regulation of gene expression changes that are permissive for memory formation.
One fascinating finding from our studies is that HDAC inhibition significantly altered histone methylation levels. Specifically, treatment with the non-selective class I HDAC inhibitor NaB significantly attenuated H3K9 di-methylation in hippocampus after fear conditioning. This suggests the new idea that a possible mechanism for the permissive actions of inhibiting HDACs, resulting in memory enhancement, is through the negative regulation of hippocampal H3K9 di-methylation. However, the specific HDAC isoform (s) inhibited by the HDAC inhibitor NaB to mediate this effect on H3K9 di-methylation is unknown and remains an intriguing focus of ongoing studies. Promisingly, the HDAC2 isoform has recently been identified as a modulator of dendritic spine density, synapse number, and synaptic plasticity which negatively regulates memory formation (Guan et al., 2009). Although very speculative at this point, an intriguing hypothesis is that inhibition of HDAC2 negatively regulates H3K9 di-methylation to allow memory enhancement. Whether this scenario is true or not, our present study is consistent with the idea that inhibition of HDAC isoforms promotes active gene transcription for stable formation of enhanced long-term memories.
Another remarkable finding from these studies is that changes in H3K4 tri-methylation were associated with increased DNA methylation at the Zif268 promoter region, while Zif268 gene expression was active during memory consolidation. In addition, these changes in Zif268 DNA methylation were transient returning to baseline levels 24 h later long after the consolidation period. These observations are in sharp contrast to findings from developmental studies that suggest that DNA methylation is primarily associated with the repression of gene transcription. However, a recent study by Chahrour et al. (2008) indicates that methyl-CpG binding proteins, such as MeCP2, bind to DNA methylation sites with the transcriptional activator cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein 1 to actively regulate gene transcription. Indeed, our results suggest that MeCP2 levels increased at the Zif268 promoter region in association with increased DNA methylation resulting in transcriptional activation of the Zif268 gene following fear conditioning. Correspondingly, we found a cAMP response element (CRE; TCACGTCA) binding site for the transcription factor CREB downstream of the CpG island 1 region and upstream from the transcriptional start site of the Zif268 gene (Fig. 8C). Thus, based on previous studies by Chahrour et al. (2008) and our present data, we propose that dynamic changes in DNA methylation may serve as both a repressive or active marker of transcription in hippocampus during long-term memory formation. This new concept raises several intriguing possibilities for future research. For example, in our study we demonstrated that contextual learning induces global changes in histone methylation as well as altered DNA methylation. However, it remains to be determined what specific cell-types are involved in these observed epigenetic mechanisms.
In summary, our results continue to support a role for epigenetic mechanisms in the process of stable formation of long-term memories. Specifically, our findings demonstrate considerable histone methylation changes in hippocampus in association with memory formation and furthermore suggest that this process is necessary for long-term memory formation. In addition our results provide the first evidence for an association between differential histone methylation and DNA methylation regulation at the Zif268 gene promoter in the adult brain and implicate a possible role for these processes in the formation of long-term memories. Finally, as epigenetic mechanisms continue to be linked to cognitive dysfunctions a better understanding of the complex molecular interaction and regulation of these processes will need to be further refined.
The importance of continued investigation of such mechanisms is underscored by several prior studies. For example, similar to DNA methylation, histone methylation was once considered to be a non-plastic process. The concept of the irreversibility of histone methylation arose from the belief that histone demethylases did not exist. However, this view has recently been challenged with the identification of several histone H3 demethylases including LSD1 and JHDM1 (Shi et al., 2004; Tsukada et al., 2006; Whetstine et al., 2006; Tahiliani et al., 2007) which can specifically demethylate lysine-4 within histone H3. Thus, the discovery of these enzymes may lead to more selective therapeutic interventions that include the use of histone demethylase activators or inhibitors as well as HDAC inhibitors for treatment of cognitive impairments associated with neurological disorders.
(A) Locomotor activity. Open-field assessment of overall locomotor activity and anxiety-related responses displayed no significant difference among genotypes. (B) The center distance ratio score. There was a significant difference amongst the genotypes. Specifically, analysis found that eed+/+;Mll+/− or eed+/−;Mll+/− mice displayed reduced center exploration, suggesting greater anxiety-related responses. (C) Rotarod test. When mice were given four trials on two consecutive days, the overall analysis confirmed that the eed+/+;Mll+/− or eed+/−;Mll+/− mice performed significantly worse on the rotarod compared with eed+/+;Mll+/+ and eed+/−;Mll+/+ littermates. Moreover, this difference was present on trial 2–8. (D–E) Mini-motor test of wire-suspension, vertical-pole and stationary dowel tests. (D) Along with significant differences among the four genotypes in the wire test, the eed+/−;Mll+/− mice performed worse than eed+/+;Mll+/+ and eed+/−;Mll+/+ littermates. (E) The vertical pole test also demonstrated a main effect of genotype and significantly lower scores for eed+/+;Mll+/− animals compared with eed+/+;Mll+/+ and eed+/−;Mll+/+ littermates. (F) Composite motor score of overall performance on four motor tests (rotarod, wire suspension, pole test, and stationary dowel). The composite motor score confirmed both the main effect of genotype in various tests and the overall deficient performance of eed+/+;Mll+/− and eed+/−;Mll+/− mice in contrast to eed+/+;Mll+/+ and eed+/;Mll+/+ littermates. (G) Acoustic startle and prepulse inhibition of the acoustic startle response. Both acoustic startle response and the increase in the level of prepulse inhibition with the increase of the prepulse sound were comparable and not significant between genotypes. (H) Behavioral memory formation measured by contextual and auditory-cue conditioned fear. The left side of the figure indicates the level of freezing for the four genotypes 24 hours following training. In addition to significant differences among the genotypes for the context test, eed+/−;Mll+/+ and eed+/+;Mll+/− animals displayed significantly reduced freezing compared with wild-type littermates. Note the additional decrease in freezing of double heterozygotes in comparison to eed+/−;Mll+/+ and eed+/+;Mll+/− animals, although the latter difference did not reach statistical significance. The right side of the figure displays the level of freezing during the CS test (defined as freezing in the presence of the auditory CS minus the freezing during the pre-CS period). In contrast to the context test, the differences between the genotypes during the CS test did not reach statistical significance. Error bars indicate standard error of mean (SEM).
We thank members of the Lubin laboratory for their thoughtful comments, Dr. M. Han and the University of Alabama at Birmingham Genomics Core Facility for assistance in DNA sequencing, and Dr. A. Schumacher for providing the eed; Mll mutant mice. This work was supported by the National Institute of Health (NS048811, MH082106, MH57014, NS37444, NS013546, AG031722, and NS057098), the Federation of American Societies for Experimental Biology, the National Alliance for Research on Schizophrenia and Depression, the Rotary Clubs Coins for Alzheimer's Research Trust Fund, and the Evelyn F McKnight Brain Research Foundation.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text: On Tuesday, May 14,2013 Rob was diagnosed with Peripheral T-Cell Lymphoma. Only four weeks later, Rob passed away on Tuesday, June 11, 2013. He is no longer suffering from this terrible cancer and is now resting in peace in heaven. He was surrounded by his family and friends. Rob was only 42 years old and though most of us cannot understand why he was taken so young, we all can agree, Rob lived his life to the fullest. Looking through pictures and videos, there is a common theme, Rob was always smiling. Rob was a wonderful Husband, Dad, Son, Brother and Friend. The day Rob was diagnosed he had a positive outlook and continued to with an occasional thumbs up during the four weeks. We were blessed and fortunate to have a brilliant team of expert doctors and nurses caring for him. They wanted a different outcome just as much as we did. We miss him dearly. In Rob's honor, let's raise some money to help the doctors learn more about lymphoma and find a cure! All the proceeds that the Rally For Rob team raise go towards the Leukemia & Lymphoma Society!

subset: commoncrawl


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- Peptic
- Peptic Pep"tic, a. [L. pepticus, Gr. ?. See {Pepsin}.]
1. Relating to digestion; promoting digestion; digestive; as,
peptic sauces.
[1913 Webster]
2. Able to digest. [R.] [1913 Webster]
Tolerably nutritive for a mind as yet so peptic. --Carlyle. [1913 Webster]
3. (Physiol. Chem.) Pertaining to pepsin; resembling pepsin in its power of digesting or dissolving albuminous matter; containing or yielding pepsin, or a body of like properties; as, the peptic glands. [1913 Webster]
The Collaborative International Dictionary of English. 2000.


text:
Everyone knows that fairness couldn't be achieved in a day. But, there are certain effective natural beauty tips and face packs that can make your skin fair in a few days. But, before talking about beauty tips using natural ingredients to get quick fairness; one should keep certain important points in their mind to protect the natural skin tone from damage. It's important that we should care about our skin and protect it from any potential and known damage. The following points will be helpful in keeping the skin fair or retaining the natural skin tone:
There are many other natural ingredients that could help in achieving fair skin and glow. All one has to do is to look for proper remedies and solutions on time. Natural ingredients will never harm the skin in any way and will give natural and long lasting results. In spite of choosing clinical solutions and using harsh chemicals or solutions over the skin, try the natural remedies first. So, instead of asking doctors about how to get instant fairness, give a glance at nature's wonders. Natural remedies will not even cost much like the doctors and clinically treatments and will surely help out in some or the other. So, don't skip the natural and simple way to get the fair and glowing skin.
Also read
Home Remedies For Tan Removal
Home Remedies For Glowing Skin In Summer
How to Get Fair Skin in Summer
How to Get Fair Skin Naturally
10 Best Home Remedies for Tan Removal from Face
Home Remedies For Instant Glow
April 21, 2016 11:15AM
Thanks a ton for this amazing article Rinku. These face packs will be really great for me.
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April 04, 2016 11:27AM
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Steven Wayne Morris: East Texas Cop Gets 8 Years for Repeatedly Sexing Teen in Back of Her Car
Steven Wayne Morris: Cop sent away for eight years.
A police officer who had repeated sexual encounters with a 15-year-old girl in the back seat of her car has been sentenced to eight years in prison.
Steven Wayne Morris, 28, was an officer for the East Texas town of Bullard when he began texting a student at Bullard High School. Within a year, the two began having sex in the back of her car, according to media reports.
Morris initially denied the charges but confessed to Texas Ranger investigators. He entered a guilty plea as part of a plea bargain, KLTV reports.
He had been suspended from the force once rumors began circulating about the relationship in 2009, and later was fired.
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The opening of the massive Comic-con convention last week provided days of comic book related news coverage. However, few of the images from that event were of African Americans. The dearth of African American perspectives in mainstream comic books inspired many black artists to create webcomics. Webcomics are online sites that present a story in comic book form. The success of Boondocks and the current global recession were motivators for many of these artists to try their hand at starting an online webcomic business. These five webcomics present a sample of African American entrepreneurs who are presenting a different perspective on the web.
Created by Houston based artist Fave, A Pug Named Fender chronicles the adventures of a pug as he enjoys the thrills of barbecue, music, technology, and other essentials that make life worth living. This recently launched webcomic has already featured guest appearances by soul music artists like Questlove. New episodes of A Pug Named Fender are posted every Tuesday and Thursday.
Michelle Billingsly created JOE! to capture the life the title character, a rambunctious 10 year old. This webcomic doesn't just focus on Joe and has created a cast with well developed characters. There is no regular update schedule, but new strips come out about twice a month.
George Ford publishes Addanac City which depicts the shenanigans of Hank Addanac. It's an interesting mix of Calvin and Hobbes and Phineas and Ferb. Ford keeps a rigorous schedule of publishing seven comics a week that goes back to August 2008. The cast is very diverse and both the writing and art show a high degree of quality.
Charles Arrington's Redux Deluxe covers the adventures of three boys named CJ, Chris, and Rob as they try to retrieve a lost basketball from a neighborhood girl named Angela. Containing many references to comic book and video game culture, new episodes of Redux Deluxe come out twice a week.
Phoenix artist Frank Jordan publishes a new Company Man strip five days a week. I offers a humorous look at the lives of a diverse cast of characters. The content of the humor make it a webcomic for mature readers.
The artists behind these five webcomics are using new media to present the diverse perspectives of African Americans through the comic art form. Both the comic book and webcomic industries tend to be representative of white culture, and these webcomics offer a refreshing dose of color commentary.
@Tystarr: Thanks for suggesting Twisted Comix. Unfortunately, I was unfamiliar with your webcomic until you mentioned it. However, one reason I wrote this post was to hear from other African American webcomic creators. I will definitely check out your strip.
Oops I posted wrong….
Lol wow some of my fav comics are up there!
But yeah… Twisted Comix is a great strip too.
Woah woah woah, What about Twisted Comix? http://www.Twistedcomix.com
@Amani: Thank you! I really appreciate that!
@Charles: You're very welcome. I hope that other African Americans use the web to present their art to the world and add their voices to the public forum of the internet. Not only that, but all of you have the opportunity to become successful entrepreneurs by selling ads, merchandise, and doing public speaking.
Anjuan, thanks for the shout out of my webcomic, Redux Deluxe. It means alot to me, probably more than you ever know. I was concerned that my webcomic was getting far too few views and not too many were checking my site out, so its beyond great that someone has actually taken the time out to read my comic and suggest it to others as well.
And these are a great selection of webcomics by African Americans on the web. Most of them I'm already familiar with. We definitely need more webcomics from other African Americans and other minority cultures as well in general.
Great article. I need more color in my comics so I am going to subscribe to all five of these. I love the different topic you wrote about. Nice work.
@Fave: Absolutely! Webcomics are like the "funnies" in newspapers that many people still use to get a quick chuckle before starting their day (or, as you said, at some point in their day).
@George: You're welcome! I definitely hope that more people appreciate the great work that you guys are doing in this online artform!
@Michelle: You are very welcome! I think what you are doing with your webcomic is fantastic! You made the research I did looking for artists to feature very enjoyable!
@Brenda: I'm glad to have the opportunity to hopefully give Michelle a little more shine!
@Frank: You're welcome! I'm glad that you have some more webcomics to check out, and I know that others will check out yours!
Thanks for including Company Man with these awesome artists Anjuan! I really appreciate it. Thanks for turning me on to Joe! and Redux Deluxe, as I'll be checking those out soon.
Thank you so much for including my beautiful talented Niece Michelle Billingsley. We are so very proud of her and her talent. We are JOE FANS FOR LIFE. Again, we thank you for representing for all of these wonderful artists.
Awesome article! I absolutely appreciate being one of the 5 to be recognized. This means more to me than you would ever know!
Me along with the cast of "Joe!", thank you, Anjuan!
Thanks for including Addanac City and I in your article. I'm familiar with a lot of these guys (and gal) and it's an honor and pleasure to be included in their company.
Hopefully, with spotlights like the one you have shone upon us, African-American artists and cartoonists will get more notice and have the world drop by our sites to appreciate our hard-working endeavors.
Fantastic post on AA webcomic creators. I appreciate the love you showed to APNF and, while I had most of these on my radar, I now have (Redux Deluxe) to check out as well.
What's great about these webcomics is their ability to entertain (and perhaps) inspire without being overly intrusive or demanding on a reader's time schedule. It's a quick dose of humor that we all need at some point during the day.
@Fredric: I'm glad you liked it! I hope that everyone supports these and other webcomics created by African Americans. We need to honor our artists!
@Latoicha: No, I had not heard of Onyxcon. I'll be travelling on the 13th and 14th, but I will try to make it next year!
Great post! Have you heard of Onyxcon? Onyxcon is a comic book and pop culture convention specifically for African-Americans. Here is a link: http://www.onyxcon.com. It is taking place in Atlanta, August 13 and 14th. You should check it out!
awesome find! i already subscribe to a pug named fender. thanks for the others!


text:
Rihanna and Ryan Phillippe have been dating for months
Don't bother to hide! Rihanna and Ryan Phillipe have been very discreet about their relationship as several sources told Us Weekly they have been dating for months.
Apparently it all started when the singer and her ex Matt Kemp split up. Since then, the beautiful singer and the actor have been regularly, but quietly, seeing each other.
The singer is said to have blown off Ryan Phillippe at a Gucci/Roc Nation brunch in L.A. on Feb. 12, while other sources claim the two spent that night together.
"She thinks he's hot," an insider says. "They totally had sex. And it wasn't even the first time!"
"There was no PDA," an attendee of the Los Angeles party says. "They hung out a bit, but were discreet. It is still very casual."
"Clearly they're keeping it on the downlow!" adds another source.
Image source: Celebrity Paradise


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Big Cupcake
Decorating By veronica970206 Updated 27 Jan 2012 , 4:14pm by cashley
I see on here that many make the BIG cupcake pan cupcake and the liner is made of chocolate...How is this done? If you use a metal pan, do you grease it or use cooking spray and then move around the chocolate to coat it? I just LOVE how it looks and I really want to try it Thank you for any responses in advance
Here is the tutorial from cakecentral for it hope this helps
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text:
I made these red velvet cupcakes for Valentine's Day
I made the heart decorations on top by piping royal icing onto a Silpad and letting them dry for several days before peeling them off. The frosting is my cream cheese frosting but I added some vanilla bean paste, Love that stuff!
Cream Cheese Frosting
8 oz cream cheese softened
1 stick butter softened
2 pounds powdered sugar
1 Tbsp vanilla
1-2 Tbsp milk (if needed)
1 tsp vanilla bean paste
Beat cream cheese and butter for 1-2 minutes, add vanilla. Start adding powdered sugar ~1/2 cup at a time until consistency and flavor is achieved, may add milk to thin icing if needed
Happy Valentine's Day Everyone!


text:
Netsuite has released their 2013_1 version of their PHPToolkit (php library for accessing Netsuite Backend API). Since I had last looked into their toolkit, the Netsuite developers have completely rewritten their toolkit, so I thought I should mention some of my thoughts on the new version:
The Good Stuff
- Object Orientated. The new toolkit makes much better use of objects. Rather than one object (nsComplexObject), there are now objects for logical units like "items" or "customers".
- Easier to Explore: It is now easy to see what properties an object has. You used to have to look at the webservices documentation to see what properties an object had (and what name they used). However, now you can just look at the php object's properties.
- Some backwards compatibility: they created a method called setFields() which allows you to set object values via arrays like the older toolkits. A nice touch to reduce code rewriting.
- More Support: now there are more objects and properties accessible via the API
The Not So Good Stuff
- Not backwards compatible: I had to rewrite almost every call to the Netsuite API. The old methods of using the toolkit are broken. The setFields method is nice, but is still used differently than the old toolkit.
- setFields() fails to handle nullFieldList — Looks like a simple bug in the code (not hard to fix though).
- Not namespaced: The toolkit defines hundreds of objects with names like "Customer", "Item", and "Task". Chances are pretty good that at least one of these names will cause a collision. Note: the toolkit does check for existence of a class before defining their own, but that prevents the use of their class. Namespaces would be a much cleaner solution.
Overall I would say the change is for the better. There is still work to be done, and it would have been nice if there was more backwards computability, but overall the change is a good one.


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Sambhalo Ae Watan Walo - Part2 - Subhash Ghai - Bollywood Songs - Mahendra Kapoor MP3
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Ke Ghar Ab Aaoge - Sandese Aate Hain [Full Song] - Border (1997) MP3
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Des Mere Des - The Legend Of Bhagat Singh | Ajay Devgan | A.R. Rahman & Sukhwinder Singh MP3
Watch Ajay Devgn in the song 'Des Mere Des' from the movie 'The Legend Of Bhagat Singh'. Song Credits: Singer(s): A. R. Rahman & Sukhwinder Singh Music ...
Hindustan Meri Jaan - A scene from Movie Border MP3
Last fighting scene from the Bollywood flick Border with Sunny Deol. Hindustan meri jaan, meri shaan, meri aan. The fellowship of Dignified Indians. Join us on ...
Aye mere watan ke logo jara aankho main bhar lo pani . ( Live ) MP3
Purane supper dupper hit old sada bahar indian hindi songs . ( old is gold )
watan walo MP3
Aab tumhare hawale watan saathiyo MP3
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10 Indian Patriotic Songs & Facts You Should Know | Independence Day Special MP3
On India's 69th Independence Day, Saregama presents a unique compilation of some lesser known patriotic songs sung by famous singers such as Sonu ...
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text:
The board of trustees of the Walter Anderson Museum of Art has appointed Rosemary D. Roosa as interim director. Roosa is from Biloxi, and her career includes adjunct professor of continuing studies at Tulane University (2005-present), estate manager (2000-2007), vice president and then later president of Gulf Coast Coors Inc. (1988-2000) and U.S. House of Representatives as a member of the Capitol Hill staff (1985-1988).
She graduated from William Carey University with a master of science, psychology (industrial and organizational) in 2004 and has also attended Harvard Business School as well as Sophie Newcomb College/Tulane University. She also studied at the American University in Paris, France, with the Junior Year Abroad Program.
Roosa is the daughter of the late Apollo 14 astronaut Stuart Roosa.


text: In the Recent Status Updates widget I use on CycleChat there's a box at the bottom to make a new profile post: Could this option be added to the new XF 1.4 profile post widget please? It would complete the set; quickview , full view ... , comment , add new profile post. Cheers, Shaun


text:
Would you like to save as your currency permanently or for this visit only?
Clean hotel, good service and breakfast at a very good price Nothing, will stay there again
It was quiet. I could relax. The breakfast was good. The staff was very friendly. The tv channels were limited just like a cheap motel. I didn't care for the overall smell of the room, hall, and everywhere.
Most of the staff was very friendly, the breakfast which was included had plenty of options and it was very close to shopping and the university. The room was very clean upon our check in. And in the evening they offered fresh baked cookies. The night staff male was rude and not very helpful. While the breakfast which was included was good, I would have liked to see more fresh fruit options. Also the gym was very small and not appropriately stocked (no towels, no water). The equipment wasn't maintained too well, but it was still usable (noisey, like it needed oiling).
I liked the room set up I was traveling with a boat and management told me I could park around back and take up several spots. So I parked liked management wanted me too and when I woke up there was a car blocking me in. I told the girl at the front desk and she was making every atempt to locate the owner after an hour went by no luck, she the. Calls her boss (the owner) and was ready to have it towed, but her boss said "we can't just tow a guests car, even if they are blocking another guest" he wanted the girl to try again to locate the owner but at that time I was running an hour and a half behind. So at this point i my self had to call the police and the owner didn't want to do anything. The police said the owner needed to call a tow truck. It was suck an inconvence for me to get the truck and boat out. Me being he guest i should not have to call the police and a tow truck. The owner needs to grow a spine and realize that when a guest does something like that and can't be located to remove the vehicle from property. I will not be staying there ever again due to the rude manager/owner.
Location, location, location. The bed was very comfortable. The bathroom was roomy. Huge refrigerator to use. Plenty of drawer space. Decent coffee in the breakfast room. Non-smoking floor smelled like cigarette smoke. Our room did not, but right off the elevator it did and all in the hall. The pillows were too small and soft, offered no support for my neck. I don't like using multiple pillows (they move around too much) but I did here just to get to sleep. One of the towels in the bathroom (when we first arrived) was put on the towel rack obviously unwashed from a previous user...gross. Grateful there were other clean towels to be used. Didn't use the coffee machine because it is stored on the bathroom counter. Who uses a food/beverage item in the bathroom?
It was comfortable! My 8 years daughter loves it! The shower needed longer curtains because water leaked all over the bathroom floor and into the closest. The cleaning crew could use a bit more hospitality!
Friendly Missing cleaning my room the first night
Nothing The place smelt like drugs and there was clearly some illegal activity happening outside. The window to the room had been broken and could be opened from the inside or outside. Definitely wouldn't recommend
My kids loved the indoor pool hotel located close to interstate for easy travel Room was non smoking but you could smell the smoke.
The staff was superb! Very helpful and assisted with everything to my request. The room was equiped with refrigator, security deposit box, ironing, and washing facilities. Wifi was also a great amenity. Well, its appears the hotel is slightly outdated. The carpet and walls could use a facelift to present a more appealing, contemporary look.
Room was big and comfortable , lots of space for just the two of us . Bed was huge , sheets and comforter were clean and I was satisfied with that , the bathroom was clean as well and that was another plus . The towels were small and useless for drying off , that was the only thing that annoyed me . Other than for the price , this place was amazing and definitely more than I expected .
Nothing It was dirty. The carpets felt so disgusting you couldn't walk on in. The furniture was dirty, beds uncomfortable, and the ac was very noisy
The hotel clerk Derrion was the sole redeeming feature of this place. He deserves a raise and a transfer to a facility that is much more worthy of his competence and williningness to help. He quickly refunded our money when it was evident we would not be able to stay. The first room assigned to us was clearly already occupied. The swimming pool had a light fixture loose and floating in the water but still attached to its outlet by its wires. The swimming pool area has no heat, so the area and the water are practically unusable this time of year. The next room assigned to us had a sliding closet door that was completely off its track. The safe was locked and inoperable. The TV had 1 channel. I was lured in by the seemingly low price, but with so much wrong, it was clear that any amount of money was too high. We left after 40 minutes and received all of our money back.
The lady at the front desk was very friendly. She also seemed concerned genuinely when I told her the elevators were broken. The place was not clean. There were towels on the floor and someone's dessert plate on an entry table at 10 o'clock at night when we arrived and the same towels and plate were there when we left in the morning. The rooms were poorly cleaned and the general air was of an old, decrepit hotel. We almost got stuck on one of the elevators and then both stopped working when we tried to go back up to our room. It seemed like it was a beautiful hotel at one point, but was allowed to rot away.
was a great place to stay whiel I was seeing family, central to everything I needed to do while I was in town, and a welcome respite, a quiet place to retreat, when done with the day. I know they're in the middle of a remodel, but there was a pervasive smell of mildew on the first floor near the pool and near my room.
This place was disgusting. The smell in the lobby was odorus and musty. My room was mediocre at best. I would NEVER stay at this place again. Needs to be closed down.
Bed was comfy. Shower was hot. Towels were clean. Had a pullout couch and desk. I was by the elevator and could hear everyone coming off and going on.
Close vacinity to activities and main roads. Lack of HD TV and a microwave.
Large room, comfortable, quiet No decaf coffee in room, tub drin was not working
The location was great. Children enjoyed to indoor pool area. The bed was very comfortable.
Room was excellent except tub which was peeling paint. Front desk said hotel was currently in progress to refinish tubs Breakfast would have been better with scrambled eggs
It was a beautiful place located in an exclusive safe environment. Me and my husband visited with a co worker and his wife and we were all satisfied with our stay. Enjoyed so much that me and my husband extended a night.. We are already planning on going back this summer. My husband smoke and I reserved a smoking room and once arrived it was a non smoking hotel. This was the only thing that we had an issue with.
Easy to get around from and to the hotel. Beds were ok and bathrooms were ok clean. The can use a new vacuum all over te hotel. Rooms can get undusted and their housekeepers can do a better job cleaning the rooms better.
The bath tub was old and the pair was pealing. It was a total turn off when it came to showering. Other than that it was good for the money spent.
-The bayview room with great bay windows -Location near nice shops -Very friendly and helpful staff -Good wifi; not too slow for a change -Breakfast was pricey, and not normally included (but it was good, and the staff was great). -There's some evidence that the rooms themselves are dated but generally the latest remodel was good.
The hotel was impressively decorated and comfortable at the same time. I was greeted by name when I checked in. Despite being a younger traveler I was not talked down to or shown any disdain. The location on the marina offered wonderful views. The breakfast offered was delicious and more than ample. I will definitely return the next time I am staying in the area. The room thermostat would reset to 70 automatically and the TV would not have sound every other time it was turned on. Nothing that was too important or that would keep me from coming back.
actually a beautiful hotel in a awesome waterfront location with a brewery opening next door. place needs some work from the manic elevator to the semi clean shower stall and the clogged air return on the ac and loud whining output vent .
Hotel and room was furnished with beautiful nautical theme. Restaurant and bar downstairs had delicious food and proved to be very convenient. Staff was very friendly and helpful. Proximity to the marina and the Air and Space Museum also very convenient. Parking garage was well maintained and easy to use/ access for guests. Minor cosmetic issues in the room, some menu items in restaurant were overpriced.
I love the look of this hotel as well as the views. The location is pretty perfect as well. I was also very impress with the staff. Very friendly! The room was a little less clean than i would prefer. But this has never been a problem before at this hotel.
The location of my room, the view. I didn't like the fact that I couldn't stay longer. Had to return to work.
The people at the front desk were very nice and inviting. Good value and a decent clean room for the price. The breakfast set up could be better.
the price all was fine
The room was spacious and the hotel was in a very quiet location. The staff was very helpful, especially the guy at the front desk. You will hear people walking above you if your room is on the 2nd or 3rd floor.
This has to be the best staff I've ever seen. Very friendly and helpful. They were never pushy or rude. I had my service animal with me and no problems at all. Unlike others I've stayed at. I will stay there again
Staff was extremely friendly. Had smiles on their faces, held doors open, were on top of it Great to see coming from a customer service background. Housekeeping was accommodating and interactive. If you passed by them in the hallway they would acknowledge your presence and say a friendly hello. Loved having a great display of food for their complementary breakfast which never ran out of food. No fan in the bathroom?...doomed for black mold. Did not care to have the A/C unit on either. Cleaned the front of it because it was filled with dust. Was a nightmare for those who have allergies like myself. The 2 inch gap of fresh air from the window was not cutting it for fresh air flow. Luckily did not have to be in the room as much as we had planned.
I loved the room spacing and location to local eateries. I stayed in a Spring Hill hotel in Vegas and this hotel in Hampton (short of the Vegas flare) was not short stopping. I would highly recommend this as a hotel option. We had to switch rooms because the television was not working (recently remodeled and may have not been connected properly). Thx.
Good location, exceptional rate. Appears to be in a safe neighborhood. Shower stall leaks water on floor. Be careful getting in and out of shower. Clearly it's a problem as our floor had been patched and repaired several times from the looks of it.
We stayed for two days.. The initial front desk staff was awesome; everyone else was nice. the back patio was nice. The pool area was a little small for the four families that were there at once. Lounge area & computer access was great as well. We checked out, then back in the folllowing day so we had different rooms. The first night was great. Second room had a shower drainage issue, which led to loose tiles & i think may have attracted small flies ( i saw about 3-4 that day).
Location is good for upper Peninsula actually it is in Hampton Roads The restaurant staff is Mediocre, the front desk is squished into a corner due to a remodel and it requires you to be right on top of other guests that are checking in, they advertise free wifi but charge and they charge to use the garage which baffles me because the city gives them Use of it(I used to be the city Assessor here so I know)
I like the location of the Newport News Marriott at City Center. There were places to eat, shop, take a leisure walk, beautiful scenery, and accessible.
Came in for the One City Marathon, easy access to freeways and lots of nice food places in walking distance.
Clean well appointed rooms. Excellent breakfast. Friendly staff. Hotel was under construction so not everything was available and difficult to find your way around the hotel. Internet and parking were extra.
Excellent customer service Broken side entrance/exit doors.
The bathroom was clean. Room was cheap enough. Lots of loud neighbors. The room didn't feel super clean and smelled a bit stale
The building needs to be updated. Iron in the room fell apart when picked up, will not stay there again
Staffing was very good bed linen not really clean...housekeeping needs some work...
Clean and comfortable stay. Great location to all things in the area. Breakfast could use an update,. The breakfast was lacking in options and flavor.
There was absolutely NOTHING to like There was a pair of boxers that were already in the room when my girlfriend and I got there. No available steamer, had weird funky smell, oh and the roaches were disgusting. Too bad they were inconsiderate enough to still charge me for the first damn night after all this, the front desk staff are RUDE and CARELESS and I think the manager does a terrible job running it. She's never around anyway. 0 stars, I'd just burn the place and invest in something else if I were the staff.
Nothing The staff and management
Great room for the price, staff was friendly and helpful.
Location and the staff rooms were older and it smelled liked the carpet and furniture smelled like cigarette smoke
Friendly staff and a reasonable breakfast each morning with lots of choices. That was a treat. dID NOT LIKE THAT THE SMOKE FROM cigartette smoke near THE BBQ pit came straight into the room and the whole room smelt of smoke. had to keep windows open. A door snake across the door would help stop this from happening.
Pretty nice accommodations for little money. Breakfast was simple, but good. Better than a typical continental breakfast. The walls and floors were dirty. Floors in both hallways and in our rooms were very dirty / stained. Upstairs hall smelled very bad all 4 days we were there. Our bathroom had plumbing issues. Tub faucet leaked steadily. Toilet ran on it's own about every 15 min. Toilet was loose and wobbled on the floor.
It was very close to Ft. Eustis. It was very clean. No elevator. Had to carry luggage up a flight of stairs.
The Mulberry Inn was a great value for a family vacation. The rooms were clean, the property was well-kept and the location was convenient. The staff was great - from the receptionists to housekeeping to the wonderful ladies at the breakfast - all were exceptionally professional and friendly & made an effort to accommodate the guests. While this hotel was not a romantic retreat or exclusive resort, it was an excellent place to stay for my family - the grounds and the pool were well-managed, the breakfast was great and the rooms were clean - and for less than any of the other neighboring hotels. We weren't expecting high-end accommodations, and we didn't get that. Instead we received a practical, clean and well-managed hotel for a great price. The biggest "issue" was the hallway of our building was a bit worn - it could've used a new carpet & maybe lighting less harsh than the cold LEDs.
The room was clean and the location was definitely a plus. We had a great time during this stay. The front desk staff was very helpful and we enjoyed our stay. We came down to get some breakfast and they started cleaning up 30 minutes before they were set to close. I couldn't understand that. I know they want to leave, but if its supposed to be until 10, why close @ 9:30. One drawback I did not like.
Seems to be rather new and price is good. Sound insulation is not that good.
Customer service was awesome
In room Kurig coffee. Room was previously a smoking room. They tried to cover it up but is smells bad.
The hotel was under construction and I drove 13 miles to spend sometime with my fiance. So much noise for the room I reserved I got a headache so I upgraded and paid for the suite instead. The pool was unavailable because keycard readers didn't work.
Easy check. Nice staff. Great location to my destination. There were a few spider webs on the ceiling. Other than that the stay was great.
Clean room and Friendly Staff. Was chosen as Customer of the Day and given a 'Free Upgrade to a Suite'. Loved the experience that I had at the hotel. Will definitely be staying there again.
clearness, the staff was great and very helpful. When i checked in late on Thursday night, the lady who checked me in was very nice and pleasant. I've stayed here before and I really enjoyed my stay! nothing that i didn't like my stay was great.
It was clean and it was smell great and the staff was very helpful will stay there the next time I go pick up my grandkids No everything was good the hotel was clean and the floor that I was going was just renovated
suite was amazing great breakfast
Spacious room. great for family. conveniently located off the highway. very satisfied. yes, the one bathroom in the private sleeping quarters was a bomber, but we managed. no general closet space for luggage and coats.
The renovations were beautiful!
Breakfast started at 7:00. Earlier would have suited us better. Used a debit card so they held funds for both nights plus about $50 for incidentals for 48 hours after our checkout.
My husband and I have frequently visited Williamsburg. One year we had a difficult time getting a hotel reservation in Williamsburg. We decided to try Newport News. We have been staying in Newport News for the past 10 years whenever we visit Williamsburg. We love it! This is the first time we stayed at a Residence. We were thoroughly impressed! The room was very modern, comfortable and super clean. The breakfast was delicious! The staff was helpful and friendly. The only reason that I didn't give a perfect score is the mattress was a little firmer than what I am used to. We will definitely return and would highly recommend staying there.
It was new, spacious, well-appointed rooms
Friendly staff, clean rooms, great location will stay here again!
Location Expensive
This guest did not leave comments about this hotel.
This guest did not leave comments about this hotel.
location and breakfast shower spigot did not work correctly. Only turned 90degrees, was either scalding or off. I reported to front desk staff, offered to 'have someone come up', it was late, my family was tired. We sponge bathed, should have had a discount
Room and bathroom space the nicest of any Ha,ton Inn we've been at.
It met all of my needs. The room was very clean and quiet, convenient if I needed to pick up anything forgotten at home and very close to where I was visiting and my reason for coming to Virginia in the first place. This was my second time staying at this very hotel and the staff at the reception desk did not seem as friendly and engaged as they did the first time I stayed. My first visit you had a very friendly, cordial woman working the desk that day.
Pool and complimentary breakfast
I love the staff they were very helpful and nice. The room was great and very clean. The breakfast was also great as well. The fresh cookies and milk for guest when you walk in was amazing. The is nothing bad I can say about my stay.
Helpfulness of front desk staff in getting to the site from about two miles away when I got turned around getting there in the first place. Someone put used soap in a new soap box in the bathroom. Only later when my wife opened another box of soap did we discover that my bar of soap was well used already!
Convenient and super-comfy beds The eggs at breakfast were disgusting¿¿
Free continental breakfast, cleanliness of facility
I did find a few mold spots near our tub, and one of the underwater pool lights/wires was yanked out. Otherwise, great. There were a few pieces if broken drywall at one corner of the room, and lots of painted-without-patching screws, if that sort of thing bothers you.
I had a quick stay at this hotel and was so pleased with every aspect. I was departing on a very early flight so I asked if the airport shuttle would be running at 5am. When I was told it wasn't, the front desk employee, Lee told me he would have a taxi waiting in the morning and the hotel would pay for it. Staff was friendly, location was convenient, amenities were lovely. They didn't have a clock in the room and I was told they are in the process of removing them. Odd.
The hotel was very nice on the outside as well as the inside. The staff was very professional, nice and accomadating. The smell in our room was really bad. The room was cleaned thoroughly but it still had an unpleasant odor that we could not quite identify. However, upon informing the front desk, not only did they move us to another room while we we out for the day, but they also moved every single one of our things into our new room with not such much of a hair missing. Thank you!
Excellent location. Terrific professional staff. Clean, safe and quite comfortable. Would stay there again and highly recommend. Minor thing - parking is limited and only one way in and out.
I was able to really enjoy the spa tub! The way it was angled you could also relax and watch TV There was only decaf coffee in my room
The staff was really friendly, rooms were clean and a good sizes
Staff was fantastic, this was the highpoint of the stay Lounge area was very comfortable. The mattresses were very, very uncomfortable. Stayed in a 'lower tier' hotel the previous two nights and the mattresses were far superior. Limited hours for Starbucks .. wondering why it was even there. Limited breakfast offerings ... liked the old style courtyard breakfast restaurant much better.
The staff was extremely nice and the location of the hotel was ideal.
They were ready for us. We got there late and the lady at the desk had everything ready to go. We we were in our room in minutes. Very nice and comfortable stay. Nothing. The visit was nice.
Everything was clean, the shower was good, and the bed was comfortable. There were only three pillows, and they were soft to the point of uselessness (fortunately, we'd brought some of our own). The room was not well insulated - we could hear every word of the people walking past, the TV next door, and every footfall from upstairs. Overall, a disappointing experience.
Nice place, large rooms and beds. Carpet is probably the exact carpet that was installed the day the building was erected in 1840. Staff was very friendly, it's just...dingy like the back side of a casino. It's more of a $49/night hotel but it will suffice and hey...the breakfast room was large so that's always a plus. As explained above